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The incorporation of radioactive carbons of glucose into the protein-bound amino acids of one-day-old mouse brain

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The incorporation of radioactive carbons of glucose into the protein-bound amino acids of one-day-old mouse brain

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Content .THE INCORPORATION OP RADIOACTIVE CARBONS OF GLUCOSE INTO THE PROTEIN-BOUND AMINO ACIDS OF ONE-DAY-OLD MOUSE BRAIN by Howard H. Sky-Peck .A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (Biochemistry) October 1956 U N IV E R S IT Y O F S O U T H E R N C A L IF O R N IA GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES 7 Pb D 'S7 S This dissertation, w ritte n by Howard. H_._.i>ky.;TPeck...... under the direction of.%}A§Guidance Com m ittee, and approved by a ll its members, has been p re sented to and accepted by the F a c u lty of the G raduate School, in p a rtia l fu lfillm e n t of the requirem ents f o r the degree of D O C T O R O F P H I L O S O P H Y Dean Guidance Committee Chairman ACKNOWLEDGEMENTS It is with the utmost respect and gratitude that I acknowledge the invaluable guidance and assistance of Dr. i Donald W. Visser and Dr. Harold E. Pearson, without whom this project would have been a much more difficult task. To Miss Dorothy Lagerborg, for her generous and valuable technical assistance in preparing all of the tissue incubations used in this work, my sincere thanks and appreciation. I wish to thank the National Institutes of Health for the award of a Predoetoral Public Health Service Fellowship, and the Laboratory Division of the Los Angeles County Hospital for the facilities that were made avail able . I should also like to express my appreciation to the Faculty of the Department of Biochemistry and Nutrition for their many kindnesses, understanding, and guidance during my graduate career. For the initial inspiration, constant advice, and confidence, my grateful acknowledgement and sincere thanks to Dr. Biehard J. WInzler. Finally, to my wife, Bernice, I express my appreel- ' ation for her unlimited supply of patience, understanding, and help. TABLE OF CONTENTS CHAPTER PAGE I. INTRODUCTION . ........................... 1 II. HISTORICAL REVIEW .................... 3 III. METHODS AND MATERIALS ................ 10 i Preparation of cultures and Incubation conditions ...... ............... 10 Fractionation of tissues.......... . . 13 Hydrolysis of protein fraction . .... Ik Chromatographic procedures . ........... 16 Preparation of ion-exchange columns . 16 Preparation of buffers............... 1? Operation of the 100 cm. column . . . 19 Operation of the 15 cm. column .... 22 Operation of the Dowex-1 x 8 column . 22 Collection of effluent fractions . . . 2k Analysis of effluent fractions .... 2k Apparatus . . . . ; ............... 25 Reagents............................ 25 Procedure.......................... 2? Calculations . . L *................ 29 Identification of amino acids .... 29 De-salting of amino acids ........... 33 Nature of radioactive materials investigated ..... Jk V. CHAPTER PAGE Biosynthesis of uniformly labeled i lysine..................... 34 Biosynthesis of glucose-3,^-C^ . . . 3? Uniformly labeled glucose ........... 40 Glucose-l-C1^ .......... 40 Glueose-6-C^................ 40 4 Glucose-S-C1*1 , ................. 40 analysis ............. . 41 Coincidence ...... ............. 41 Self-absorption . . ............... 41 Counting er r o r s .................. 44 Method of reporting radioactivity . . 45 Amino acid degradations.............. 45 Degradation of serine ............... 45 Degradation of phenylalanine ..... 48 IV. EXPERIMENTAL RESULTS ...................... 50 Lysine metabolism ............. 50 Glucose metabolism ........... ..... 54 Incorporation of C ^ from glucose into tissue fractions ................... 54 Adsorption ; ............... 57 Incorporation of radioactive carbon from glucose-U-G^^ into the protein- bound amino a c i d s .............. 59 CHAPTEB Demonstration of radioactivity in amino acids ................. Incorporation of radioactive carbon from glucose-l-C1^ Into the protein- bound amino a c i d s . . Incorporation of radioactive carbon from glucose^-Gl*1 ' into the protdin- bound amino a c i d s ........... . . . The fraction of glucose metabolized via glycolysis ...................... Incorporation of radioactive carbon from glucose-2-C1^ into the protein- bound amino acids ................. Incorporation of radioactive carbon from glucose-3into the protein-bound amino ^cids Amino acid degradation .'. . Serine degradation . . . . Phenylalanine degradation V. DISCUSSION OP RESULTS ......... VI. SUMMARY............... .. BIBLIOGRAPHY ...........'. ............ PAGE 66 66 70 76 79 82 83 83 86 89 130 13^ LIST OP TABLES TABLE PAGE I. Composition of Simms' solution........... 11 II. Preparation of buffers.............. ..... 18 III. Relation of absorbance to leucine equiva lents with the Coleman junior spectro photometer ................................ 3® IV. Color yields of amino acids relative to leucine.......................... 32 V. Composition of Chlorella medium ...... 35 VI. The effect of Theiler's GD VII virus on the incorporation of radioactive carbon from lysine into various fractions of minced one-day-old mouse brain ............ 51 VII. Concentrations and specific activities of protein-bound amino acids from one-day-old mouse brain Incubated with uniformly labeled L-lysine in the presence and absence of v i r u s ................. 53 VIII. Incorporation of radioactive carbon from glucose into various fractions of minced one-day-old mouse brain ................. 55 IX. Adsorption of radioactive glucose on the acid-insoluble fraction of mouse brain mince.............................. 58 TABLE X. XI. XII. XIII. XIV. XV. Concentrations and specific activities of protein-bound amino acids from one-day- old mouse brain incubated with glucose- U-C1^ ............................................ Recrystallization to constant specific activ ity of protein-bound amino acids isolated after incubation with radioactive glucose ........... ...................... Concentrations and specific activities of protein-bound amino acids from one-day- old mouse brain incubated with glucose- i-cl* .... ..................... Concentrations and specific activities of proteIn-bound amino acids from one-day- old mouse brain incubated with glucose- 6-C11* ............. ...................... Concentrations and relative specific activ ities of protein-bound amino acids from one-day-old mouse brain incubated with glucose-U-C1^, glucose-l-C1^, glucose- 6-d4, and glucose-2-Cl^............... Oxidation of glucose-l-C^ and glucose-6- C ^ to CO2 by minced one-day-old mouse brain..................................... TABLE XVI. XVII. XVIII. XIX. XX. ix PAGE Concentrations and specific activities of protein-bound amino acids from one-day- old mouse brain, incubated with glucose- 2-C^ .............. .......... 81 Concentrations and specific activities of protein-bound amino acids from one-day- old mouse brain incubated with glucose- 3 ,A-Cli+.......................... 8^ Degradation of radioactive serine isolated from the protein fraction of one-day-old 1 l l r -mouse brain incubated with glucose-U-CA^, glucose-l-C1*1 ', and glucose-6-Clif' .... 85 Degradation of radioactive phenylalanine isolated from the protein fraction of one-day-old mouse brain Incubated with glucose-U-C1^ ............................ 8? Comparison of the incorporation of acetate- l-C1^ and glucose-Z-C1^ into the protein- bound amino aeids from one-day-old mouse brain...................................123 LIST OF FIGURES FIGURE PAGE 1. Schematic representation of tissue fraction ation ..................................... 15 2. Chromatogram of protein hydrolysate from minced one-day-old mouse brain on Dowex- 50, 100 cm. column........................ 20 3. Chromatogram of protein hydrolysate from minced one-day-old mouse brain on Dowex- 50, 15 cm. c olumn.......... 21 4. Separation of glutamic acid from proline on Dowex-1,10 cm. column............... 23 5• Absorption spectra for the reaction of proline and leucine with nlnhydrin .... 28 6. Relation of absorbance to leucine equiva lents ............... 31 ?• Chlorella incubation assembly ....... 36 1 j L i * 8. C-1^ self-absorption curve with Q-gas counter ................................ 42 9. Coincidence correction curve with Q-gas counter................................... 43 10. Apparatus for amino acid degradations . . . 46 11. Chroma,togram of a radioactive ninhydrin- negative fraction from a Dowex-50 treated protein hydrolysate (100 cm. column) . . . 63 X. FIGURE PAGE 12. Chromatogram of protein hydrolysate from minced one-day-old mouse "brain incubated with glucose-U-cl^ (100 cm. column) . . . 64 13. Chromatogram of protein hydrolysate from minced one-day-old mouse brain incubated with glucose-U^C1^ (15 cm. column) .... 65 14. Hypothetical mechanism for the virus Inhibition of lysine incorporation into protein..................................... 91 15. Suggested routes of serine biosynthesis. . . 103 16. Suggested biosynthetic pathways for methionine ................... ...... 106 17. Biosynthetic pathway for aromatic amino a c i d s ........................................116 CHAPTER I INTRODUCTION In recent years considerable Interest has developed in the study of the amino acid metabolism of one-day-old mouse brain in vitro. The investigations on- the incor poration of uniformly labeled glucose into the mixed proteins of minced one-day-old mouse brain showed that C1^ was incorporated into all of the protein-bound amino acids except threonine and prollne (1, 2). It was also shown that the propagation of Theiler's GD VII strain of mouse encephalomyelitis virus resulted in an Increased Incorporation of radioactivity into all of the amino acids labeled with C1^ except lysine and histidine, which de creased in concentration and specific radioactivity (1, 2, 3). Threonine and proline showed no changes in con centration and incorporated no radioactivity from uni formly labeled glucose in the presence or absence of virus. It has been the main purpose of this Investigation to consider the incorporation of radioactive carbon from variously labeled glucose substrates into the protein- bound essential and non-essential amino acids of normal one-day-old mouse brain minces. By using the chromato graphic methods of Moore and Stein (4, 5) the quanti tative separation of amino acids on Dowex-50 columns, 2 it has been possible to determine the extent to which radioactive carbon from glucose is Incorporated into the individual amino acids of the proteins. Such studies, and Information gained from chemical degradation of Individual amino acids, may be of value in elucidating some pathways of mouse brain metabolism. Of particular interest is the study of the mechanisms for the incorporation of from glucose into the essential amino acids of mouse brain. The "essential amino acids" are defined as those amino acids which cannot be synthesized by the mouse and have to be supplied in the diet in adequate amounts in order to pre vent nutritive failure. In the growing or adult mouse these essential amino acids are threonine, valine, methio nine, Isoleucine, leucine, phenylalanine, tryptophane, histidine, and lysine. However, it should be kept in mind that amino acids which are essential nutritionally for a growing or adult mouse are not necessarily essential in a one-day-old mouse brain. The mouse brain tissue used in these investigations was selected for two major reasons: 1. The incorporation of radioactivity from glucose into the essential amino acids of one-day-old mouse brain in vitro from uniformly labeled glucose is unique. 2. The system is reproducible, yielding good quantitative results from experiment to experiment (1). CHAPTER II HISTORICAL REVIEW Parker and Hollander (6) have shown that Theiler's GD VII virus could be grown in Simms1 solution with ox serum ultrafiltrate or with rabbit serum. Pearson (7) described in detail factors affecting the propagation of Theiler's GD VII strain of mouse encephalomyelitis virus in Simms' solution with minced mouse brain preparations. Brain tissue from mice one to two days of age generally yielded more virus than did older brain preparations, although tissue from mice up to nine days old could support virus growth. The temperature range for growth was found to be 31° to 37° and the optimal pH from 7 to 9, the higher pH being more favorable for rapid virus propagation. Pearson and Winzler (8) studied the relationship of oxygen consumption, glucose utilization, and lactic acid production in minced one-day-old mouse brain in vitro in the presence and absence of Theiler's GD VII virus. It was found that the virus propagation had no significant influence on the oxidative or glycolytic metabolism of the host tissue. It was also observed that the oxidative and glycolytic metabolism of mouse brain minces fell off considerably after 12 to 24 hours. In spite of this diminished metabolism, one-day-old mouse brain was still capable of propagating the virus. Thus it appeared that the rate of carbohydrate utilization was not of primary importance in determining the growth of the virus. Rafelson, Winzler, and Pearson (9) studied the rate 3 2 of incorporation of radioactive phosphate (P 0^) into the various phosphorus-containing fractions of minced one-day- old mouse brain since it seemed possible that synthetic reactions might be directly influenced by the virus. It | was found that the uptake of was markedly stimulated ! ! in the phospholipid and phosphoprotein fractions during the period of maximal viral growth. The increased turn over in the phosphoprotein fraction was found to be due primarily to the increased turnover of ribonucleic acid phosphorus. The deoxyribonucleic acid fraction was not affected. These and other studies (10, 11) suggested that the propagation of the virus was intimately associated with the.turnover of phosphorus in the acid-insoluble fractions of the host tissue. In subsequent studies (12) the extent of incorpo ration of radioactive carbon from uniformly labeled glu cose (glucose-O-C-^) into the various fractions of minced one-day-old mouse brain was investigated. The incorpo- 14 ration of glueose-U-C fragments into the protein frac tion was increased, and the incorporation into the lipid fraction was decreased during virus propagation. These observations were interpreted as a possible redirection 5 of earbon metabolism in the virus-infected tissue. Viral growth had no influence on the relatively slow fixation of I C02 into the lipid and protein fractions. The discrepancy | lit between the decreased incorporation of C and the in creased incorporation of into the lipid fraction dur ing virus propagation may have been more apparent than real, since the carbon and phosphorus components of the phospholipid molecule may have been metabolizing independ- i ently at different rates. j I i Rafelson et al.(1) showed that radioactive carbon j from uniformly labeled glucose was incorporated into all of the protein-bound amino acids except proline and threo nine when minced one-day-old mouse brain was incubated for 24 hours with uniformly labeled glucose. During propa gation of virus the amounts and specific activities of lysine and histidine were reduced, whereas the incorpo- 14 ration of C from glucose into the other amino acids except threonine and proline was slgnificantly increased. The unexpected observation that the essential amino acids incorporated radioactive carbon from glucose in amounts roughly equivalent to those found in the non-essential amino acids suggested that the essential amino acids may exist in equilibrium with metabolites derived from glucose. Such incorporation probably does not proceed via carbon dioxide fixation, since it was previously shown (12) that there was relatively small radioactive earbon dioxide i 6 fixation, in the protein fraction of minced mouse brain Incubated in vitro. The above series of investigations showing a signif icant effect of viral propagation on the incorporation of C1* from glucose were conducted in 1949 and 195©* Early j in 1951» in experiments of a similar nature by Winzler et al. (2) it was observed that the effect of the virus on the Incorporation into the lipid fraction was no longer evident and that the total uptake by the protein and lipid fractions was lower than previously reported. The reasons for loss of this virus effect on the lipid fraction and J the decreased total incorporation of radioactivity into I the protein and lipid fractions in these later experiments remain unexplained. The studies carried out previously by Rafelson et al. (1) using the procedures for amino acid chromatography on starch columns were confirmed by Moldave et al. (3) and extended by the use of Dowex-5© resin columns which per mitted a greater resolution of the amino acid mixture. As previously reported, all of the protein-bound amino acids except proline and threonine of minced,one-day-old mouse brain incubated for 24 hours contained appreciable amounts of radioactivity from g l u c o s e I n the presence of virus there was a greater incorporation of radioactive carbon into all of the amino acids which in corporated Cl* except histidine and lysine, which 7 decreased In concentration and specific activity in the presence of virus., The observations that the essential and non-essential amino acids of incubated mouse brain contained appreciable amounts of radioactivity stimulated the investigation on 14 the incorporation of C into the protein-bound amino acids in mouse brain iri vivo to determine whether this phenomenon might be unique to an in vitro system (2). In the mouse brain incubated in vitro, the essential amino adds contained radioactivity roughly comparable to that in the non-essential amino acids; in the in vivo mouse brain, obtained from one-day-old mice injected intraperi- toneally with glucose-U-C1^, the radioactivity associated with the essential amino acids was markedly lower than that associated with the non-essential amino acids. Qual itatively, however, the experiments in vitro and in vivo were similar in that radioactivity was found associated with most of the essential amino acids. A further inves tigation to determine how general the phenomenon of radioactive incorporation into essential amino acids might be was conducted by Incubating one-day-old mouse liver in vitro and in vivo with glucose-U-C^ (2, 3)* Only the non-essential amino acids from liver contained any appreciable amount of radioactive carbon. Proline iso lated from liver following the incubations in vivo showed incorporation of from glucose-U-C-1 -^. The proline 8 fraction from liver incubated in vitro was lost. In order to gain further information as to how the essential amino acids became labeled and to determine the effect of the virus on the mouse brain system in vitro. Moldave (3) used simpler radioactive substrates such as cl^-acetate, C ^-formate, and C^-bicarbonate. From carboxyl labeled acetate, radioactivity was found associ ated largely with glutamic acid, aspartic acid, prollne, 14 histidine, alanine, serine and glycine. C -formate was incorporated mainly into serine, methionine, histidine, and tyrosine plus phenylalanine, and to a lesser extent into cystine, glutamic acid, and aspartic acid. Radio active bicarbonate was incorporated into aspartie acid and glutamic acid only. Degradations yielding the aloha carboxyl groups from the labeled amino acids isolated from the mouse brain proteins following incubations with these various substrates gave information as to mechanisms of labeling of some of the amino acids which is consistent with known metabolic pathways. However, the mechanisms whereby valine, leucine, isoleucine, tyrosine, phenyl alanine, histidine, and lysine incorporated radioactive earbon from giucose^U-C^ remained a mystery. While the catabolic pathways of protein and amino acid metabolism have been extensively investigated, the detailed mechanism of the synthesis of certain of the amino acids is still a matter of great uncertainty, and Is being pursued more actively in microorganisms than in animal tissues. It cannot be assumed that conclusions derived from experiments with microorganisms apply to animal tissues, unless data are available from experiments consistent with such conclusions. The previous investigations compared the incorporation of uniformly labeled glucose into protein-bound amino acids of adult mouse brain, one-day-old mouse brain, and liver. The one-day-old mouse brain incubated in vitro was found to be unique in that all of the essential and non-essential amino acids, except proline and threonine, were signifi cantly labeled from glucose-U-C^. In other systems, radioactivity was largely limited to aspartic acid, glutamic acid, alanine, serine, and glycine. While these studies did not indicate a net synthesis, the appearance of radioactivity in the essential amino acids could be interpreted as synthesis de novo from intermediates of glucose metabolism, unless some unknown exchange reactions are taking place between catabolic products of metabolism and amino acid precursors. CHAPTER III METHODS AND MATERIALS Preparation of Cultures and Incubation Conditions Brain tissue was aseptleally removed from one-day-old Swiss mice and minced at room temperature with scissors so that the volumes of the"tissue fragments were about 0.5 c. mm. Fifty to 75 mg. of minced tissue were placed in sterile, 50 ml. Erlenmeyer flasks containing 2.5 ml. of Simms1 solution previously adjusted to pH 9 with dilute NaOH. The metabolite to be investigated was added to make a total volume of 3.0 ml. The composition of Simms1 solu tion is given in Table I. The flasks were closed with rubber stoppers and incubated without shaking at. 35° for 24 hours. At the end of the incubation period all flasks were tested for sterility after removal of the free CO2 by aeration for 15 minutes into 15 ml. of 0.01 N NaOH. The metabolites investigated under these conditions were glucose-U-C^, glucose-l-C^, glucose-6-C^, glucose- 1 Is 2-C , and glucose-3,4-C . . The labeled glucose to be investigated was added to non-labeled glucose to give a total of 1.0 mg. of glucose per ml. of Simms' solution. The amounts of radioactivity and the total amount of glu cose added to each incubation flask were approximately the same in each Instance except for glucose-3,4-C^\ the specific activity of which was much lower than the other TABLE I COMPOSITION OF SIMMS' SOLUTION Constituent Grams per liter NaCl 8c00 KC1 0.20 CaCl2.2H20 0.15 MgCl2.6H20 0.20 NaHCO^ 1.00 Na2HP04.12H20 0.20 Glucose 1.00 Phenol red 0.0025 12 glucose metabolites Investigated. In one series of experiments, the incorporation of uniformly labeled lysine into one-day-old mouse brain in ! the presence and absence of virus was investigated. To each flask 0.2 mg. of G^-labeled L-lysine per ml. of Simms’ solution was added, in addition to the 1.0 mg. of non-labeled glucose already present. A strain of Theiler's GD VII mouse encephalomyelitis virus which is readily propagated under the above conditions (?) was added to a i series of flasks. The virus was obtained from the super natant fluid of virus-infected, one-day-old mouse brain in Simms’ solution (7). This supernatant was diluted and added in a final dilution of 1 to 1000 so that the minced tissue suspension had a starting titer of 10“2, i..e., a 100-fold dilution would kill half of a group of 5 to 7 mice intracerebrally injected with 0.03 nil. of the super natant. The control flasks received the same amount of supernatant fluid from non-infected tissue. The virus titer was determined in the supernatant fluid, after eentrifugation for 10 to 20 minutes at 1500 r.p.m. In a horizontal head, by the intracerebral injection into mice 3 weeks of age (7). The titer was expressed as the high est dilution that was lethal to at least half of the mice within 21 days after Injection. Fractionation of Tissues 13 The fractionation was carried out essentially by the methods of Schmidt and Thannhauser (13) as previously de scribed (9, 10, 11>). The contents of the incubated flasks were combined and transferred quantitatively to 6 x 1 inch Pyrex test tubes, and 5° per cent trichloroacetic acid was added to a final concentration of 5 per cent. The evolved COg was collected in the standard NaOH solution containing the previously collected free COg, by aeration from the stoppered tubes for 3© minutes with shaking. The amount of C02 was determined by titration of the excess NaOH with HC1. The trichloroacetic acid-soluble (TAS) was separated from the acid-insoluble residue (R) by centrifugation in an International Refrigerated Centrifuge, Model PR-1 with trunnion head, at 2,400 r.p.ra. and 4°. The residue (R) was washed three times for 15 minutes each with 10 ml. portions of 5 per cent trichloroacetic acid. The washes were combined and saved for determination of per cent recovery of initially added radioactivity. The lipid fraction (TL) was extracted from the residue (R) by shak ing for 2 hours with 10 ml. of 3;1 ethanol-ether and refluxing twice for 30 minutes each with 5 nil® portions of 1:1 chloroform-methanol. The remaining protein fraction (TP) was washed twice with 5 nil. portions of ether and dried in vacuo. A weighed portion of the protein fraction was analyzed for total radioactivity by solubil izing with a minimal amount of 1 I NaOH (usually 1.0 ml.) for 14 hours at 35°• No insoluble material was found • | j after this treatment. This fraction is not. completely f - protein and contains any nucleic acids or glycogen present in the tissue. Figure 1 is a schematic representation of the fractionation procedure. Hydrolysis of the Protein Fraction The hydrolysis was carried out as described by Hirs | et al. (14). Approximately 50 mg. of the dried protein i j fraction, derived from about 10 culture flasks, were j placed in a 15 x 125 mm* Pyrex test tube with 200 times the sample weight of doubly distilled 6 N HC1. The tube was vacuum sealed with the aid of an oxygen torch. The protein was hydrolyzed for 20 hours at 110 ± 2° in an oven. Little humin formation was observed during hydrolysis. The hydrolysate was evaporated to dryness and the excess HC1 removed under vacuum with the aid of a Einco rotating evaporator. The hydrolysate was washed into a 10 ml. volumetric flask with water and made up to the appropriate volume with water. This solution was stored at -5° until used. Generally, hydrolysis was completed just prior to the start of a chromatographic analysis. 15 FIGURE 1 REPRESENTATION OF TISSUE FRACTIONATION PROCEDURE r Brain + Simms 1 C^^-Labeled Substrate Aeration C02 -*------ Precipitate with * 5 per cent trichloroacetic acid. C02 **------- f Acid Soluble (TAS) Residue (H) Extract with ethanol-ether and chloroform- methanol Llpld (TL) Y Protein (TP) Chromatographic Procedures 16 The chromatographic methods of Moore and Stein for the separation of amino acids on Dowex-5© resin using citrate and phosphate buffers (5) have been used in these studies. Preparation of Ion-Exchange Columns The chromatographic tubes used in these experiments were of the Zechmelster-Cholnoky type with ground glass joints and coarse sintered glass plates, and had an inner diameter of 0.9 em. The tubes for columns of resin 100 cm. in height were jacketed to permit temperature control by circulation of water from a constant temperature bath. The jacket, similar to that of a condenser, was 108 cm. long and was constructed of 2 cm. (outer diameter) glass tubing. The tubes for columns of resin 15 cm. in height were operated without a jacket at room temperature. Dowex-50 x 8 (200 to 400 mesh) was purchased from the Dow Chemical/.Company, Midland, Michigan, and was washed repeatedly with HC1, distilled water, and NaOH as de scribed by Moore and Stein (5)* For preparation of the columns, a portion of the resin was washed on a filter with small amounts of 0.1 M sodium citrate buffer, pH and suspended in 2 volumes of the buffer. All bubbles were removed from the slurry by gentle stirring after it had been allowed to stand for an hour. To obtain uniformly efficient 100 cm. columns, it was found desirable to pour the slurry into the chromatographic tubes in five portions of approximately equal volumes. Each portion was poured through a funnel, the tip of which was bent to direct the stream against the side of the tube. Each portion of the resin was allowed to settle under air pressure of 15 cm. of mercury until no further drop in the height of the surface of the resin occurred and the liquid had fallen to within 10 cm. of the surface of the resin. The 15 cm. columns were similarly poured in two portions. These columns may be reused indefinitely. Preparation of Buffers The composition of buffers is given in Table II. Since the reproducibility of the results on lon-rexchange columns is dependent on pH, the pH values of the buffers were accurately determined. A 0.05 M solution of potassium acid phthalate was used as a pH 4 standard. The various buffers were prepared in 5 liter quantities and stored in the cold with thymol. Immediately prior to use 1.6 ml. of a 50 per cent solution of BRIJ-35 (Atlas Powder Company, Wilmington, Delaware) was added per 100 ml. of a measured quantity of all buffers, sufficient to make one complete chromatographic determination. The detergent in the eluant permitted faster flow rates without the broadening of peaks in the effluent curves. Five-tenths ml. of TABLE II 18 PREPARATION OF BUFFERS pH Composition ^ 3.42 + 0.01 2/ 500 ml. pH 5 sodium citrate + 110 ml. 1.0 N. HC1 + 390 ml. water. Before use add 0.5 ml* thiodiglycol per 100 ml. 4.25 ± 0.05 500 ml. pH 5 sodium citrate 50 ml. 1.0 N HC1 + 450 ml. water. Before use add 0.5 ml. thiodiglycol per 100 ml. 5.0 ± o.i 500 ml. pH 5 sodium citrate & + 5°0 ml. water. Before use add 0.1 gm. disodium versenate 2/ and 1.5 ml. benzyl alcohol per 100 ml. 6.8 ± 0.03 500 ml. 0.1 M Na2HP0^ + 450 ml. 0.1 M NaH2P0^.H20. Before .use add 0.1 gm. disodium versenate 2/ and 1.5 ml. benzyl alcohol per 100 ml. 6.5 ±0.05 42 gm. citric acid monohydrate + 580 ml. 1.0 N NaOH diluted to 1 liter with water. Before use add 0.1 gm. disodium versen ate 2/ and 1.5 ml. benzyl alcohol per 100 ml. 1/ To all buffers, 1.0 ml. of BRIJ-35 solution per 100 ml. is added. 2/ The buffer, pH 5 (0.2 M), is prepared from 21.008 gm. of citric acid (reagent grade) and 200 ml. of N NaOH diluted to 500 ml. 3/ Analytical grade. Contains negligible amounts of ninhydrin*-posltive material when tested at 0.1 per cent concentration. 19 thiodiglycol (purchased from Eastman Kodak Company, Roches ter, N. Y.) were added to every 100 ml. of citrate buffer i J used on the 100 cm. column as an antioxidant to prevent loss of methionine. i Operation of the 100 cm. Column Approximately one hour prior to use, the column was mounted on a fraction collector and water at 38.0 ± 0.5° was circulated through the jacket. A 2.0 ml. aliquot of the protein hydrolysate, equivalent to approximately 10 mg.,| was added to the top of the resin column using a pipet with | a bent tip to facilitate the addition of the sample to the column without disturbing the surface of the resin. The pH of the hydrolysate was usually 2.5 to 3*9* which is considered optimal for samples added to the. 100 cm. column. With columns operated at 50®, It was necessary to use air- free buffers in order to prevent the formation of bubbles on the column as the temperature was raised. Therefore, all buffers were heated to boiling just prior to use, poured into the separatory funnel used as a reservoir, and while still warm, covered with a layer of mineral oil to prevent access of air. The rate of solvent flow through the column was adjusted to about 4 ml. per hour by using the reservoir as a leveling bulb. One-ml. fractions were collected with the automatic fraction collector. Figures 2 and 3 are schematic representations of the separations MICROMOLES O F AMINO ACID FIGURE 2 CHROMATOGRAM OF PROTEIN HYDROLYSATE FROM MINCED ONE-DAY-OLD MOUSE BRAIN ON D0WEX-50 100 CM, COLUMN .proline glutamic glycine aspartic Ithreonine leucine alanine serine 0.1 isoleucine methionine valine lbo pH 3.42, 37-5 2 00 EFFLUENT ML. MICROMOLES O F AMINO ACID FIGURE 3 CHROMATOGRAM OF PROTEIN HYDROLYSATE FROM MINCED ONE-DAY-OLD MOUSE BRAIN ON D0WEX-50 15 CM. COLUMN mixture lysine .2. tyrosine- phenylalanine .1 histidine arginine NH i5o pH 6.5 citrate 20® 100 K-pH 5 4 - pH 6.8 phosphat EFFLUENT ML. 22 obtained on the 100 cm. column and the 15 cm. column, re spectively, showing solvents and temperature changes. The 100 cm. column was stopped after the emergence of leucine. jThe remaining amino acids were separated on the 15 cm. column. At the end of the fractionations the 100 cm. column was cleaned with 100 ml. of 0.2 N NaOH and 100 ml. of citrate buffer, pH 3*^2. The column was then ready for reuse. Operation of the 15 cm. Column The shorter column employed for the determination of tyrosine and phenylalanine and the basic amino acids was operated at room temperature. For this fractionation, a second aliquot of the hydrolysate was taken up in citrate buffer of pH 5*0. The resultant pH was usually about 4-, which is optimal for the 15 cm. column. The column was run under gravity and 1.0 ml. fractions were collected. The rate of solvent flow through the column was adjusted to approximately 4 ml. per hour. The column was cleaned with 0.2 N NaOH before reuse. Operation of the Dowex-1 x 8 Column Prodlne isolated from the 100 cm. column was found to be contaminated with glutamic acid. The proline fraction was further separated on a Dowex-1 x 8 column (0.9 x 10.0 cm.) as shown in Figure 4 by a modification of the method of Partridge and Brimley (15). The column was washed with MICROMOLES O F AMINO ACID FIGURE 4 SEPARATION OF GLUTAMIC ACID FROM PROLINE 0. ON DOWEX-1 10 CM. COLUMN proline 0. 0- . glutamic acid EFFLUENT ML. 24 2 N HC1, followed by distilled water until the effluent was negative for chloride. The proline fraction was brought from pH 3.42 to pH 6.0 with N NaOH and was then added to 1 j the top of the resin column. The column was washed with j 10 ml. of water followed by 5 ml* of 0*1 N HC1. Proline was not retained on this column, but passed through, while glutamic acid was held until the column became acid from i the HC1 added. Collection of Effluent Fractions Effluent fractions were collected automatically with a drop-counting fraction collector manufactured by the Technicon Company, New York. One-ml. fractions were collected in 18 x 150 mm. Pyrex culture tubes as described by Moore and Stein (16). Analysis of Effluent Fractions Each effluent fraction from the columns was spot- tested on filter paper in order to locate the individual amino acids. The color was developed by Immersing the dried filter paper in a solution of 100 mg. of ninhydrin in 10 ml. of n-propanol and 20 ml. of 0.2 M citrate buf fer of pH 5.0, and heating in an oven at 90° for 10 min utes. All amino acid peaks were discernible with nin hydrin except proline. The fractions suspected of contain-- ing proline were spot-tested on filter paper and immersed in a solution of 0.2 per cent isatin in acetone containing 4 per cent acetic acid, dried, and heated in an oven for 10 minutes at 100 as suggested by Saifer and Oreskes (17)• The effluent fractions corresponding to amino acid peaks jwere pooled and their total volume determined. One-ml. aliquots of these pooled fractions were developed colorl- metrically with ninhydrin as described by Moore and Stein, (18). Apparatus. The Coleman Junior Spectrophotometer, Model 6A, was used throughout these studies for the read ings of,the color produced with ninhydrin. For use in the Coleman apparatus, 18 x 150 mm. Pyrex culture tubes were calibrated according to the procedure suggested by Moore and Stein (16) using a solution of methyl red. Reagents. Ninhydrin. The ninhydrin (Nutritional Biochemicals Corp., Cleveland, Ohio) was recrystallized by heating 100 gm. of ninhydrin in 250 ml. of water with 5 gm. of activ ated carbon, filtering the hot solution, and storing the filtrate overnight at 4°. The recrystallized ninhydrin was washed 5 times with 20 ml. portions of cold water and the air-dried crystals stored in a dark glass bottle. Hydrindantin. 80 gm. of ninhydrin in 2 liters of water at 90° were added with stirring to a solution con taining 80 gm. of ascorbic acid (Merck) in 400 ml. of water at 4f d?. Crystallization started immediately and proceeded for 30 minutes. When the solution cooled to room temperature the hydrindantin was filtered off, washed with cold water, and dried over P2O5 i» & vacuum desic cator. The crystals were stored in a dark bottle away from light. Sodium Acetate Buffer pH 5.5. gm. of CH3COONa.3H20 were added to 400 ml. of water and brought into solution by stirring on a steam bath. When this had cooled to room temperature, 100 ml. of glacial acetic acid were added and the solution diluted to one liter. The solution was 4 N and had a pH of 5*51- O.03. The buffer was stored at 4°. Methyl Cellosolve. Methyl cellosolve* purchased from the Mefford Chemical Company, Los Angeles, was doubly distilled in glass. The product gave a negative peroxide test with 4 per cent aqueous KI. Ninhydrin Solution. 2.0 gm. of ninhydrin plus 0.3 gra. of hydrindantin were added to 75 ml. of methyl cello solve. To this solution were added 25 ml. of the pH 5*5 buffer and the two were mixed without incorporation of air bubbles. The reddish reagent was immediately trans ferred to a brown bottle and stored under nitrogen in the cold. Reagent stored in this manner was reliable for one week only. Diluent Solution♦ A mixture of equal volumes of ethanol and water was used for dilution of the reaction mixture. 27 Standard Amino Acid Solution. A 1.0 ml. aliquot of a 1.0 mM aqueous solution of leucine was diluted with 9 v-ol- j umes of water prior to each set of analyses. Procedure. Triplicate samples of the blank and stand ard were pipetted into photometer tubes. For analysis of the effluent fractions from the ion-exchange chromatograms, 1.0 ml. was used followed by 1.0 ml. of the ninhydrin solution. The buffer was sufficiently strong that prelim inary adjustment of the pH of the samples was unnecessary. Aluminum caps were placed on the tubes, and the tubes were shaken briefly for 15 seconds by hand prior to heating for 15 minutes in a boiling water bath. To each tube were then added 5*0 ml. of the diluent solution, and the tubes were allowed to cool below 30° before thoroughly shaking for one minute. The tubes were then read in the spectro photometer, starting 15 minutes after removal from the water bath. No more than 50 tubes were analyzed at a time in order to permit completion of readings within one hour. Samples were read at an absorption maximum of 570 np except for proline, which was read at *+40 np (Figure 5)* If the absorbance was above 1.00 on the Coleman spectro photometer, 5*0 ml. of additional diluent were added to the solutions. The blank readings for 1.0 ml. samples and 1.0 ml. of ninhydrin solution were 0.05 to 0.10 on the optical density scale. ABSORBANCE 28 “f i FIGURE 5 ABSORPTION SPECTRA FOR THE REACTION OF PROLINE AND LEUCINE WITH NINHYDRIN l.O- leucine proline 0.2. 400 600 WAVELENGTH mju 29 Calculations. A standard curve was prepared for 1.0 ml. of aqueous solutions of leucine at nine concentrations, varying from 0.1 to 2.0 millimoles per liter. Prom Table III a graph was prepared giving the mlllimolar concentra tions corresponding to absorbance readings. Figure 6 shows that the color development with concentration and dilution obeys Beer's law. The color yields for the individual amino acids per mole can be expressed relative to the leucine value as 1.00. The apparent millimolar concentrations as calculated from Table III for 1.0 ml. of aqueous samples were divided by the actual millimolar concentrations of standard solu tions of the individual amino acids to give the color yields shown in Table IV. For calculation of results, the apparent concentrations of the amino acids obtained from Table III were divided by the color yields from Table IV. Identlficatlon of Amino Acids Filter paper chromatography was carried out on the peaks isolated from the columns, and the amino acids were identified from their values by comparison with known amino acids. Two solvent systems were used, 4:1:5 butanol- acetic acid-water and phenol saturated with pH 9*3 borate buffer (19). The comparison of curves obtained from a hydrolysate of bovine serum albumin with those obtained by Moore and Stein (5) and from mouse brain hydrolysates 30 TABLE III RELATION OF ABSORBANCE TO LEUCINE EQUIVALENTS WITH THE COLEMAN - JUNIOR SPECTROPHOTOMETER 1/ Spectrophotometer Readings Millimoles of Leucine per Liter Volume of Diluent Added 5 ml. 10 ml. 15 ml. 0.10 0.07 0.25 0.1? 0.10 0.50 0.33 0.20 0.11 0.75 0.51 0.31 0.18 1.00 0.68 0.41 0.25 .1.25 O.85 0.51 0.31 1.50 I.05 0.62 O.36 1.75 1.20 0.70 0.44 2.00 1.30 O.83 0.51 1/ 1.0 ml. aqueous sample Of leucine of the concentration indicated, treated with 1.0 ml. of ninhydrin solution. ABSORBANCE FIGURE 6 RELATION OF’ ABSORBANCE TO LEUCINE EQUIVALENTS 31 5. ml. Diluent 10 ml. Diluent 15 ml. Diluent 0.2 2.0 MILLIMOLES OF LEUCINE PER LITER 32 TABLE IV COLOR YIELDS OF AMINO ACIDS RELATIVE TO LEUCINE Amino Acid Relative Color Yield Aspartic acid 0.9^ Threonine 0.9^ Serine 0.95 Glutamic acid 0.99 Proline ^ 0.225 Glycine 0.95 Alanine 0.97 Cystine 0.55 Valine 0.97 Methionine 1.02 Isoleucine 1.00 Leucine 1.00 Tyrosine 1.00 Phenylalanine 1.00 Histidine 1.02 Lysine 1.10 Arginine 1.03 1/ Read at kkO millimicrons. 33 showed that the positions of the amino acid peaks were ^ almost identical. Moore and Stein have shown that in a large series of chromatograms integration of the effluent j curves gives recoveries of 100 ± 3 per oent. The accuracy of the method as employed here, on the basis of recoveries of amino acids from synthetic mixtures, was approximately 100 ± 5 per eent. De-Salting of Amino Aeids Radioactivity in amino acid samples containing low amounts of activity was difficult to measure due to the self-absorption effect of the large amounts of salt in the eluting solutions. Buffer salts, detergent, and thiodi glycol were removed quantitatively from individual amino acid eluates by adsorbing these on Dowex-2 x 8 columns 0.9 cm. in diameter and 10 cm. in length, by the procedure of Dr£ze (20). Each column was washed with 20 ml. of 2 N NaOH (COg-free), followed by water until the effluent was neutral to litmus. The sample containing the amino acid to be eluted was added to the column, followed by 20 ml. of water. Ten ml. of 1 N acetic acid were then added to elute the amino acid. The movement of the acid front could be observed by the color change of the resin. The amino acid was collected as the effluent became acid. Before reuse the column was washed with 10 ml. of 1 N HC1 and 5 ml. of water. 3k I j The de-salting of the basic amino acids was better achieved with a Dowex-50 x 8 column (0.9 x 10 cm.) which was washed with 10 ml. of 1 N HC1 followed by.water until neutral. The amino acid solution was then added to the column and the solution washed through with 20 ml. of 0.5 N HC1. The basic amino acids emerged after 3 ml. of k N HC1 had been added. Nature of Radioactive Materials Investigated Biosynthesis of Uniformly Labeled Lysine The alga Chlorella pyrenoldosa was used for the bio synthesis of radioactive lysine because the alga could be grown on a simple salt medium with CO2 as sole carbon source. The composition of the medium (21) is given in Table V. One liter of the medium, without glucose or agar, was prepared in a 2 liter Erlenmeyer flask, stoppered with a cotton plug, and sterilized for 30 minutes at 115 pounds pressure. When the medium had reached room temperature it was inoculated by means of a platinum loop with Chlorella frQm a stock culture (containing glucose and agar). The flask was then sealed with a sterile rubber stopper con taining a glass tube and stopcock. A CO2-generation chamber (B) containing BaC^Oj was then attached to the side arm of the incubation flask. The apparatus is shown in Figure 7. The system was closed to the outside atmos phere by elosing stopcock 3, and air was removed from the COMPOSITION TABLE V OF CHLORELLA MEDIUM ^ 35 Constituent Grams per Liter KNO3 1.01 MgS04.?H20 0.98 KH2PO4 0.55 NH4H2PO4 0.12 C a (NO3) 2 0,50 H3BO3 0.0057 1/ MnCl2.4H20 0.0035 2/ ZnSOij,. 7H2O 0.0005 2/ CuS0^.5H20 0.0002 2/ HM0O4.H2O 0.0001 2/ Ferric citrate 0.20 Glucose & 20.00 Agar ■£/ 20.00 1/ Adjusted to pH 6.0 with 0.1 N H2SO4. 2/ Used for stock cultures only. 2/ These trace elements were prepared as a separate stock solution and an aliquot of this was added to the medium. FIGURE 7 CHLORELLA INCUBATION ASSEMBLY Aspirator A» INCUBATION CHAMBER B. C02-GENERATION CHAMBER C. 10 PER CENT PERCHLORIC ACID D. 2 N NaOH AS C02 TRAP incubation chamber (A) with the aid of an aspirator. Stop cock 1 was closed, and.C-^O^ was generated by leaking 10 per cent perchloric acid onto the BaC^O^. Stopcock 3 was opened briefly to allow the system to attain atmospheric pressure. The culture was allowed to stay at room temp- jerature in diffuse sunlight until no further growth was * observed. This took approximately two weeks. At the end of this time the incubation flask was aerated and the excess C^02 wap trapped in a solution of Ba (OH) 2. The Chlorella was fractionated, and the protein hydrolysed as previously described (page 14). The hydrolysate was added to the 15 cm. Dowex-50 x 8 column and the lysine isolated. Two milligrams of radioactive L-lysine were obtained in this manner from 100 mg. of protein. The specific activity of the lysine was 4 juc per mg. The lysine was then diluted with non-labeled L-lysine to give a specific activity of 250,000 c.p.m. per mg. of lysine. Biosynthesis of Glucose-3.4-C^ The method adopted for the biosynthesis of glucose- 3,4-C1^ was a modification of the techniques of Wood (22), Solomon (23), and Zilversmit (24). A 250 gm. adult rat was fasted for 48 hours. At the end of this time 600 mg. of glucose in 10 ml. of distilled water was given by stomach tube. Immediately following the administration of glucose a total of 5.0 ml. of O.35 molar NaHC1* ^ - -3 g - j containing 5*0 millicuries was injected Intraperitoneally in five equal doses of 1.0 ml. each at half hour intervals. The rat was kept in a closed system so that the respiratory could be trapped in Ba(0H)2* At the end of 2.5^ours the rat was sacrificed and its liver placed in a 15 ml. Pyrex centrifuge tube containing 30 per cent KOH. The liver glyeogen was isolated by the method of Good (25). The mixture of liver and KOH was heated in a boiling water bath for one hour until the solution became a clear red. After cooling the solution was diluted with 1.2 volumes of 95 per cent alcohol and heated until the mixture started to boll. The mixture was then cooled in an ice bath and centrifuged. The mother liquor was decanted and the remaining alcohol removed by evaporation. This isolation of glycogen was repeated to give a yield of 68 mg. of glycogen. The glycogen was then hydrolysed to glucose by heating in a boiling water bath for 3 hours with 0.6 N HG1. The excess HC1 was removed under vacuum, leaving an amber syrup. The radioactive glucose was further purified by ascend ing paper chromatography (26), by adding the glucose as a long thin band about 5 cm. from one edge of a 50 em. x 50 cm. sheet of Easton-Dlkeman No. 627-030 filter paper, using a mixture by volume of 5 parts ethyl acetate, 2 of pyridine, and 5 of water (27). A 1 cm. wide strip was cut from the chromatogram, and the sugars present were 39 identified by developing a fluorescent yellow color with 0.2 M m-phenylenediamine.dihydrochloride in P®n cent alcohol, heated to 105° for 5 minutes (28). A standard glucose chromatogram was used to identify the radioactive glucose band. The paper chromatogram was then exposed to i Eastman Kodak medical "no screen" X-ray film and developed after 6 hours exposure.(29). The glucose band was traced from the X-ray film, cut out and eluted from the paper and dried. The glucose-3finally isolated had a specific activity of 101,000 c.p.m. per mg. of glucose, with a yield of 56 mg. 1 h The distribution of C in the glucose molecule was determined by incubating an aliquot of glucose containing, 10 mg. and 100,000 c.p.m. with a culture of Lactobacillus easel (2*0. The resultant lactic acid was subjected to oxidation with pota.ssium permanganate to yield aeetaldehyde lA and carbon dioxide. The G Og was trapped as BaC Qj and counted. The aeetaldehyde was oxidized to iodoform and formic acid by the action of sodium hypoiodite. The iodoform was plated directly:.and counted, while the formic acid was plated as its sodium salt and counted. The GO2 fraction represented carbons 1 and 6, and formic acid represented carbons 2 and 5* The results indicated that approximately 95 pez* cent of the radioactivity resided in carbons 3 and The glucose-3,4-0^^ used in the incu- batlon experiments had a final specific activity of_________ 97,000 c.p.m. per mg. of glucose. 40 Uniformly Labeled Glucose Uniformly labeled glucose (glucose-U-G^) was obtained from Isotopes Specialties Company, Burbank, Calif., who 14 prepared it photosynthetically from BaC 0^ in sweet potato leaves by the method of Putman et al. (30). The material was diluted with D-glucose to approximately ?40,000 c.p.m. per mg. of glucose for the incubation experiments. Glucose-l-C^ Glucose-l-C-^ was obtained from Dr. Isbell at the National Bureau of Standards, Washington, D.C., and was diluted with D-glucose to approximately 73° >000 c.p.m. per mg. of glucose for the incubation experiments. Glucose-6-C1^ Glucose-6-C^ was obtained from Dr. Isbell at the National Bureau of Standards, Washington, D. C., and was diluted with D-glucose to approximately 760,000 c.p.m. per mg. of glucose for the incubation experiments. Glucose-2-C1^ Glucose-2-C1^ was obtained from Tracerlab Inc., Boston, Mass., and was diluted with D-glucose to approx imately 700,000 c.p.m. per mg. For the tissue fractions (CC>2, TAS, TL, and TP), duplicate aliquots of each fraction were dried directly on aluminum planehets over an area of 5*3 sq. cm. and counted with a,Q-gas counter (Model D-46, Nuclear, Chicago). The sealer used was an "Auto-sealer” manufactured by-Tracerlab Inc., Boston. The amino acids isolated by chromatography,, were plated directly over an area of 5-3 sq. cm. and counted in the same manner. All samples were corrected for self-absorption to 0.5 mg. per sq. cm. (Figure 8) and a minimum of 2048 counts per sample was taken. The following corrections in counting rates were considered. Coincidence Coincidence error was determined for the gas-flow counting tube by preparing a series of samples of radio active glucose of various activities. The data are shown in Figure 9, and indicate that a linear relationship exists between the amounts of glucose present and the counts per minute up to 10,000 counts per minute. Therefore, it was not necessary to apply coincidence corrections in any of the experiments reported. Self-Absorption It has been reported by Kamen ( - 31) that a self- absorption correction Is necessary when the thickness of FIGURE 8 C1/f SELF ^ABSORPTION CURVE WITH Q-GAS COUNTER 10- s o <y i —i o o o 0.8 RATIO OBSERVED COUNTS PER MINUTE COUNTS PER MINUTE AT 0.5 MG. PER SQ.. CM COUNTS PER MINUTE OBSERVED 43 FIGURE 9 COINCIDENCE CORRECTION CURVE WITH Q-GAS COUNTER 15000 12000. 9000- 6000- lodoo i4doo 2(500 Counts per minute Actual " kk a C^-sample is greater than 0.5 mg. per sq. em.; however, j | the data presented In Figure 8 seems to indicate that a self-absorption correction is necessary for C-^-samples of weights less than this. The ratio between the observed counts and sample weights .was determined for. the Q-gas counter and the 5*3 sq. cm. aluminum planchets. This was done using a constant amount of radioactive glucose dil uted with variable amounts of added non-labeled glucose. Figure 8 was obtained by plotting the log of sample weight per sq. cm. against the ratio of observed counts to the observed counts at 0.5 mg. per sq. cm. thickness. All samples were corrected to 0.5 mg. per sq. cm. by dividing the observed count by the ratio shown in Figure 8. Both background and radiation standard (C1^ Standard, Nuclear, Chicago) were measured before and after each series of experimental samples, and were found to be sufficiently constant that no corrections for variation in the scalar sensitivity were necessary. Counting Errors All samples were counted for sufficient time to pro vide a standard counting error of less than 5 P©*1 cent. The background was counted long enough to give a standard error of less than 2 per cent. The standard error (S.E.) was determined by the equation: S.E. = y ~ ' t 4-5 where N is the total number of counts for the sample, and t is the counting time in minutes. Method of Reporting Radioactivity j t Specific Activity. Specific activities of amino acids\ 1 I (SA) are calculated as the observed counts (corrected) per j I micromole of amino acid. I \ i I Relative Specific Activity. The relative specific | activity (RSA) is defined as the counts per minute per micromole of amino acid divided by the counts per minute i per micromole of glucose added, multiplied by 100. I 5 'i Amino Acid Degradations \ ] Degradation of Serine j 11 1 , n ""I r i "" - i The radioactive serine, isolated from incubation ex periments by chromatography, was degraded and its compo nents analyzed by a procedure adapted from Sprinson and Chargaff (32) and Sakami (33)- The serine fraction was de salted, non-radioactive carrier serine added, and the ma terial reerystalllzed to constant specific activity. Approx imately ^30 jaM of serine were dissolved in 9*0 ml. of 0.5 M sodium phosphate buffer pH 5.8. Three ml. of this solution containing 1^3 (15 mg.) of serine were used in each degradation. The buffered serine solution was placed in the reaction vessel, shown in Figure 10, and aerated to remove excess CO2. Three ml. of 0.1 M NalOlj. were added and the system aerated for one hour to remove the CO2 formed during the reaction: /' 46 FIGURE 10 APPARATUS FOR AMINO ACID DEGRADATIONS A, c°2 trap reaction vessel safety trap vacuum B. "steam generator a t. condenser distillation vessel 47 HOCHoCHCOOH NalO^ ^ HCOOH + HCHO + COo 2| pH 5 . 8 nh2 The C02 was trapped in 10 ml. of G.l N NaOH. An aliquot of this base was titrated with standard HC1 to determine the amount of CO^ evolved. The remaining C02 was precipitated as BaCO^ for the determination of radioactivity. The 'reaction mixture containing HCHO and HCOOH was steam-distilled (Figure 10) into 0.5 M sodium phosphate buffer of pH 7.8. To one-half of this mixture were added 2.0 ml. of an alcoholic solution of dimedon. The mixture was allowed to stand overnight to insure complete precip itation of the formyl dimedon derivative. The formyl dimedon was plated directly on aluminum planchets and counted. ( j ) H_n / HCHO ch3 CH^ ^3 The second half of the distillate was placed in the reaction vessel with enough HC1 to lower the pH to The system was aerated to remove excess C02• Three ml. of an 8 per cent solution of mercuric chloride in 2 per cent CH^C00Na.3H20 and 2 per cent CH^COOH were added to the system and the temperature raised to 100° for one hour. The C02 evolved from the oxidation of formic acid was trapped in 10 ml. of 0.1 N NaOH and determined as de scribed above. Degradation of Phenylalanine Radioactive tyrosine and phenylalanine isolated from the 15 cm. column were de-salted, separated by paper chromatography, and the phenylalanine eluted with water. Non-radioactive phenylalanine was added, and the material recrystallized to constant specific activity. The side- chaln of this amino acid was degraded by a modification of the methods of Vernon (3*0 » Ginger (35) > and Aronoff (36)„ The reactions are as follows: Eighty juM of phenylalanine In 3 ml. of citrate buffer at pH 2.5 plus 150 mg. of ninhydrin were added to the reaction vessel (Figure 10 A) and the system aerated to remove excess CC^. The chamber was heated in a boiling water bath for 3° minutes. The C02 evolved was trapped in 10 ml. of 0.1 N NaOH and the concentration and radioactiv ity determined as previously described. One hundred sixty juM of phenylalanine were added to the reaction chamber with 0.5 gm. of potassium dichromate plus 3 ml. of 25 per cent H2S04 (by weight) and the NHo I ^J^CH2CHCOQH nlnhvdrin^ ^"^CHgCHO 4. NH3+CO2 ^ 3 C00H NHoOH ^ O ' 000 H + 2C02 + NH3 NHoOH mixture heated in an oil bath to I3O0 for 5 hours. The C02 evolved was trapped and determined as above. After cooling, the benzole acid which remained was extracted 3 i | times with benzene and recrystallized from hot water . A small portion was plated directly on an aluminum planchet and counted. One hundred sixty /aM of benzoic acid were decarbox- ylated to aniline by the method of Snyder (37)* To the benzoic acid in the reaction vessel were added 20 mg. of NH2OH.HCI and 250 mg. of polyphosphoric acid. The reaction mixture was maintained for 20 minutes at 160° by means of an oil bath. The C02 was trapped in 0.1 N NaOH as before. The product was adjusted to pH 12 with 5© per cent NaOH and the aniline extracted three times with benzene. Decarboxylation of the benzoic acid obtained as a degradation product of radioactive phenylalanine was not carried out due to the very low specific activity. CHAPTER IV < EXPERIMENTAL RESULTS ! i i Lysine Metabolism j I Previous investigations by Rafelson (1) and Moldave | (3) have shown that the ineorporation of C-^ from uniformly! j labeled glucose into protein-bound lysine of one-day-old } i mouse brain is greatly reduced in the presence of Theiler'sj GD VII virus in vitro. It was also shown that as little j i as 0.5 rag. of lysine per ml. of medium inhibited propaga- j s s { tion of the virus. In order to elucidate further the j relationship of lysine to the Virus, uniformly labeled i - ' - ' ! lysine was added to the medium at a concentration of 0.2 j l*f mg. of C -lysine containing 156,000 c.p.m. per ml. with 1.0 mg. of unlabeled glucose per ml. of medium. This concentration of lysine-did not prevent virus propagation as determined by in vitro hemagglutination titers. 1 L l Table VI gives data for the incorporation of C from C^-lysine Into the CO2, lipid, protein, and acid-soluble fractions from minced brain tissue incubated in the presence and absence of virus. Each figure in Table VI represents a mean of four determinations with its standard deviation. The amount of radioactivity recovered as CGL, was greater for the controls than for the virus-infected Incubates. This difference was shown to be significant by obtaining a P value corresponding to a t value for 51 TABLE VI THE EFFECT OF THEILER1S GD VII VIRUS ON THE INCORPORATION OF RADIOACTIVE CARBON FROM LYSINE INTO VARIOUS FRACTIONS OF MINCED ONE-DAY-OLD MOUSE BRAIN Per cent added "i h C -lysine activity recovered Fraction Control Infected P -i/ co2 0.29 ± .©3 0.15 ± .03 P < 0.01 Lipid 0.17 .03 0.1? HP- .03 I I » o Protein 3.^0 ± .30 2 -7© ± .1^ 0.01< P< 0.02 Acid-soluble 9*K0 ± 2.5 96.0 ± 3.0 0.3 < PC 0.4 1/ The level of significance (P) was obtained from the Table of t, after calculating t by the method of Fisher for small sample statistics. 2/ Standard deviations. Statistical Methods for Research Workers. R. A. Fisher, Hafner Publ. Co., N. Y., (1950). 52 three degrees of freedom. These P values, which are shown in Table VI, were approximated from the Table of t. The lipid fractions showed no significant difference between i - ; |the control and infected incubations. However, there was ! > 1 it ' a significantly greater uptake of into the protein I fractions of the control experiments than in the virus- infected incubations. The data presented in Table VII show the incorporation of G into the individual protein-bound amino acids in the presej&jpe and absence of virus. Hadioaetivity was located in only two of the amino acids isolated, aspartic acid and lysine. The specific activities of aspartic acid from the control experiment were higher than those found in the virus-infected incubates. In either ease the specific activities of aspartic acid were very low. The specific activity for lysine was found to be practically identical in the presence and absence of virus. It can be seen, however, that the virus infected tissue contained lesser amounts of histidine and lysine in the proteins. j This is in accord with previous findings by Rafelson (1) and Moldave (3)* The total incorporation of C^-labeled lysine into the protein-bound lysine was significantly less in the case of the virus-infected tissue. 53 TABLE VII CONCENTRATIONS AND SPECIFIC ACTIVITIES OF PROTEIN-BOUND AMINO ACIDS FROM ONE-DAY-OLD MOUSE BRAIN INCUBATED WITH UNIFORMLY LABELED L-LYSINE IN THE PRESENCE AND ABSENCE OF VIRUS Amino acid juMoles per 100 mg. protein %J Control Infected Specific Control activity & Infected Aspartic acid 20.6 22.0 16 9 Threonine 10.8 12.2 0 0 Serine 18.2 15.5 0 0 Glutamic acid 20.0 19-6 0 0 Proline 34.2 32.7 0 0 Glycine 19.2 19.5 0 0 Alanine 21.3 20.6 0 © Valine 18.5 17.9 0 0 Methionine 10.1 8.7 0 0 Isoleucine 11.5 10.3 0 0 Leucine 20.8 19.1 0 0 Tyrosine and phenylalanine 16.8 15.1 0 0 Histidine 11.8 5.6 0 0 Lysine 29.9 l6.4 1640 1650 Arginine 15.^ 13.9 0 © 1/ Average of two determinations each. 2/ Counts per minute per jumole of amino acid. 5^ Glucose Metabolism " 1 ^ Incorporation of Cj_\ from Glucose into Tissue Fractions ! j In order to gain more information concerning the con- ! i version of glucose to the essential amino acids of one-day-j old mouse brain, a series of more specifically labeled ! ! glucose substrates was used, in addition to the uniformly labeled glucose already employed in this laboratory (2). The variously labeled substrates employed in this inves- 1 h. 1 it . 1 L l tigation were: glucose-U-C . / glucose-l-C , glucose-6-C , glucose-2-G1* 4,, and glueose-3,4-G1^. In all investigations approximately the same specific activity was us<3d In the 14 incubations except for glucose-3,4-C , which had a much lower specific activity due tot-the method of synthesis. Table VII gives data from four different experiments for the incorporation of radiocarbon from glucose-U-C^, glucose-l-C1^, and glucose-b-G-^into the CO2, acid-soluble, lipid, and protein fractions of minced one-day-old mouse brain incubated at pH 9 for 24 hours with 3 mg. of variously labeled glucose substrates of the same specific activity. About 88 per cent of the original C14 activity remained in the trichloroacetic acid-soluble fractions at the end of the incubation period, and from previous experiments would be expected to be present largely as lactic acid and glucose (8). Approximately 3-1 per cent of the initial radioactivity 55 TABLE VIII INCORPORATION OF RADIOACTIVE CARBON FROM GLUCOSE INTO VARIOUS FRACTIONS OF MINCED ONE-DAY-OLD MOUSE BRAIN Per cent added C J^-Klucose activity recovered Fraction Glucose-•U-C1^ 14 Glucose-l-C Glucose-6-C^ | ! Acid-soluble 86. 0 + 86.7 ± 5.2 96.5 ± 6.0 e©2 3.1 ± .©4 1.9 + .02 1.9 ± .03 Lipid ©.? + .03 0.9 ± .03 0.8 ± .08 Protein 1.9 ± .14 1.1 ± .15 1.0 + .06 1/ Average of four determinations. 2/ Standard deviations. 56 was found to be in the carbon dioxide formed during the incubation with glueose-U-C1^. Both glucose-l-C1^ and j l/j. j glueose-6-C evolved approximately the same quantity, 1.9 i 1^ 1 per cent ©f C in the carbon dioxide, during the experi- i mental period. The interesting differences between glu- J cose-U-C1^ on the one hand and. glucose^-C1^ on the other j hand led to a comparison of the radioactive carbon dioxide iii 1 it evolution from glucose-l-C and glucose-6-C as a function of time, and will be presented later in this report. A significant difference is apparent between glucose- lit 1 it - l^ U-C and glucose-l-C or glucose-6-C in the extent of incorporation of radioactive carbon from glucose into the protein fraction of the incubated brain tissue. This difference will be more apparent when the data for the Individual protein-bound amino acids are presented later. Comparison of the per cent uptake of C1^ into the 1 it 1^ lipid fractions from glucose-U-CA and glucose-l-G showed that there is a significant difference.correspond ing to a probability of less than 0.01. The lesser amount of C1^ incorporated into the lipid fractions from 1% glucose-U-C is consistent with the greater evolution of 1 it 1^ COg from the glucose-U-CA incubations. Glucose-l-C and glucose-b-C1^ Incorporate C1^ into the lipid fractions to approximately the same extent, as is indicated by a P ~ 0»2» 5? Adsorption The possibility that radioactivity was adsorbed from acid-soluble material onto the acid-insoluble fractions I was investigated, and the results are presented in Table j IX. Minced one-day-old mouse brain was incubated with j unlabeled glucose for 2k hours. At the end of the incubation period, radioactive glucose was added and the tissue fractionation immediately carried out. Less.nthan 0.05 per cent of the initial radioactivity added was adsorbed by the acid-insoluble fraction, as shown by Table i IX. This would indicate that adsorption of glucose was 1 not a factor which would interfere with any conclusions I reached. The possibility that radioactive acid-soluble compo nents other than glucose might have a greater adsorptive affinity was investigated by Rafelson (12) under the identical conditions presented in this text and in the same laboratory. He found that less than 0.05 per cent of the initial G ^ activity was found in the acid-Insoluble residue after mixing a neutralized acid-soluble fraction 1 k from tissue incubated with glucose-U-C with an unlabeled incubate and fractionating. This author felt that a repetition of this work was unnecessary. 58 TABLE IX ADSORPTION OP RADIOACTIVE GLUCOSE ON THE ACID-INSOLUBLE FRACTION OF MOUSE BRAIN MINCE Per cent of C^-glucose activity recovered Acid-soluble Acid-insoluble 99.57 0.0^ 99.68 0.05 99.97 0.02 99. ^6 0.05 Incorporationof Radioactive Carbon from Glucose-U-C^ Into the Protein-Bound Amino Acids It was shown in Table VIII that minced one-day-old ik% mouse brain incubated with glucose-U-C incorporated significant amounts of radioactivity into the protein ! fraction. The nature of the radioactive components in this protein fraction was known, due to the work of Bafelson (1) and Moldave (3)* The experiment described below, using lb glucose-U-C , was undertaken to verify these previous findings. Table X presents data from this experiment, in which minced one-day-old mouse brain was incubated for 2b hours l i j , with glucose-U-G . The protein fraction was isolated, hydrolyzed, and chromatographed on the Dowex-5© x 8 column as already described In Chapter III. In this experiment 1.0 ml. effluent fractions were collected and the individ ual aipino acids located by spot-testing on filter paper as described by Moore and Stein (b). The individual amino acids were then pooled and aliquots taken for quantitative estimation with ninhydrin and for radioactive analysis. It may be seen from Table X tha,t there was radioactivity * associated with all of the amino acids isolated except threonine, and that entirely comparable amounts of radio activity appeared in the essential and non-essential amino acids in this system. This observation has caused much comment, since the essential amino acids valine, isoleucine, 60 TABLE X CONCENTRATIONS AND SPECIFIC ACTIVITIES OF PROTEIN-BOUND AMINO ACIDS FROM ONE-DAY-OLD MOUSE BRAIN INCUBATED WITH GLUCOSE-U-C14 Amino acid >uMoles per 100 mg. protein Specific activity —^ Aspartic acid 22.1 6080 Threonine 16.7 0 Serine 18.5 2080 Glutamic acid 19-0 4080 Proline & 33-6 2120 Glycine 19.0 1900 Alanine 19.4 5440 Valine 1?.8 2600 Methionine 4.5 1330 Isoleucine 14.9 3150 Leucine 21.1 3290 Tyrosine and phenylalanine 14.9 2570 Histidine 16.4 1090 Lysine 33-2 670 Arginine 14.6 1810 I/ Counts per minute per jomole of amino acid. 2/ After Dowex-1 x 8 purification. 61 leucine, methionine, phenylalanine, lysine, and histidine ! cannot be synthesized rapidly enough in the intact growing j { animal to permit normal growth. Another finding apparent i h_ in the above data is the Incorporation of into proline, which had not been obtained by the previous investigators, j | For this reason further purification of the pooled proline fractions was undertaken by the use of Dowex-1 x 8 columns and by paper chromatography. The data for proline present ed in Table X is for the material purified on Dowex-1 x 8. Except for the presence of radioactivity in proline, the concentrations and radioactivities found in the amino acids did not differ significantly from those found by the previous investigators. It was noted early in the investigation that only 70 to 80 per cent of the total radioactivity in the protein hydrolysate added to the chromatographic columns was recovered in the amino acid fractions. In order to ascertain the cause of this loss, the protein hydrolysate was added to a Dowex-50 column, 10 x 0.9 cm., in the hydro gen form, which retained all of the amino acids. The effluent which passed through the column was ninhydrin- negative, but contained approximately 30 per cent of the initial radioactivity of the hydrolysate. The effluent fraction was then added to the 100 cm. chromatographic column and chromatographed in the prescribed manner. This was done to determine whether radioactivity associated with 62 the amino acid peaks could be due to this fraction. It may be seen in Figure 11 that over 95 per cent of the radioactivity was collected from the first 50 ml. of effluent. This radioactive fraction emerged before the first amino acid fraction (aspartic acid) was eluted from i 5 the column, as shown in Figure 12, which indicated that it had no appreciable part in contributing radioactivity to the amino acids. It has been shown by Dustin et al. (38) that interaction between amino acids and carbohydrates during protein hydrolysis leads to an acidic product from the carbohydrate which is eluted prior to aspartic acid under the same chromatographic conditions as described above. Figures 12 and 13 present data for a preliminary ex periment conducted under the guidance of Dr. Kivle Moldave, in which one-day-old mouse brain was incubated for 24 hours with glucose-U-C^^. The protein fraction was Isolated, hydrolyzed, and chromatographed on the 100 x 0.9 cm. and the 15 x 0.9 cm. Dowex-50 columns. In this experiment 1.0 ml. effluent fractions were collected. Gne-half of an effluent fraction was used for ninhydrin analysis, and one-half for the determination of radioactivity with a gas- flow counter. It may be seen from Figures 12 and 13 that a radioactive peak was associated with all of the amino acids except threonine. These results were the same as those shown in Table X where the amino acid fractions were COUNTS PER MINUTE PER ML. EFFLUENT FIGURE 11 400 300 200 100- CHROMATOGRAM OF A RADIOACTIVE MINKfBRIM-NEGATIVE FRACTION FROM A D0WEX-50-TREATED PROTEIN HYDROLYSATE (100 CM. COLUMN) 50 100 pH 3.42 37.5° 150 r 20 EFFLUENT ML. 00 “ 350 3^0 pH 4.25 50°— >| MICROMOLES O P AMINO ACID FIGURE 12 Q.3- 0.2 - O.fc CHROMATOGRAM OF PROTEIN HYDROLYSATE FROM MINCED ONE-DAY-OLD MOUSE BRAIN INCUBATED WITH GLUCOSE-U-C (100 CM, COLUMN) proline Ih glutamic aspartic threonine serine ninhydrin negative glycine alanine 50 100 . 150 pH 3.42 37,5° valine leucine isoleuclne methionine 200 -1000 ^500 1 250 I pH It. 25 50° — EFFLUENT ML.. o\ COUNTS PER MINUTE FIGURE 13 O M b < o E M s < • 6 cn w b O £ o P3 o H E Of 3 0.2 0.1 - CHROMATOGRAM OF PROTEIN HYDROLYSATE FROM MINCED ONE-DAY-OLD MOUSE BRAIN INCUBATED WITH GLUCOSE-U-C (15 CM:. COLUMN) lb mixture lysine tyrosine- phenylalanine histidine arginine NH? 5T : 16S 7 I5 o 20 | < r pH 5 — pH 6.8, phosphate >| & ■ pH 6.5 citrate EFFLUENT ML. 1000 g H E 500 03 w co b o o O n 66 pooled and determined. After the initial experiment of Figures 12 and 13, it was felt that it would be more prac tical to pool the amino acid fractions and use aliquots I for analysis. In this manner material was always available; I for further analyses, such as degradation. Henceforth, | data pertaining to the amino acids Isolated by ion-exchange chromatography will be based on pooled fractions. Demonstration of Radioactivity in Amino Acids The presence of radioactivity associated with the amino acid peaks was not conclusive proof that the radio activity was contained in the amino acid. This question was examined for a few of the amino acids. Pooled amino acid fractions were analyzed for their specific activities and evaporated to dryness, taken up In a minimal amount of hot water and racemized with M NaOH. Crystalline non radioactive racemic carrier was added and the amino acid recrystallized to a constant specific activity. The results of the recrystallizations are given in Table XI, and indicate that the radioactivity measured in these amino acids probably represents actual incorporation of C from glucose into the amino acid molecule. ik Incorporation of Radioactive Carbon from Glucose-l-C into the Protein-Bound Amino Acids The distribution of radioactivity into the various amino acids from uniformly labeled glucose (Table X.________ TABLE XI 6? I i RECRYSTALLIZATION TO CONSTANT SPECIFIC ACTIVITY OF PROTEIN-BOUND AMINO ACIDS ISOLATED AFTER INCUBATION WITH RADIOACTIVE GLUCOSE Specific activity —^ Directly Calculated kmino acid .uMoles from , after Recrystallization column-2/ addition of carrier 1 2 3 Serine 18.5 2080 46 47 44 45 Phenyl alanine 2/ 2.9 1084 9 11 10 9 Leucine 21.1 3290 133 132 134 133 Lysine 11.0 670 29 31 29 29 1/ Counts per minute of C1^ per jumole of amino acid. 2/ Isolated by paper chromatography. 3/ Specific activity of pooled fractions. 68 Figures 12 and 13) was extensive in both essential and non- essential amino acids, and made difficult the interpret ation of intermediary pathways by which this incorporation may have occurred. It seemed that a study of the incor- ! poration of a more specifically labeled glucose into the protein-bound amino acids might be more easily interpreted. ! The major objective was to investigate which of the carbons j s from glucose contributed to the synthesis of essential amino acids. Table XII presents data for the incorporation of 1 it radioactive carbon from glucose-l-C Into the protein- bound amino acids of minced one-day-old mouse brain Incub ated for 2k hours at 38°. The experimental conditions were Identical to those already described for glucose-U-C^ Incubations. The amount and specific activity of glucose- 1 it l-C added to the medium was the same as that used in the previous experiment (Table X). The data presented are the averages of four separate experiments and had a standard error of the mean of less than 5 P®** eent. It can be seen that radioactivity was incorporated to a significant degree Into aspartic acid, serine, glutamic acid, prollne, and alanine, with a very small incorporation into methionine and tyrosine-phenylalanine. Except for the very small amount of radioactivity associated with phenylalanine and methionine, none of the essential amino acids was labeled from the number one carbon of glucose. It was also noted TABLE XII CONCENTRATIONS AND SPECIFIC ACTIVITIES OF PROTEIN-BOUND, AMINO ACIDS FROM ONE-DAY-OLD MOUSE BRAIN INCUBATED WITH GLUCOSE-1-C1^ Amino acid uMoles per 100 rag. protein Specific activity ^ Aspartic acid 22.3 1995 %/ Threonine 15-0 0 Serine 16.7 804 Glutamic acid 20.3 2?80 Proline 34.6 1604 Glycine 20.2 0 Alanine 21.0 2550 Valine 19.6 0 Methionine 9.8 146 Isoleucine 12.8 0 Leucine 20.2 0 Tyrosine and phenylalanine 15.7 109 Histidine 13.7 0 Lysine 32.3 0 Arginine 15.6 0 3J Counts per minute per jumole of amino acid. 2/ Average of four determinations. 1 2x ^ that glycine contained no radioactivity from glucose-l-CA^,j 1 ! whereas serine Incorporated an appreciable amount of ; i 1 il The data obtained with glucose-l-CA^ caused much speculation as to why glycine and the essential amino acids were not labeled. There was a possibility that the essen- j tial amino acids incorporated C from glucose by some j i alternate metabolic scheme other than by way of the Embden- Meyerhof pathway. If this alternate pathway involved the shunt or "oxidative" mechanism, carbon one of glucose would be lost as C1^03. This would leave the five unlabel ed carbon atoms from glucose as precursors for glycine, valine, isoleucine, leucine, histidine, lysine, and arginine. To test whether this was the case, glucose-6- was employed as substrate. Conclusions from these studies will be discussed in detail in a later section. ' 1 h . Incorporation of Radioactive Carbon from Glucose-6-C into the Protein-Bound Amino Acids Glucose-6-C^^ was Incubated with minced one-day-old mouse brain for 2k hours at 38°. At the end of this period the brain proteins were isolated, hydrolyzed, and chromatographed on Dowex-50 columns in order to isolate the individual protein-bound amino acids. The conditions and procedures employed were Identical to those for the glucose-U-C-^ and glucose-l-C^ experiments. The specific In activity of the glucose-6-C was approximately the same 71 as that employed for the glucose-U-C^ and glucose-l-C^ j Incubations. j Data are presented lh Table XIII showing the incorpor- " 1 t if ation of radioactive carbon from glucose-6-C into the protein-bound amino acids of mouse brain. The results obtained were not anticipated, and were essentially the l l f , same as those obtained with the glueose-l-C experiments. Radioactivity was incorporated into aspartic acid, serine, glutamic acid, proline, alanine, and to a very small extent methionine and tyrosine-phenylalanlne. None of the other essential amino acids or glycine was labeled in these experiments. The amoknts of radioactivity incorporated . 1 ji, into the labeled amino acids from glucose-6-C incubations were approximately the same as those for the glucose-l~CA^ incubations (Tables XII, XIII). However, there was some indication that the specific activities of the labeled amino acids from the glucose-6-C-^ incubations were higher i/i than those for the glucose-l-C incubations. This would be anticipated if a portion of the radioactivity from glucose-l-C^ were lost as C^Og by way of the shunt mechanism. The radioactive carbon in the six position would not be lost in this manner, hence a higher specific 14" activity could result from glucose-6-C incubations, especially for those amino acids derived from the tri carboxylic acid cycle. In order to determine whether these slight differences were genuine or due to the differences ■ n 72 TABLE XIII CONCENTRATIONS AND SPECIFIC ACTIVITIES OF PROTEIN-BOUND AMINO ACIDS FROM ONE-DAY-OLD MOUSE BRAIN INCUBATED WITH GLUCOSE-6-C Amino acid juMoles per 100 mg. Specific activity 1/ protein Aspartic acid. 20.8 ^ 2140 Threonine 15.7 0 Serine 16.0 980 Glutamic acid to 0 * G O 2500 Proline 34.1 1500 Glycine 20.0 0 Alanine 21.3 3250 Valine 19.2 0 Methionine 10.7 108 Isoleuclne I3.O 0 Leucine 20.6 0 Tyrosine and phenylalanine 16.3 132 Histidine 14.2 0 Lysine 31.8 0 Arginine 15.6 0 1/ Counts per minute per jumole of amino acid. 2/ Average of four determinations. 73 in the initial specific activities of the glucose sub strates employed, Table XIV was prepared to present data for the incorporation of into the amino acids from glucose-U-C1^, glucose-l-C3’ *1 ', glucose-6-Cli+, and glucose- 2-0^ incubations calculated as "relative specific activ ity" . The relative specific activity as used here is the counts per minute per micromole of the amino acid divided by the counts per minute per micromole of glucose sub strate, multiplied by 100. It is evident from Table XIV that there is no significant difference between the amount of radioactivity incorporated into the amino acids from the glucose-l-C^ and glucose-6-C^ incubations. The data presented for the mieromolar concentrations of the amino acids represent the average and the standard deviation from nine individual experiments. The relative;.specific activities from the glucose-l-C^ and glucose-S-C-^ incubations represent the average of four individual experiments each. The data presented for the glucose-2- incubations represent the average of two independent experiments. The deviations about the average are also presented to indicate the extent to which quantitative interpretations may be made from these data. The large difference between the glucose-U-C^^ incubations and the glucose-l-C^ or glucose-6-Cli|' incubations will be discussed in a later section of this dissertation. Table XIV also serves the purpose of showing the reproducibility TABLE XIV i CONCENTRATIONS AND RELATIVE SPECIFIC ACTIVITIES.OF PROTEIN-BOUND AMINO ACIDS FROM ONE- 1 DAY-OLD MOUSE BRAIN INCUBATED WITH GLUCOSE-U-C14 GLUCOSE-1-C14, GLUCOSE^-C^, AND GLUCOSE-2-C1^ Amino Aoid juMoles per Relative Specific Activity ^ 10Q mg. Protein Glucose-U-C^ Glucose-l-< Glucose-6-C1^ Glucose-2-C1^ Aspartic acid 22.1 ± 0.5 ^ 5.o4 *2/ 1.67 ± .09 2/ 1.6? + .08 1.52 (.21)^ Threonine 15.1 ± 1.1 0 0 0 0 Serine 16.1 + 0.8 1.62 0.6? + .02 0.69 ± .03 0.73 (.14) Glutamic acid 20.1 + 0.9 3.^ 2.12 ± .22 1.83 ± .24 2.49 (.13) Proline 34.6 + 1.0 1.75 1.22 + .12 1.15 ± .10 1.01 (.08) Glycine 19.3 t 1.0 1.59 0 0 0.43 (.11) Alanine 20.8 ± 0.8 4.50 1.92 + .09 1.93 ± .08 2.48 (.36) Valine 19.5 ± 0.7 2.15 0 0 © Methionine 10.4 ± 0.6 1.10 0.09 ± .01 0.08 ± .01 0 Isoleucine 12.9 ± 0.9 2.60 0 0 0 -0 TABLE XIV (continued) CONCENTRATIONS DAY-OLD MOUSE AND RELATIVE SPECIFIC ACTIVITIES OF PROTEIN- BRAIN INCUBATED WITH GLUCOSE-U-C14.. GLUCOSE- GLUC0SE-2-C14 -BOUND AMINO ACIDS FROM ONE- -1-C14, GLUCOSE-6-C-^, AND ——— —— — — ---- Amino Acid ;uMoles per Relative Specific Activity ^ 100 mg. Protein Glucose-U-C^^ Glucose-l-C1* * Glucose—6—C^^ Glucose-2-C-^ i i Leucine i 20.7 ± 1.0 2.71 0 0 0 Tyrosine and phenylalanine 16.2 ± 0.7 2.13 0.08 + .01 0.09 ± .01 0.13 (0) Histidine 13.9 ± 0.9 0.90 0 0 O.31 (.01) Lysine 31.7 ± 0.6 0.55 0 0 0 Arginine 15.8 + 0.8 l.k$ 0 0 0 1/ Relative Specific Activity represents the counts per minute per pmole of amino acid divided by the counts per minute per ^imole of glucose, multiplied by 100. 2/ Standard deviation. 3/ One determination only. . kj Average of two determinations with their deviations about the mean in parenthesis. -o VjV 76 of the data from experiment to experiment. The Fraction of Glucose Metabolized Via Glycolysis In an attempt to evaluate the contribution of the glycolytic pathway, in contra.st to non-glyeolytic routes, * 1 ^ to the incorporation of C into the amino acids, glucose- j l-G1^ and glucose-6-C^ were compared as precursors of Gl2f02. Glucose-l-C1^ or glucose-6-C1^, if eatabolized via the glycolytic path to would yield C1^'©2 with the same specific activity due to the fact that the first and sixth carbon atoms of glueose would become the methyl carbon of pyruvate. If another route were operative by which carbon one of glucose appeared as CQ2 more rapidly than did the average of the remaining carbon atoms, the specific activity of C^^02 from glucose-1-C-^ would be 1 j U higher than the specific activity“from glucose-6-C . This approach has been employed in experiments using glucose- l-G^ and glucose-6-C^ with rat diaphragm and liver (39, * 4 - 0), where it was shown that glueose was eatabolized in rat diaphragm to CO2 predominantly via the glycolytic path while rat liver eatabolized almost half of its glucose via the "oxidative" route. While a value less than one in the ratio of C-^02 derived from glucose-6-C^i* ' to the derived from glu- cose-l-C^^ would indicate the existence of a pathway other than glycolytic, the rati© of one does not preclude the 77 possibility that an alternative pathway is being utilized without an increased rate of G1^02 evolution from carbon one. This could be the case if the glucose were metabol ized to fructose-6-phosphate instead of 6-phosphogluconlc acid, and entered the "oxidative" pathway by means of the - transketolase and transaldolase reactions which have been demonstrated in animal tissue by Horecker et al. (41. 42). By this means an alternate pathway could exist, leading to the same intermediary products but without the excess evolution of CO2 from carbon one of glueose, which is associated with the shunt mechanism. With these considerations in mind, minced one-day-old 14 mouse brain was incubated with glucose-l-CA^' and glueose- 6-C1^ of the same specific activity for increasing periods of time. The time intervals chosen were 1, 3» 9, and 24 hours. The shorter time periods were of greater import ance for the interpretation of results, for at longer time periods the system would tend to approach an equilibrium with regard to C1^ (because of recycling), and a ratio of one would be approached. Table XV presents data for the per cent of added substrate recovered as C-^Og for increasing periods of time. The ratios at various time intervals for the from glucose-6-C^ to the from 14 ' glucose-^-l-C are given. It can be seen from the ratios 1 i i * that O2 is evolved at a slightly higher rate from glu eose- l-C^jth^_from glucose-6-C^ at the initial time_____ 78 TABLE XV OXIDATION OF GLUCOSE-1-C12* AND GLUCOSE-6-C1^ TO C02 BY MINCED ONE-DAY-OLD MOUSE BRAIN Per cent of ^ 1JL, Time added substrate C~~. from glucose-6-Cry s G14 from glucose-l-C1^ j Hours Substrate recovered c»o2 Glucose-S-C1* * 0.028 1 Glucose-l-C1^ 0.034 3 Glucose-6-C^ 0.12? Glucose-l-C1^ 0.148 14 Glucose-6-C 0.237 6 Glucose-1-C1** 0.301 Glucose-6-C^ 0.390 9 Glucose-l-C3'^ 0.408 Glucose-6-C1^ 1.89 24 14 Gluoose-l-C 1.96 0.82 0.86 0.79 0.96 0.97 79] | period and approaches a ratio of one within the 24 hour j i period. The data obtained from short incubation periods \ i Indicate that only a very small portion of glucose is metabolized via the shunt mechanism in one-day-old mouse brain; the major portion is metabolized via glycolysis. j I Data for the 24 hour incubation experiments, presented in j i Table VIII, showed no evidence of a shunt mechanism. A | j comparison of the relative specific activities of aspartic acid, glutamic acid, serine, and alanine from 2k hour incubations with glucose-l-C1^ and glucose^-C^ (Table XIV) also showed no evidence of a shunt mechanism. Data obtained under the conditions of these experiments by Pearson and Winzler (8) indicated that the production of lactic acid was slightly in excess of the glucose which disappeared. These data also indicate that glucose was metabolized predominantly via the glycolytic pathway. Incorporation of.Radioactive Carbon from Glucose-2-Cx into the Protein-Bound Amino Acids Incubation of minced one-day-old mouse brain with glucose-l-C^ or glucose-6-C^ gave no incorporation of radioactivity into the essential amino acids. Thus, carbons 2, 3, or 5 of glucose must be responsible for the incorporation of radioactivity into these amino acids, i L l which were labeled from glucose-U-CA incubations. The investigation of Horecker e_t al. (43) of the 80 1 mechanism of pentose phosphate conversion to hexose mono phosphate has shown the formation of an "active glycol- aldehyde" which can condense with aldoses to form longer carbon-chain sugars when catalyzed toy the enzyme trans- ketolase. This new form of two-cartoon fragment, which differs chemically from acetyl-CoA, presents a possibility 14 for a mechanism which might incorporate C into the essential amino acids. To investigate this possibility, 14 glucose-2-G was incubated in the onerday-old mouse brain using the experimental procedures described previously. Table XVI presents data from duplicate experiments showing the incorporation of into the protein-bound amino acids of mouse brain after incubation with glucose- 2-0^. The specific activity of the glucose^-C1^ used was approximately the same as that used in the incubations with glucose-U-C^, glucose-l-C^, and glucose-S-C1* * ’ (Tables X, XII, and XIII). A significant amount of radio activity was incorporated into aspartic acid, serine, glutamic acid, proline, glycine, alanine, histidine, and to a lesser degree tyrosine-phenylalanine. None of the essential amino acids was labeled except for the unexpect ed incorporation into histidine and the small incorpor ation into phenylalanine. While identical qualitative ’ patterns were obtained in the experiments with glucose- 14 14 1-G and giucose-6-C , the results' of this experiment 14 1 4 with glucose-2-C differed in the finding of C I n _____ 81 TABLE XVI CONCENTRATIONS AND SPECIFIC ACTIVITIES OF PROTEIN-BOUND AMINO ACIDS FROM ONE-DAY-OLD. -MOUSE BRAIN INCUBATED WITH GLUC0SE-2-C14 I E ! Amino acid juMoles per 100 mg. protein Specific activity ^ Aspartic acid 22.4 & 2190 ^ Threonine \ 15-0 0 Serine 16.9 1110 jGlutamic acid 20.3 3310 JProline 33-7 1180 blyeine 18.9 685 Alanine 21.3 3580 Valine 18.5 0 Methionine' 12.0 0 Isoleucine 13.4 0 Leucine 21.4 0 Tyrosine and phenylalanine 16.7 175 Histidine 15.1 402 Lysine 30.8 0 Arginine 15-9 0 1/ Counts per minute per punole of amino acid. 2/ Average of two individual experiments. 82 glycine and histidine and the absence of in methionine. The data presented in Table XVI resemble qualitatively those obtained by Moldave (3) for the incorporation of I j from carboxyl-labeled acetate into the protein-bound amino [ ' " I acids. The similarity of G1^ incorporation into the amino j } lit t acids between acetate and glucose-2-C will be discussed, j I . ; | Incorporation of Radioactive Carbon from _Glucose-3 .4-C^ | into the Protein-Bound Amino Acids J While glueose-U-C1^ labeled all of the amino acids j I i investigated except threonine, there was no incorporation j of Cx into valine, isoleucine, lysine, leucine, and arginine from glucose-l-C^, glueose-b-C1^, or glucose- 2-C^. Therefore, it was concluded that carbons 3, 4, or 5 of glucose were responsible for the incorporation of C*^ into these amino acids. Methods for the synthesis of glucose-5, glucose-3-C^, or glucose-4-C^ have not as yet been developed. However, a biologically synthesized glueose-3,4-C1^ was prepared: and incubated in the presence of one-day-old mouse brain for 24 hours at 38°. The amino acids were isolated and analyzed as previously described in this text. The specific activity of the glucose-3 ,**— C-^ used was approximately one-seventh of that used in the previous labeled glucose experiments, due to the small over-all incorporation of from NaHC^O^ into the 3,k position of glucose. For this reason, all interpretations 83 made from the results obtained from glucose-3,4-C^ incu bations will be speculations based om the assumption that the data presented are significant. However, this assump tion may not be valid for quantitative comparisons with the other glucose substrates under investigation. The incorporation of from glucose-3into the protein-bound amino acids is presented in Table XVII. It would appear that radioactivity was associated with all of the essential and non-essential amino acids except threonine. The amino acid with the highest specific activity was alanine, followed by serine. The relatively low incorporation of G ^ into aspartic acid and glutamic acid in comparison to alanine will be discussed later. Amino Acid Degradation Serine Degradations The investigation of the incorporation of from 1 Li, lh ’ glucose-l-G-1^ and glucose-6-C indicated that serine was labeled, and to the same extent, from both precursors (Tables XII and XIII). In order to gain more information as to the primary origin of glycine and serine from glucose, degradations were performed on the radioactive serine isolated from the glucose-U-C^-^, glucose-l-C1^, and glucose-6-C1^ incubations. The data presented in Table XVIII represent duplicate degradations of serine Isolated from the glucose-U-C^, _____ 84- TABLE XVII CONCENTRATIONS AND SPECIFIC ACTIVITIES OF PROTEIN^BOUND AMINO ACIDS OF ONE— DAY-OLD MOUSE. BRAIN INCUBATED WITH GLUCOSE-3,4—C1^" Amino acid juMoles per 100 mg. protein Specific activity^/ Aspartic acid 21.5 ^ CO Threonine 150 0 Serine 17.0 204- Glutamic acid 20.? 59 Proline 31.6 28 Glycine 20.5 61 Alanine 21.1. 550 Valine 19.8 105 Methionine 5-7 174 Isoleucine 12.9 79 Leucine 20.9 68 Tyrosine and phenylalanine 16.6 31 Histidine 13.2 111 Lysine 30.5 36 Arginine 16.8 90 1/ Counts per minute per jumole of amino acid. 2/ Average of two independent incubations. TABLE XVIII DEGRADATION OP SERINE ISOLATED FROM THE PROTEIN FRACTION OF ONE-DAX-OLD MOUSE BRAIN INCUBATED WITH GLUCOSE-U-C14, GLUCOSE- 1-C14, AND GLUCOSE-6-ClZ<' Glucose-U-( 314 1/ Glucose-1-( 'lk V Glucose-6- -O1 4 !/ ,/uMoles of . . . C.p.m.. carbon 2J Perc!Snt 2/ juMoles of C.p.m. carbon "2d Per cent @14 2/ ;aMoles of C.p.m carbon 3/ Per cent C1^ 2/ 1 Serine 429 650 100 429 1000 100 429 1400 100 -C00H 133 195 32.8 135 34 1.0 131 9 0.6 -chnh2 122 198 33.1 123 22 0.9 124 14 1.0 -ch2oh y 131 203 34.1 131 84? 98.1 134 1392 98.4 Total recovery 386 596 389 903 390 1415 Per cent recovery 90.0 91.7 90.8 90.3 91.0 101.0 1/ Duplicate determinations with an average deviation of +2.5 per cent. 2/ Per cent radioactivity in each fraction corrected to 100 per cent recovery: total c.p.m. recovered_________100_____ _____ total c.p.m. in serine per cent recovery of ci^ Jd Counts per minute represents total radioactivity of each fraction isolated, corrected for self-absorption and back scattering. 4/ Recovered as formyl dimedon. 86 glucose-l-C**1 ', and glucose-6-Cli* ' experiments. The over-all recoveries of the various fractions were approximately 90 ( per cent. The final isolation of the alpha carbon as BaCG^i ■ i resulted in an 85 per cent recovery, the added loss being | i due to the oxidation of formate to CO.?. Approximately 98 i j per cent of the amount of incorporated into serine from! j glucose-l-C1^ and glucose^-C1* * - was contained in the beta carbon of serine. The incorporation of radioactivity into the serine isolated from glucose-U-G1^ incubations was evenly distributed throughout the molecule. Phenylalanine Degradations Table XIX presents data from duplicate degradations of phenylalanine which had been isolated from the protein- bound amino acids of mouse brain after incubation with i I I glucose-U-C* . Degradations of phenylalanine from glueose- ^i|r * i h - t £r 1-C , glueose-6-C , or glucose-2-C were not attempted because of the low specific activities of the tyrosine- phenylalanine fractions (Tables XII, XIII, and XVI). Ninhydrin oxidation of the carboxyl carbon of phenyl alanine resulted in an 85.7 per cent recovery of C02, and indicated that 4-2.1 per cent of the radioactivity was contained in this carbon. This is in close agreement with Rafelson et al. (1), who found 4-6 per cent of the total activity in the carboxyl carbon. The chromic acid oxid ation of the carboxyl and alpha earbons accounted for TABLE XIX DEGRADATION OP RADIOACTIVE PHENYLALANINE ISOLATED PROM THE PROTEIN FRACTION OF ONE-DAY-OLD MOUSE BRAIN INCUBATED WITH GLUCOSE-U-C1^ juMoles Total e.o.m. juMoles Recovery Per cent C.o.m. C.p.m. corrected to 100 per cent recovery Per |gnt Phenylalanine —^ 91 900 -COOH ?8 85.7 324 378 42.1 Phenylalanine ^ 152 1500 -CHNH2GOOH 241 . 79 1084 1370 91.5 -O-C00H 61.5 40.5 49 121 8.1 1/ Ninhydrin degradation of carboxyl carbon and isolation as BaCO^. 2j Chromic acid oxidation of aliphatic carbons one and two to CO2 and isolation as BaCO^. Per cent radioactivity in carbon two is obtainable by difference. CO -o 88 91*5 per cent of the total activity. The benzole acid residue isolated from this oxidation accounted for 8.1 per cent of the total radioactivity after correction for recovery. By obtaining the difference between ninhydrin and chromic acid oxidations, ^ +9 per cent of the total radioactivity was estimated to be in the aloha carbon. CHAPTER V, DISCUSSION OP RESULTS 1 i The amount of lysine incorporated into mouse "brain proteins was significantly less in the virus-infected tissue than in the non-infected, although the specific activity of the protein-bound lysine remained the same (Table VII). This would seem to indicate that there was no greater dilution from ndn-radioactive sources in the free lysine pool in the presence of virus, but that the free lysine became less available for brain protein ineor- i I poration. The smaller amount of lysine incorporated could still contain the same specific activity, If it were not diluted by mixing with unlabeled precursors or lysine prior to entry into the protein. It has been shown by Borsook et al. (44, 45) that lysine is decarboxylated in rat liver homogenates to form glutaric acid via rf-aminoadipic acid and <*-ketoadipie acids. By analogy with the biosynthesis of ornithine, it is likely that the semi-aldehyde of <*-aminoadipic acid is the primary precursor of lysine in much the same way that the semi-aldehyde of glutamic acid is a precursor of the ornithine carbon chain (46). The significantly lower ‘ evolution of in the presence of virus may Indicate a change in the metabolic pathways of lysine due to the presence of virus. This Is reflected in the incorporation 90 of radioactivity into the protein-bound aspartic acid in; the presence and absence of virus. With a smaller evolu tion of there is a lower specific activity in aspartic acid. This is possibly due to the C^Og of lower specific activity incorporated into oxaloacetic acid by C02 fixation of pyruvate, which is then transaminated to aspartic acid. However, this mechanism is limited by the very low rate of C0£ fixation which was shown to take place in this system (1). The very low specific activ ities obtained for aspartic acid (Table VII) agree with this finding without being in disagreement with the mech anisms suggested here. Figure 14 shows a possible interpretation of the 14 results obtained. The lower values obtained for G 0g iu the presence of virus may be explained if i:t is assumed 14 that the free C -lysine is shunted to a hypothetical compound X, formed in the presence of virus, leaving less free lysine available for decarboxylation. This would also leave a smaller pool of lysine available for incor poration into the proteins, without changing its specific activity. This explanation is strengthened by the data of Rafelson et al. (1) for the free, amino acids isolated after incubation with uniformly labeled glucose in the presence and absence of virus. It was shown by these authors that the amount of free lysine in the presence of 91 FIGURE lb HYPOTHETICAL MECHANISM FOR THE VIRUS INHIBITION OF LYSINE INCORPORATION INTO PROTEIN Protein containing C^-lysine It C^-compound-X ^v3- ruP C^-lysine Glutaric acid Pyrmvate + c*^02 It Oxalacetate Aspartate 92 virus was much lower than in the non-infected system. The finding of less lysine in the proteins may also Indicate that the virus is causing a change in the type of protein synthesized without affecting the availability of free lysine. It has been shown that radioactive carbon from glu cose is incorporated into most of the amino acids of the protein fraction of minced one-day-old mouse brain after Zh hours of incubation with uniformly labeled glucose. Of interest is the observation that all of the essential amino acids, with the exception of threonine, exhibit appreciable amounts of radioactivity derived from glucose- U-G1^. Thus it would seem that the metabolic behavior of one-day-old mouse brain as used in our studies is differ ent from adult mouse brain, liver, or other tissues investigated. These observations suggest that in this system the essential amino acids may exist in equilibrium with metabolites derived from glucose. It is possible that in a one-day-old mouse brain system in vitro such as the one employed here, the primary breakdown products of essential amino acids are less rapidly disposed of than in the intact animal, and are available for reactions with metabolites derived from glucose, leading to the re-form ation of essential amino acids with high specific activ ities not ordinarily associated with the intact animal. According to Eafelson et al. (1), no net increase in . ____ 93 amino ac:id was demonstrated in any of their in vitro experiments, although the extent of radioactivity of the protein-bound amino acids would indicate a replacement of I 5 to 12 per cent of the amino acids originally present in j ! the mouse brain. It is possible, of course, that the i brain has the ability to synthesize essential amino acids de novo: however, such a postulation would not be in j accord with the results previously reported on the de carboxylation of the essential amino acids obtained from f ■ i these tissue cultures, which seem to suggest a partial { re-synthesis rather than de novo synthesis (1). It is j i difficult, to interpret the intermediary pathways by which j incorporation of radioactive carbon from glucose-U-C^ may have occurred. The extent of incorporation of glucose- 1-C^, glucose—2— , glucose-6-C-^, and glucose-3,^-C^, which might be more simply interpreted, has been investi gated, and the results are presented here for discussion. The presence of radioactive carbon from C -glucose in the non-essential amino acids (except serine) may be explained, in general, by well-demonstrated biochemical reactions. The carbon precursors of the amino acids alanine, aspartic acid, and glutamic acid in the form of their corresponding cC-keto acids pyruvic, oxalacetie, and cC-ketoglutaric, are readily provided from the glycolytic and citric acid cycles. The conversion by transamination 9^ | ■ of the cC-keto acids, arising from glucose, to the corre- j ^ i I spending amino acids has "been well established in mammalian tissue by Krebs et. al. (k-7), Gammarata (**-8), and Meister (^9). If it is assumed that one-day-old mouse brain forms alanine, aspartic acid, and glutamic acid in the same manner as other mammalian tissues, the distribution of radioactive carbon in these amino acids from the various < 1 ft C substrates may be predicted. Uniformly labeled glu cose would be metabolized to uniformly labeled alanine, aspartic acid, and glutamic acid. Glucose-l-C1^ or glu- i jit eose£6£C would give rise to methyl-labeled pyruvate, whieh would undergo transamination to methyl-labeled alanine. Prom the methyl-labeled pyruvate, methyl-labeled acetyl-GoA would be formed. Since acetyl-GoA has been shown by Heidelberger and Potter (50) and Utter (51) to condense with oxalacetate to form biologically asymmetric Ik citric acid, the C would eventually be recovered in carbon four of cC-&©toglutarate, and transamination would result in the correspondingly labeled glutamate. Aspartate would be labeled in the alpha and beta carbons because of its origin from biologically symmetrical succinate. The reaction leading to the production of pyruvate from oxal acetate as described by Wood and Werkman (52), or from malate as described by Ochoa (53) could result also in 95 carboxyl labeling of pyruvate, which through transamin ation could result in carboxyl labeled alanine with a much j lower specific activity. The labeling of these three j 14 ! amino acids using glucose-2-C as substrate would result j * 1 I in C incorporation into the carbonyl group of pyruvate, the carboxyl group distal from the carbonyl group of Of-ketoglutarate, and both earboxyl groups of oxalacetate, which would yield carboxyl-labeled pyruvate. Glucose- i L l 3,4-C would give rise to carboxyl labeled pyruvate, but a lower incorporation into oC-ketoglutarate and oxalacetate would be expected due to a loss of C as C0£ prior to i entry into the citric acid cycle. A comparison of the relative specific activities of those amino .acids associated with the glycolytic pathway and citric acid cycle (aspartic acid, glutamic acid, and alanine) obtained from glucose-l-G^, glucose-2-C^i j ', and glucose-6-G1^ incubations (Table XIV) shows a similar 14 incorporation of G for the three amino acids. A com parison of the specific activities of these amino acids labeled from glueose-3,4-C1^ (Table XVII) indicates that less was incorporated into aspartic acid and glutamic acid than was incorporated into alanine. These results are consonant with the mechanisms described in the previous paragraph. 14 In these investigations an excess of glucose-U-C , glucose-l-G1^, and glucose-6-C1^ was used, each labeled 9 6 compound having approximately the same specific activity. 14 Thus carbon one or six, respectively, from glucose-1-C or glucose-S-C'1 '^ contains six times as much radioactivity i it as the corresponding carbon from glucose-U-C . The data obtained for CO2 evolved (Table VIII) during incubations of minced one-day-old mouse brain show a greater evolution of 0^02 from glucose-U-C^ than from either glucose-1-C^ or glucose-6-Cli|'. This is consistent with a metabolic process whereby a mole of glucose metabolized gives rise to two moles of CO^ prior to entry into the tricarboxylic acid cycle. Thus glucose-U-C^ loses labeled C02 from carbons 3 and h, while unlabeled COg is formed from these carbons in glucose-l-C1^ or glucose-6-C-'-i t '. Aspartic and glutamic acids formed directly from the tricarboxylic acid cycle Intermediates were expected to have lower specific activities from uniformly labeled glucose than from glu- cose-l-C1^ or glucose-6-C1^ incubations because of the 14 1 h . greater loss of C from glucose-U-G as CG>2 resulting in a lower specific activity of acetyl-CoA available for entrance into the tricarboxylic acid cycle. The uptake of G1^ into alanine from the three glucose precursors was expected to yield approximately the same specific activ ities. The finding that the total incorporation of radio activity into aspartic acid, glutamic acid, alanine, and other non-essential protein-bound amino acids was higher from glucose-U-C^ than from gluoose-l-C*^, glucose-2-C^^, 97 1^- or glucose-6-C was unexpected and cannot be accounted for by any of the known metabolic pathways. Although only one experiment was performed using ! 1 it j I glucose-U-C , the results obtained were in close agree ment with previous investigators (1, 3). . The data obtained from glucpse-l-C • * - * * ■ and glucose-6-Cl^ incubations (Table XIV) which represent four experiments for each sub strate, were reproducible. While the two experiments 1 h- employing glucose-2-C show some variation, the incor poration of C1^ into aspartic acid, glutamic acid, proline, i serine, and alanine show relative agreement with the results obtained from glucose-l-C1^ and glucose-6-C^. The results obtained from glucose-3, may be subject to great error due to the very low specific activities in the isolated amino acids. A quantitative comparison between glucose-3and the other labeled glucose substrates is not justified at this time. Further lp.ves- i i j , tlgation using glucose-3,^~C of greater specific activ ity is now in progress. The metabolic interrelationship, of ornithine, pro line, and glutamic acid was early suggested not only from their structural similarity, but also from nutrition experiments of Hose et al. (5^)* Direct evidence for the conversion of glutamic acid to proline and ornithine in the rat was provided by the experimental findings of Sallaoh et al. (55). who found relatively high_____________ 98 1 i t concentrations of CA in proline and arginine isolated from the carcasses of rats fed DL-glutamic acid-5-C^. ! ] The major paths of proline synthesis in E. coll. N. crassaJ and T. utllis have all been shown to involve the same sequence: glutamate, glutamic r-semi-aldehyde,A -pyrrol- ine-5-carboxylate, proline (56). It is of course recog nized that ornithine is not a constituent of proteins, and in the mammal appears to serve mainly as a component in the urea cycle leading to arginine synthesis. The close relationship of proline and arginine to the tricarboxylic acid cycle, as described above, may well explain the Incorporation of into these amino acids 1 I j. from the various C-1 -glucose precursors. Therefore, the i h, finding that the C precursors which labeled glutamic acid also labeled proline is easily rationalized. It Is difficult to explain, however, why these data are in contrast to the findings of Rafelson (1) and Moldave (3), who found proline unlabeled after incubations with uni formly labeled glucose. This difference between the present and previous investigations is unexplainable. A comparison of the data obtained for the incorpor ation of C1^ into the protein-bound amino acids from glucose-U-C^ and glucose-l-C1^ incubations (Tables X, XII) 1A indicates a possibility that the incorporation of C from glucose into the essential amino acids may be derived from some metabolic pathway other than glycolytic. It has been 99 shown "by Corl and Lipmann (57) that the enzymatic oxidation of glucose-6-phosphate proceeds in two steps, through i l 6-phospho-X-gluconolactone to 6-phosphogluconic acid. j j Horecker et al. (58) isolated and identified the product j i of 6-phosphoglueonate oxidation as ribulose-5-phosphate. j It wa.s also shown (59) that the equilibrium of 6-phospho gluconate oxidation is reversible as demonstrated by j i enzymatic fixation of into earbon one of 6-phospho- j I gluconate and by the reductive earboxylation of ribulose- I ! 5-phosphate in the presence of TPNH and CO2• The loss of j carbon one from glucose-l-C^ by the aforementioned meeh- j I i , j anism could account for the non-labeling of the essential j I J amino acids, if the shunt mechanism Is of major importance in the mouse brain. The extent to which this "oxidative" pathway contrib utes to the metabolism of glucose in one-day-old mouse brain has been investigated by comparing the ratio for the per cent evolved during the incubation of glucose- 6-C^ and glueose-l-C*^. The data for the ratio, per cent C^Oz from glucose-6-C1^ ---- per cent C ©2 from glueose-l-C^ as shown by Table XV, indicate that the oxidative or "shunt" pathway plays a minor role In mouse brain. Data obtained under the conditions of these experiments by Pearson and Wlnzler (8) indicate that the production of lactic acid Is slightly in excess of the glucose which 100 disappears. These data also indicate that glucose Is metabolized predominantly via the glycolytic pathway. . | The similarity with respect to the degree and spec- j • i it j trum of labeling in the amino acids from glucose-l-G and j / glucose-S-C1^ incubations (Tables XII, XIII) also leads to a conclusion that the shunt mechanism is not an important pathway under the conditions of these experiments. If the oxidative mechanism involving 6-phosphoglueonate and pentose were a principal mechanism, the quantitative differences between the amounts of incorporated into the amino acids would be greater due to the loss of carbon ! one as C02 from glucose. The question of the primary pathway whereby glucose gives rise to glycine and serine has been the subject of much controversy. Experiments by Shemin in 19^6 (60) clearly established the direct formation of glycine from the oK-carbon, carboxyl carbon, and amino group of serine. Later studies by Elwyn et al. (6l) showed that the ^S-carbon of serine is split off as a one-carbon fragment at the oxidation level of formaldehyde. A considerable number of experiments (62) support the view that from a quantitative standpoint serine is the most important pre cursor of glycine in the mammal. Ghargaff and Sprlnson (63) assumed that pyruvic acid added ammonia to form amino-acrylic acid, which then added water across the ._doubXe_Jbu_nd_tjD._y;ij3!ld._s_erine This-_me_ohanism_was bas_ecL,---- 101 however, on the scheme for the catabolism of serine to pyruvic acid. Arnsteln (64) investigated the synthesis of glycine and serine in folic acid-deficient rats using L-^C"^-alanine, glucose-U-C^, and -glycine as precur sors. In the normal rat, serine and glycine were found to be equally labeled, and to have 8© per eent as much radioactivity as alanine, glutamic acid, and aspartic acid 14 when glucose-tJ-G was fed, but only about 14 per cent in i I I the experiments in which L-CA -alanine was fed. Thus glucose was found to: be a better precursor of serine and glycine than alanine or pyruvate. When glucose-U-C^ was fed to folic acid deficient rats, glycine alone showed a decrease in radioactivity, having only 50 per cent that of serine. Arnstein concludes that the biosynthesis of gly cine from glucose involves serine as an intermediate and that serine is synthesized from a three carbon precursor more closely related to glucose than pyruvate. Snell (65) has shown that serine can be formed non-enzymat1cally from /0-hydroxypyruvate with the aid of pyridoxamine at pH 5 and 100°. Sallaeh and Hose (66) have found that carboxyl labeled glyceric acid is incorporated into carboxyl labeled serine in rats. These authors have also demonstrated the formation of serine from an alanine-hydroxypyruvate trans amination, using an acetone powder extract from dog liver. The various proposals for the nature and origin of serine 102 from glucose are presented in Figure 15 for the purpose of discussion. i In the one-day-old mouse brain system, serine and glycine are labeled similarly from glucose-U-C^ (Table X). Neither glucose-l-C***' nor glucose^-C^ contributes to the labeling of glycine; however, both are equally effective i it as precursors of C -labeled serine (Tables XII, XIII). Radioactive carbon is incorporated into both glycine and serine from glueose-2-S (Table XVI). The specific activ ity of serine appears to be greater than that of glycine | when calculated in terms of micromoles of amino acid; how- • 3 ever, when calculated in terms of micromoles of carbon, j both glycine and serine have the same specific activity. j ' This similarity would be a further indication of the dir ect interconversion of glycine and serine. It appears that both glycine and serine are labeled from glucose- 1 3,^—C ; however, no quantitative interpretation should be made from these data due to the large errors Introduced by the low specific activity of the substrate employed. The distribution of radioactive ca.rbon in serine derived from glucose-1-C^ and glucose^-C^ as shown by degradation (Table XVIII) indicates that all of the radio activity in serine from glucose-and glucose-6-Cl^ is located in the beta carbon atom. This suggests that serine is produced from a three carbon precursor FIGURE 15 SUGGESTED ROUTES OF SERINE BIOSYNTHESIS GLUCOSE CO, TRIOSE-POZj, PENTOSE-PO4 /tf-HYDROXYPYRUVATE PYRUVATE transamination SERINE 1 GLYCINE originating from glucose and. that glycine is derived pri marily from serine or is formed independently from carbons 2, 3, 4, or 5 of glucose. Weinhouse (62) has shown that the intact rat converts carbon one of ribose to hippuric acid glycine as rapidly as it does the carbon of glucose. These findings, according to Weinhouse, suggest a possible synthesis of glycine via glycolaldehyde arising from car bons one and two of pentose through a transketol&se reac tion. It would appear, however, that in animal tissue there is as yet no experimentally established source of a 2-ca.rbon precursor of glycine.. While threonine, choline, glycolaldehyde, and betaine can be converted to glycine, serine remains from a quantitative standpoint the most important if not an obligatory precursor (62). The similarity in the incorporation of into the beta carbon of serine from glucose-l-G^ and glucose-6-C^ (Tables XIV and XVIII) suggests that the three carbon pre cursor of serine is not formed by way of the oxidative or shunt mechanism, but is formed from some intermediate of the glycolytic pathway. Many routes for the synthesis of serine from glucose have been suggested. Some of the major suggestions are presented in Figure 15. From the above results and dis cussion, a conclusion is reached as to the major metabolic pathways by which serine is synthesized from glucose. 105 These pathways are Indieated in Figure 15 "by heavy arrows leading from glucose to serine. Two pathways are suggest ed which involve /^-hydroxypyruvate on the one hand, and some triose phosphate on the other. The data, however, do not justify a choice "between the two alternatives. It has "become evident that the biosynthesis of methio-j nine may follow at least five different pathways: by direct transfer of methyl groups from N-onium compounds such as betaine; by direct transfer of methyl groups from S-onium compounds such as thetins; by the process of trans- methiolation involving thiomethyl-adenoslne and o(-amino- butyric acid; by the reversal of the pathway of synthesis of cysteine from methionine; and by synthesis de novo of the methyl group of methionine from the one carbon units originating from numerous metabolites such as the /f-carbon of serine and the oC-earbon of glycine (6?). Some of the mechanisms leading to the synthesis of methionine are presented in Figure 16. The finding of i/i- lit radioactive carbon from glucose-U-C , glucose-l-C , and glucose-6-C-^ in methionine is not surprising, since it has been established that labile methyl groups may be synthesized both in vivo and in vitro (68). The /£-earbon of serine has been shown by Arnstein (69) and Stekol (70) to be incorporated into the methyl group of methionine in young rats (reaction 1 of Figure 16). In the mouse brain FIGURE 16 SUGGESTED BIOSYNTHETIC PATHWAYS FOR METHIONINE GLUCOSE PYRUVATE SERINE NH3 — y f ASPARTATE ASPARTYL-/&-PHOSPHATE oC-AMINOACRYLATE ‘ ASPARTIC-yd’ -SEMIALDEHYDE 1 HCHO CYSTATHIONINE CYSTEINE + HOMOSERINE +NH3 ^ CC-KETO-lf- » HYDROXYBUTYRATE (C-AMINOBUTYRIC HOMOCYSTEINE 11\\ t h20 Of-KETO-A-BUTENIC OC-AMINQ-^-BUTENIC METHIONINE H O system, the transfer.of the /5-carbon of serine to the methyl group of methionine could account for the labeling 1 ll ±h of this amino acid from glucose-U-C , glucose-l-C , and glucose-6-C^. However, the amount of radioactivity incorporated into methionine from glucose-U-C is approx imately ten times that incorporated from glucose-l-C^ or glueose-6-G1^. This difference in C1^ uptake could indi cate the amount of radioactivity incorporated into the earbons of methionine other than the methyl group. How ever, Hafelson (1) and Moldave (3) found that approximate ly one-fourth of the activity in methionine resided in the 'methyl group and one-fourth in the carboxyl group from uniformly labeled glucose. This would indicate that carbons other than carbon atoms one and six of glucose also contribute to the methyl group of methionine. The apparent absence of radioactivity in methionine from glu- cose-2-Cli|' seems to indicate that carbons one and six of glucose contribute to the methyl group of methionine by way of serine, whereas earbons three, four, or possibly five contribute to the rest of the methionine molecule. This assumption is supported by data on the incorporation of C1^ from glucose-3,4-C1^ into methionine (Table XVII). The possible mechanism for the incorporation of C*^ from glucose-U-C^ and glucose-3 ,*J~C^ into the earbons of methionine other than the methyl group may be 108 postulated from known reactions of other systems if it is assumed that the reactions are reversible. The replaceability of homoserine by or-aminobutyrjc acid has been demonstrated by Emerson in Neurospora (?1). Carroll et al. (72) have shown a rapid conversion of homo serine to cC-aminobutyric acid in rat liver (reaction 2). The presence of oC-aminobutyrie acid has been demonstrated in brain tissue (73). The synthesis of methionine in A. aerogenes (reaction 3) has been shown to be related to the transfer of the thiomethyl group from thiomethyl adenosine to cC'-am'ino'butyric acid (7^)* 1^ this case methionine is synthesized by a direct reversal of the degradative path for methionine in yeast. In animal tis sues methyl mercaptan is formed from methionine (reaction 4) and the reverse process has been demonstrated (75). Since in yeast methyl mercaptan l : s . transferred to <£-amino- butyrie acid to form methionine, it seems possible that this may also occur, in animals. As a speculative sugges tion, methyl mercaptan may add across the double bond of an unsaturated ^ carbon oC-amino acid, just as H2S is assumed (reaction 5) to add to oC-amlnoaerylic acid to yield cysteine (76, 77). Black and Wright (78, 79) have shown that in baker's yeast (reaction $) aspartic acid yields two intermediates, /9-aspartyl phosphate and aspartic-^-semialdehyde, the 109 latter being formed from the former by a TPNH-requirlng dehydrogenase. A third enzyme requiring DPNH was also isolated which converted the semialdehyde to L-homoserine. A possible synthesis of homoserine and possibly homo- | cysteine (reaction 7) has been suggested from a reaction reported to oecur leading to the synthesis, of cC-keto-Tf- hydroxybutyric acid from pyruvate and formaldehyde (80). In view of the fact that glucose-2-C^ does not incor porate into methionine, this series of reactions, Involving aspartic-^-semialdehyde, pyruvate, and formal dehyde, could not be of major importance in the one-day- old mouse brain system. This is especially true since glucose-2-C1^ shows extensive uptake of 0 ^ into aspartic acid and alanine. It has been shown in Neurosnora (81) that methionine may be formed from homoserine and cysteine via cystathionine and homocysteine (reaction 8). However, homoserine is converted in rat liver to oC-aminobutyric acid at such a rapid rate that the reactions of homoserine + cysteine cystathionine ----- > homocysteine can not satisfy the sulfur requirements of the rat (82). A sequence of reactions is presented in Figure 16 which summarizes the various suggested mechanisms for the 1 i d * incorporation of C- * - from glucose into the carbon atoms of methionine. It is suggested here that homoserine may undergo a similar series of events as does serine in its conversion to cysteine by way of cC-aminoacrylie acid (reaction 5). Hypothetically, homoserine could undergo dehydration (reaction 9) to form ”oC-amino-/5-butenic acid" (2-amlno-A^-butenoic acid). This compound could undergo the following reactions: 1) add hydrogen across the double bond to form OC-aminobutyric acid; 2) form oC-keto- /£?-butenic acid by transamination, then add water to become (£-keto-3f-hydroxybutyrate; 3) form homocysteine by the addition of H2S in the same manner that OC-aminoacrylate forms cysteine; and form methionine directly by the addition of methyl mercaptan to the terminal carbon atom (reactions 10, 11, 12, and 13, respectively). The similarity in chemical structure of the three branched chain amino acids valine, leucine, and isoleucine indicates that these substances may be biochemically related. Bonner et. al. (83) found a mutant of Neurosnora which required isoleucine and valine for growth, and further studies showed that oC-aminobutyric acid and threonine could support growth in an isoleucine-requiring mutant. Tatum and Adelberg (8^) using Cl^-labeled acetate in Neurosoora found label in isoleucine and valine, sug gesting a possible common origin of these carbon chains starting with acetate. However, Walker et al. (85) found a much lower incorporation of acetate carbons than of lactate carbons into valine, which suggested that Ill neither acetate itself nor any substance easily derived therefrom metabolically, such as components of the citric acid cycle, is involved in valine biosynthesis. These J findings suggested pyruvate as a direct precursor of val- | ine. Strassman eb al. (86) have shown by isotopic studies T. utllis that pyruvate is apparently the sole source of the carbon chain of valine. Various samples of radio active valine isolated from A. aerogenes (87) grown on acetate-l-cl\ glucose-l-C^-^, and glucose-3, subject ed to degradation gave results identical to those obtained by Strassman. / It was suggested by Strassman (86) that a ketol con densation of pyruvate and acetaldehyde occurred to yield acetolactlc acid. A molecular rearrangement such as the acyloin rearrangement transporting the 3'-Daethyl group up to the 2' or carboxyl carbon of aeetolactate would give the valine carbon chain with the observed isotopic distri bution. Adelberg (88), using 1,2-C^-acetate plus unlabeled threonine in a Neurosoora mutant requiring isoleucine found that the ethyl group of isoleucine came from threo nine earbons 3 and k, and that carbons 1 and 2 of threo nine were incorporated into carbons 1 and 2 of isoleuclne. Adelberg suggests an aldol rather than a ketol condensa tion between the carbonyl of acetaldehyde and the 112 methylene carbon of cC-ketobutyric acid to yield an inter mediary pinacol. Migration of the group from carbon 2 to carbon 3 would eventually lead to isoleucine.. Abelson (89) has found that valine, the ket© analog of valine, and pyruvate dilute the carbons of leucine in E. coli grown on labeled glucose. The acetate carboxyl carbon was exclusively and abundantly found in the carbox yl carbon of leheine (90), and the methyl carbon appeared predominantly in the leucine oC-earbon. Lactate carbon 2 was incorporated into leucine earbons 3 and b, and lactate carbon 3 in leucine carbons 5 and 5'• Abelson suggests that an oC-ketovaline condensed with a 2-carbon compound followed by a decarboxylation to yield the carbon skeleton of leucine. The present work with the one-day-old mouse brain system has confirmed the previous observations (1, 3) that glucose-U-C^ is incorporated into valine, isoleucine, and leucine. Approximately 50 per cent of the radioactiv ity in these amino acids resides in the carboxyl groups (1, 3)- It seems tempting to speculate that these amino acids are formed by condensation of some intermediate with a 2-carbon fragment derived from glucose and that the CC-carbons of these amino acids contain the remaining radioactivity. However, there is no incorporation of radioactivity into these amino acids during incubation 113 » with glucose-1-C1^, glucose-6-C^, or glucose-S-C1^. It was also shown that carboxyl labeled acetate Is not incor porated into these amino acids '(3). The data obtained using glucose-3,4—0 ^ seem to indicate that carbons 3 and i 4 of glucose are incorporated into valine, isoleucine, and leucine. The mechanisms for the biosynthesis of valine, iso leucine, and leucine described briefly above would not appear to be operative in the one-day-old mouse brain. Pyruvate would not be expected to be an important precur sor of any of these, since no isotope from glucose-2-G-^, glueose-l-G1^, glucose-6-C^, or acetate-l-C1^ is incorpo rated into them. The incorporation of C1* * - into valine 14 from glucose-3,4-G is in agreement with previous find ings (87), but it is probably by way of some pathway which does not involve pyruvate in this system. 1 i l l It is possible that C incorporation into these branched chain amino acids could be due to a 2-carbon fragment derived from carbons 3 an-<3- 4 ©f glucose. Since radioactive carbon from G^-carboxyl labeled acetate |s t not incorporated into these amino acids (3), it seems possible that this reaction, consisting of a transfer of a 2-carbon intermediate from one compound to another through the mediation of an enzyme system, is different from the acetyl-GoA type reactions suggested by Strassman (90). The incorporation of radioactivity into the carboxyl and CC-carbons of leucine from a 2-carbon fragment can be en visioned from the known reactions; however, a 2-carbon fragment can not incorporate into the carboxyl groups of valine and isoleucine if an aeyloin rearrangement occurs. The possibility that radioactivity is incorpo rated into the carboxyl groups of these amino acids by a reversal of the OC-decarboxylation which takes place during catabolism is a remote one in view of the fact that glu- eose-6-C^, glueose-l-C^, and glucose-2-C^ do not con tribute to the labeling of these amino acids. Under the conditions of these experiments in vitro. in which the breakdown products of catabolism accumulate, it is possible that the equilibrium might be shifted towards resynthesis. Coon has shown (91) that isovalerie acid which is formed during the catabolism of leucine gives rise to a 2-carbon intermediate plus an acetone or isopropyl group. If the above reactions are reversible, and a radioactive 2-carbon intermediate could combine with this isopropyl group to form an intermediate O^-keto- isovaleric acid, then radioactivity could be incorporated into valine. This mechanism could label valine in the carboxyl and alpha carbons. Abelson (89) deduced from isotopic competition experiments in E. eoli that leucine can be synthesized by the condensation of a 2-carbon 115 intermediate with isobutyrie acid which arises by decar boxylation from the QC-ketoisovalerie acid analog of valine. This reaction could result in the labeling of leucine in the carboxyl and aloha carbons. - In recent years much progress has been made in eluci- ! dating the biosynthetic pathways leading to the formation of aromatic amino acids. This undertaking has been made possible by the isolation of a wide variety of bacterial mutants requiring a mixture of tyrosine, phenylalanine, tryptophan, p-aminobenzoic acid, and p-hydroxybenzolc acid (92). Studies ©n the incorporation of variously 1 it G -labeled glucose into shikimic acid by intact cells, carried out by Sprinson (93)> have yielded certain general information concerning the earliest stages of this path way. The carboxyl carbon and carbons 1 and 2 of shikimic acid were shown to be derived from a glycolytic fragment presumed to be 2-phosphoenolpyruvate. The remainder of the molecule appeared to be derived from a 4-carbon frag ment, presumed to be erythrose-4-phosphate, in which carbons 3 to 6 of glucose are represented. The origin of this fragment can be explained by a transketolase-trans- aldolase series of reactions involving pentose and sedo- heptulose-1,7-diphosphate (42). A brief scheme for the synthesis of phenylalanine and tyrosine, based on the work of Davis et al.. (9^), is presented in Figure 1?. Prephenic 116" I FIGURE 1? BIOSYNTHETIC PATHWAY FOR AROMATIC AMINO ACIDS COOH I COPOoHp I ! 3 CHo 2-phospho- enolpyruvate CHO I CHOH' I CHOH I CH2OPO3H2 Erythrose- 4-phosphate eoocv/ch2c. I I Prephenio acid COOH I C=0 I CH~ I CHOH I CHOH I CHOH.: I CH2OP03^ HO^COOH O^lJoH 'OH S -dehydroquinic acid 1 p OOH ,COOH Compoun Z 1 OH NOH Shikimic acid CHoC. .COOH CH2CHC00H p-hydroxyphenyl- lactic acid 0 Phenyl- pyruvic I CHoCHCOOH / 2 Tyrosine 11? acid is a compound of special interest, since it is the substrate of the aromatization reaction in the biosynthesis of phenylalanine, and undergoes dehydrogenation and de- j earboxylation to yield phenylpyruvate and COg (95). Com- | ! pound Z1 is an acid-labile conjugate of shikimic acid and I » has been reported by Gilvarg (96) to yield pyruvic acid on I hydrolysis. It appears to be an intermediate between shikimic acid and prephenic acid, containing the partly saturated ring and a carboxyl group of the former compound, and also pyruvic acid in a labile attachment. Presumably, the mammal is unable to synthesize amino j acids containing the aromatic ring, but,is capable of transforming such amino acids. The amino acid tyrosine, while unessential In the diet because of its formation from phenylalanine (97), is an important precursor of i other compounds such as thyroxine, epinephrine, melanins, etc. In the. one-day-old mouse brain system there is exten- i II sive incorporation of C into tyrosine and phenylalanine from glueose-U-Clif. The uptake of C1^ into these amino acids from glucose-l-C^, glucose-2-C^, and glucose-6-C^ is significant but far less than that obtained with glu- cose-U-C^. The Incorporation of from glucose-3, Into tyrosine and phenylalanine seems comparable On a percentage basis to the uptake by the other amino acids, 118 but Its low specific activity makes interpretation ques tionable . By partial degradation of phenylalanine obtained from the glueose-U-cl^ Incubations it is found that the compar atively high uptake of is predominantly in the side chain (Table XIX). The radioactivity is almost exclusive ly in the aloha and carboxyl carbons. Thus, in this sys tem, it appears that the phenylalanine molecule is com prised o:£ an essential and a non-essential portion; the non-essential portion, the alpha and carboxyl carbons, { t may be derived from a 2-carbon fragment. This 2-carbon fragment does not originate from the metabolism of acetate since it has been shown by Moldave et al.. (3) that there is no incorporation of from acetate-l-C-^ into tyro sine or phenylalanine. The small incorporation of from glucose-l-G^, glucose-2- C a n d glucose-6-C^ into tyrosine and phenylalanine may represent the extent to which the aromatic ring is synthesized in this system, or i4 it may represent the extent to which G is distributed throughout the glucose molecule due to metabolic recycling and resynthesis of glucose. In either case, carbons 1, 2, and 6 of glucose contribute very little to the labeling of the aromatic portion of the tyrosine and phenylalanine or to the carbon precursors of the side chain. 1 il The extensive incorpotation of C into the alpha and 119 carboxyl carbons of phenylalanine is in contrast to the results obtained in bacterial systems. An observation in i E. coli (98) on the origin of the side chain of phenyl- j 1 l i r ™_ i j i alanine indicates that the /£-carbon of the side chain is i derived to the extent of 5° per -cent from carbon 1 of glucose, whereas the carboxyl carbon of shikimic acid is derived from the 3,4 carbons of glucose (99)• In the con version of shikimic acid to phenylalanine the carboxyl carbon of shikimic acid is lost. Thus it appears that in i the bacterial systems the ring and carboxyl carbon are j I derived from shikimic acid and the side chain probably i originates from some 3-ca^bon unit derived from glycolysis since the ^-carbon is derived equally and almost entirely from carbons 1 and 6 of glucose (98, 100). This does not appear to be the ease in the mouse brain system, since the ^-carbon plus the ring (benzoic acid in Table XIX) contain 14 very little radioactivity and the incorporation of G from glucose-l-C*^ and glueose-6-C^ is quite small com pared to that from glucose-U-CI^. It is suggested here that the incorporation of radioactivity into phenylalanine may occur in the mouse brain by the condensation of some 2-carbon fragment with the carboxyl carbon of shikimic acid, or some compdund derived from it, eventually to form phenylpyruvic acid. This hypothetical 2-carbon fragment would have to originate from some metabolic pathway other than glycolysis. One possibility is the formation of an active 2-carbon complex from carbons 1 and 2 of erythrose or erythrulose by a transketolase mechanism (42). Carbons 1 and 2 of erythrose correspond to carbons 3 and 4 of glu- 14 cose, and could account for some of the C Incorporation into some of the essential amino acids. Since histidine is an essential amino acid for most mammals, relatively little has been done in studying the biosynthesis of this amino acid in mammals. While histid ine is known to yield glutamate, formate and'ammonia when catabolized (101), glutamate does not appear to be a direct precursor of histidine. Aside from the fact that radioactive formate is found to be incorporated into car bon 2 of the imidazole ring of histidine in human liver slices (102), very little more is known about its synthesis in mammals. The biosynthesis of histidine in microorganisms has been worked out from the effects of blocking steps in the normal synthetic pathway. Histidine is derived in Neuro- spora. from a series of imidazole derivatives, the ring being attached to glycerol phosphate,which is converted to acetol phosphate and then transaminated (103, 104). After dephosphorylation the resulting amino alcohol, histidinol, is oxidized to histidine via the amino aldehyde (105). The compounds preceding imidazole glycerol phosphate and those 121 Involved in the mechanism of formate and nitrogen incorpo-J ration appear to "be closely related to ribose-5-phosphate j j or rlbulose-5-phosphate (10*0. Levy and Coon (106) have j shown in yeast that the isotope frbm acetate-2-C-*-i t ' is located -exclusively in carbon atom 2 of. the imidazole ring. Since formate was previously shown to be a precur sor of this carbon atom in yeast and in rat liver (102, 10* 1 -), it is concluded that the QC-carbon of acetate can serve as a source of formate. Levy and Coon concluded from their data that neither acetate and other compounds' indirectly derived from acetate nor glutamate serve as | precursors of the 5-earbon chain of histidine. The chem ical degradation of histidine by Levy and Coon (106) derived from glucose-l-C1^ incubations in yeast demon strated that the carbon chain is labeled primarily in position 5. Not only are the results obtained with one-day-old mouse brain in contrast to results obtained in other mam malian tissues, but they are also in contrast to results obtained in microbiological systems. In the mouse brain system there was a significant amount of radioactivity lit - t h incorporated from glucose-U-C and glucose-2-C into 1 it 1 it histidine. The incorporation of C from glucose-3,4—C into histidine is comparable to the amounts of Gl** taken up by the other amino acids. In contrast to this, mouse 122 brain showed no incorporation of carbon atoms 1 and 6 into histidine from glucose, which was shown to occur by Levy and Goon (1G2) in yeast. The results obtained by Moldave et al. (3) for the incorporation of G1^ from acetate-l-C1^ compare qualitatively with the results obtained with glu- cose-2-Cli* ' (Table XX), but indicate that the carboxyl group of acetate is more closely related to histidine than is carbon atom 2 of glucose. In view of the present knowledge pertaining to hist idine biosynthesis, an attempt to explain these results is difficult. It is unlikely that carbon atom 2 of glucose is incorporated directly into histidine by way of ribose- 5-phosphate, since carbon atoms 1 and 6 are not incorpo rated from glucose. However, an enzyme complex of glycol- aldehyde can be formed from carbons 1 and 2 of ribulose- 5-phosphate by transketolase reactions (43). The glycol- aldehyde may form glycine* directly, via glyoxylate, and evidence for such an occurence has been shown by Weissbach and Horecker (107), who found that extracts of spinach leaves incorporate carbon one of ribulose-5-phosphate into the alpha carbon of glycine. It has been shown by Sakami (108) and Siekevitz (109) that the alpha carbon of glycine is irreversibly oxidized to formate in vitro. These same experiments show that formate is formed more, readily from glyoxy late than from glycine.. The formate formed in this 123 TABLE XX COMPARISON OF, THE INCORPORATION OF ACETATE-l-C1^ AND GLUC0SE-2-C14 INTO THE PROTEIN-BOUND AMINO ACIDS FROM ONE-DAY-OLD MOUSE BRAIN Giucose-2-C1^ Acetate-1 _cl4 1/ Amino acid jiMoles per 100 mg. protein Specific activity ^iMoles per 100 mg. protein Specific activity Aspartic acid 22.4 2190 22.0 2195 Threonine 15.0 0 19.6 • 0 Serine 16.9 1110 19.4 80 Glutamic acid 20.3 3310 23.1 5950 Proline 33.7 1180 34.4 360 Glycine 18.9 685 20.2 40 Alanine 21.3 3580 11.6 390 Valine 18.5 0 21.1 0 Methionine 12.0 0 10.7 0 Isoleucine 13.0 0 10.9 0 Leucine 21.4 0 20.4 0 Tyrosine and phenylalanine 16.? 175 13.8 0 Histidine 15.1 4-02 16.4 1680 Lysine 30.8 0 32.1 0 Arginine 15.9 0 11.8 0 1/ Moldave, K. , J. Biol. Chem.j 200. 357 (1953). 124 manner would then be available for incorporation into car bon atom 2 of the imidazole ring without the prior neces sity of forming glycine. A comparison of the specific ; ! activities of glycine and histidine (Table XX) from glu- j cose-2-G1^ would not preclude this mechanism as a possi bility. However, the large incorporation of C1* * ' from i l l r acetate-l-C-1 -^ into histidine compared to glycine obtained by Moldave (3), would preclude such a mechanism in this instance. Further, if carbon atom 2 from glucose were j incorporated into histidine by way of formate, it would j be expected that this formate intermediate would contrib- j ute to the incorporation of into methionine, which it does not (Table XX). - The incorporation of carbon atoms 3 and 4 of glucose into histidine may be postulated from the action of the formyltransacetylase enzyme of E. coll acting on pyruvate. This reaction has been shown to occur in E. coll by Lipmann (110) and Strecker (111) in the following manner: pyruvate + CoA + enzyme — * acetyl-CoA-enzyme + formate Formate could then be available for histidine synthesis. A study of the catabolic metabolism of histidine in Pseudomonas by Tabor and Hayaishi (112) has shown that histidine is eatabolized by the following sequence of reactions: histidine histamine --- > imldazoleacetic acid — > formylaspartic acid — aspartic acid + formic acid. The administration of C^-histamine to the rat______ 125: In vivo showed 40 per cent of the initial radioactivity to % be excreted in the urine as imidazoleacetie acid (113). KarJala (114) isolated imidazoleacetie acid riboside from the urine of mice as a conjugate excretion product of histidine catabolism. If the catabolism of histidine is assumed to be reversible in the one-day-old mouse in vitro. due to the accumulation of metabolic products, then it would be pos- 1 I I sible that C from glucose could be in equilibrium with these breakdown products. Formate could exchange with the formyl group of formylaspartlc acid. This would be in agreement with the finding by Moldave (3) that formate is incorporated into histidine in this system. The reaction of G from glucose with imidazoleacetie acid to form carboxyl labeled histidine would be in keeping with the degradation data presented by Moldave (3) for C^-glucose 1 h and CA -acetate. Lysine metabolism shows so much variation from species to species that it is difficult to construct any general pattern. In E. coll.cf,^-diamlnonlmellc acid is presumed to be the precursor of lysine, producing it by decarboxyl ation. (115). In fungi, however, 0C-aminoadipic acid is a precursor of lysine. Mitchell and Houlihan (116) have shown that this amino acid can replace lysine in mutants of Neurosnora requiring lysine. A confirmation of this ” ' ' ' " " 1261 role of qf-aminoadipie acid has been provided by Windsor (11?) who showed that cl^-d£-aminoadipic acid is incorpo rated into lysine. In animals, lysine is a required amino acid, so that the steps leading to its synthesis are effectively blocked. Lysine does not contribute to the metabolic pool of nitrogen in the rat, nor does it take part in the general reversible transamination of the oC-keto acids. However, Meister (118) has found that a rat liver glutamine transaminase preparation will transfer amino groups between glutamine and ^-N-substituted af-keto analogues of lysine. Lysine has been shown by Borsook et al. (45) to be converted in liver to glutaric acid via OC-aminoadipie acid and OC-ketoadipic acid. In the one-day-old mouse brain only glucose-U-C-^ and glueose-3,^-C^ show any incorporation of into lysine. If it is assumed that the data obtained with glucose-3 are significant, then carbons 3 and/or 4 of glucose are the only carbons which label lysine. In a previous 1U ' experiment using glucose-U-C (1) it was shown that all of the radioactivity resided in the carboxyl carbon of lysine. These results might be explained by the fixation of radioactive carbon dioxide, originating from carbons 3 and 4 of glucose, through the action of lysine decarboxyl ase. Hanke and Siddiqi (119) have reported that when lysine is treated with a lysine decarboxylase in the 127 presence of C1*^, and the reaction stopped when the de carboxylation is half complete, the remaining amino acid contained appreciable radioactivity. However, if CO2 is Incorporated into the carboxyl carbon of lysine, it would have to do so without being in equilibrium with the free- CO2 pool, as the other Ci^-labeled precursors yield without incorporation of into lysine. This mechanism seems remote unless there is some specific one-carbon complex not in equilibrium with CO2. In general, the question of how radioactive carbons from glucose may appear in amino acids that presumably can not be synthesized at all, or not at rates commensurate with maintenance or growth of the organism, is one of considerable interest. It is possible, of course, that the one-day-old mouse brain, being a possible embryonic tissue, has the ability to synthesize essential amino acids de novo. However, such a postulation:.would not be in accord with degradation data for phenylalanine or results previously reported on the partial degrada.tion of several essential amino acids obtained from these tissue cultures, which seem to suggest a partial resynthesis rather than de novo synthesis (1, 3). v The data indicate that, of the six carbons derived from glucose, carbon atoms 3 and 4 are more intimately concerned with incorporation into the essential amino 128 acids. The incorporation of these carbons into the essen tial amino acids could not occur by means of CO2-fixation, j i which may occur after the decarboxylation of carbons 3 and 4 from pyruvate, unless a C02-complex is formed which is not in equilibrium with free CO2. A suggestion which fits the essential facts is the formation of a 2-carbon complex involving carbon atoms 3 and 4 of glucose. This 2-carbon . complex, if it exists, is unrelated to acetyl-CoA or com ponents in equilibrium with it. However, carbon atoms 3 and 4 could form a 2-carbon complex by means of the "Horecker cycle" in a manner similar to the formation of an "active glycolaldehyde" from carbons 1 and 2 of pentose during the transketolase and transaldolase reactions (42). If it is assumed that erythrose-4-phosphate, which is formed during this series of reactions, could give rise to a 2-carbon complex in a similar manner, this complex would represent carbons 3 and 4 of glucose. This 2-carbon complex could then be in equilibrium with the essential precursors of the essential amino acids. According to this view, protein-bound amino acids are in equilibrium with the essential and non-essential free amino acids which are in equilibrium with the essential and non-essen tial precursors. It is with this 2-carbon non-essential precursor that the intermediates derived from glucose are in equilibrium. This mechanism for the incorporation of 129 radioactivity from glucose into the essential amino acids is schematically represented as follows: glucose w non-essftntial — fructoae-6-PO^ erythrose-4-PQjf. precursor ^ . k f K I f non-essential [C3 - Cifl enzyme amino acid + protein r f ■ ■ — essential < " " • " " " essential amino acids precursor This mechanism could result in the incorporation of radioactivity from glucose into the essential amino acids without requiring that they be synthesized completely from non-essential precursors. This is in agreement with the experimental results and with the well-established fact that certain essential amino acids are required for maintenance and therefore cannot be synthesized to an extent to support life. The above scheme is subject to experimental verification, since the non-essential carbons derived from glucose-3,4-C^ should be adjacent and equally labeled. In order to do this, glucose-3 will have to be synthesized with a specific activity much greater than used here. CHAPTER VI SUMMARY | The extent of incorporation of radioactive carbon from glucose into the protein-bound amino acids of minced one-day-old mouse brain, incubated with glucose-U-C^, ill i II glucose-l-G , glucose-2-C , glucose-3,4-Cx , and glucose-- 6-0^ has been investigated. The proteins were isolated, hydrolyzed, and the component amino acids separated by ion- exchange chromatography. Radioactive peaks of approx imately comparable specific activity were found to be associated with all of the essential and non-essential 1 it amino acids except threonine from glucose-U-CA^' incubates. Both glucose-l-C1^ and glucose-6-C1^ incorporated radio activity into aspartic acid, serine, glutamic acid, pro line, alanine, and to a small extent into methionine, tyrosine, and phenylalanine. The results obtained with glucose-l-C1^ and glucose-6-C1^ did not differ signifi- 1 it cantly. Radioactivity from glucose-2-CA was incorporated into aspartic acid, serine, glutamic acid, proline, gly cine, alanine, histidine, and to a small extent tyrosine l i t - and phenylalanine. The incorporation from glucose-2-Cx resembled qualitatively the Incorporation from acetate- i i i f , 1 —C reported by Moldave (3)* Although the results obtained with glucose-3,4-C1^ were inconclusive due to the low specific activity employed, all of the amino acids 131 except threonine were labeled. The presence of radioactive carbon from glucose in the non-essential amino acids was explained, in general, by well established biochemical reactions. It was shown 1 i j , 1 that carbons from glucose-l-C and glucose-6-C1^ were incorporated to the same extent into the beta carbon of serine without being incorporated into glycine. The lij, radioactivity from glucose-U-C in serine was equally distributed throughout the serine molecule. These data plus the data obtained for the C^Og evolution during glucose-l-C-*-** and glucose-6-Cl** incubations, were inter preted to indicate that serine was synthesized from some 3-carbon fragment derived from the glycolytic pathway, and that glycine was derived predominantly from serine. Similarity between the amount of C-*-**02 evolved as a function of time from glucose-l-C^ and glucose-6-C^ was interpreted as an indication that the shunt pathway is not of major importance in the mouse brain system. Postulations were made, based on known biochemical reactions from other metabolic systems, to explain the i h, incorporation of C from the variously labeled glucose substrates into methionine, valine, leucine, isoleucine, i it histidine, and lysine. A method of incorporating Gx^ into the carbons of methionine other than the methylene group was presented, which suggested the possible involvement of 132 "oC-amino-/^-butenic acid" . This compound could be formed from homoserine and converted to methionine as in the j homologous conversion of serine through oC-aminoacrylate, followed by the formation of cysteine. lit The incorporation of C into valine, leucine, and isoleucine could not be explained by any of the establish ed mechanisms. A suggestion was made that a 2-earbon fragment derived from carbons 3 and 4- of glucose might explain the incorporation into these branched amino acids. It was pointed out, however, that this 2-carbon fragment ' could not be derived from a glycolytic intermediate, acetate, or from pyruvate per se. The radioactivity associated with histidine after glucose-U-C^, glucose-2-C^, and glucose-3incu bations was interpreted as arising from the incorporation of formate. It was indicated that formate could be formed by various means from carbons 2, 3> or 4 of glucose. By degradation of phenylalanine isolated after glu- cose-U-C1* * ' incubations it was found that over 90 per cent of the radioactivity incorporated into this amino acid was equally distributed between the carboxyl and aloha carbons of the side chain. The suggestion was made that phenyl alanine is comprised of an essential portion and a non- essential portion, and that the non-essential portion is in equilibrium with some 2-carbon fragment derived from 133 glucose. This hypothetical 2-carbon fragment may involve carbons 3 and. of glucose but is not derived from acetate or the glycolytic pathway. * 1 i t No satisfactory explanation for the mechanism for incorporation into lysine from glucose could be found. It 1 II ' was suggested, however, that C may be incorporated into lysine by a reversal of a lysine decarboxylase reaction. A hypothetical scheme was presented based partially on known biochemical mechanisms, to explain how carbon atoms 3 and 4 from glucose are incorporated into essential amino acids. A 2-carbon complex representing carbons 3 and * * - of glucose was suggested to arise from erythrose-**— phosphate by means of the transketolase-transaldolase reactions. It was suggested that this 2-earbon complex was involved with essential precursors for the formation of radioactive essential amino acids. I l l L I .0 G R A P H Y BIBLIOGRAPHY 1. Rafelson, M. E., Jr., Winzler, R. J., and Pearson, H. E., J. Biol. Chem., 205 (1951). 2. Winzler, R. 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Creator Sky-Peck, Howard H. (author)

Core Title The incorporation of radioactive carbons of glucose into the protein-bound amino acids of one-day-old mouse brain

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1956-10

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-604163

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Identifier DP21577.pdf (filename),usctheses-c17-604163 (legacy record id)

Legacy Identifier DP21577.pdf

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Document Type Dissertation

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