University of Southern California Dissertations and Theses

The metabolism in vitro of 4-C¹⁴-cytidine

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The metabolism in vitro of 4-C¹⁴-cytidine

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Content THE METABOLISM IN VITRO OP 4-C14-CYTIDINE 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 by Lawrence Grossman October 1Q54 -PK.0 ( s ; » ' SS &S1g T his dissertation, w ritte n by Lftwyeace Grossman under the direction ofQ^-.fk.Guidance Com m ittee, and approved by a ll its members, has been p re sented to and accepted by the F a cu lty of the G raduate School, in p a rtia l fu lfillm e n t o f re quirem ents fo r the degree of D O C T O R O F P H IL O S O P H Y . . . I .................. D a t e J k ^ Guidance Com m ittee Chairman A CKNOWEEDGEHENTS It is with sincere gratitude that I acknowledge the invaluable guidance and encouragement, both scientific and personal, of Dr. Donald W. Visser, I wish to express my appreciation to Dr. William C. Werkheiser for his guidance in certain phases of this work, I would like to express my appreciation to the Faculty of the Department of Biochemistry and Nutrition for their guidance during my graduate career. Appreciation is extended also to the National Institutes of Health and to the California Division, of the American Cancer society for their financial.support of this research program, and to the Allen Hancock Foundation for the facilities that were made available. My sincere appreciation to my wife, Barbara, for her help, encouragement and inspiration. TABLE OP CONTENTS CHAPTER PAGE I. INTRODUCTION . . .............. 1 II. HISTORICAL ........ 5 Pyrimidine catabolism .............. 5 Biosynthesis of pyrimidines •«•••..•• 8 The role of orotic acid ............ 12 Nucleosides and nucleotides as nucleic acid precursors .......... ........... 15 III, EXPERIMENTAL.......... . . 21 Preparation of 4-C^-cytidine.............. 21 2-Thio-4-C^4-uracil....................... 21 4-C14-Uraci l ............................... 23 2.4-Dichloro-2-C^4-pyrimidine ....... 23 2.4-Diethoxy-4-C^4-pyrimidine ....... 24 Tetraaceto-D-ribofuranose synthesis ........ 24 5-Trityl-D-ribofuranose .............. 24 1,2,3-Triaceto-5-trityl-D-ribofuranose . . . 25 1,2,3,5-Tetraaceto-D-ribofuranose ..... 27 l-Bromo-2,3,5-triaceto-D-ribofuranose . . . 28 Cytidine purification .............. 30 Preparation of Dowex-50, hydrogen-form . . . 30 Ion-exchange resolution of the cytidine mixture ........... ...................... 31 V CHAPTER PAGE Determinations of the purity of cytidine , . 32 Tissue incubations . . . . . . . . . . . . . . 32' Buffer................... . ................ 32 Preparation of tissues............. 35 Tissue fractionation . ..................... 36 Cellular fractionation ................, . 36 Preparation of the protein residue ......... 37 Preparation of the acid-soluble fraction , . 37 Removal of lipids from the protein residue . 38 Fractionation of the protein residue . . . . 38 Isolation of pyrimidine nucleotides ..... 40 Buffers for ionophoresis of nucleotides . , 40 Filter paper ionophoresis at pH 6.0 .... 40 Filter paper ionophoresis at pH 3.7 .... 42 Ion-exchange purification of labeled nucleo tides ............ 42 Isotopic analysis of labeled nucleotides • • 44 Self-absorption curve ........... 45 Methods of reporting radioactive data .... 45 Specific activity . . . . . . . . . . . . . 45 Relative specific activity *...«... 45 Spectrophotometrie assay of nucleotides . . 45 Ion-exchange chromatography.............. . 46 vi CHAPTER PAGE- Preparation of the resin.......... 46 Column preparation . . . . . . . . . . . . . 48 Sample introduction. ............ ..... 49 Ion-exchange chromatography by gradient elu- i tion .......... 49 Resin regeneration.................. 55 Preparation of 5»-cytidylic acid (CMP-5») . . 53 Chemical tests ........................... 56 Organically-bound phosphorus ........ 56 Pentose determination .......... ..... 57 Saturated pyrimidine ring and carbamyl compound determination 58 IV. RESULTS............................ 61 Incorporation studies .............. 62 Ion-exchange chromatographic analysis .... 62 Isolation and separation of labeled nucleo tides ............ 72 Qualitative analysis of the unidentified radioactive peak .................. 82 Organically-bound phosphorus.............. 85 Pentose determination . .................. 85 Nitrogenous base determination ....... 86 V. DISCUSSION..................................... 88 vii CHAPTER PAGE Incorporation studies . « . .............. 88 Tissue extract investigations ............. 91 VI. SUMMARY.................................... . 99 BIBLIOGRAPHY....................................... 101 LIST OP TABLES TABLE PAGE I. Composition of Krebs-Ringer-Bicarbonate Buffer..................................... 54 II. Preparation of Ammonium Formate Buffers . . . 41 III. Ionophoretic Separation of Ribonucleotides at pH 3.7 . . ............................... 43 IV. Extinction Coefficients and Ratios of Optical Density at 260 and 280 rap. for Ribonucleo tides ..................................... 47 V. Incorporation of 4-c^-Cytidine into Rat Liver Nuclear and Cytoplasmic RNA Pyrimidine Nucleotides in Vitro ........ 63 LIST (F FIGURES FIGURE PAGE 1. Synthesis of 4-C^-Cytidine............... , 22 2. Ion-Exchange Purification of 4-G-^-Cytldine , • 33 3. Gradient Elution Pattern.................... 52 4. Ion-Exchange Separation of Components of the Acid-Soluble Constituents from the Cytoplasm of Rat Liver Slices Incubated in the Presence of Ron-Labeled Cytidine..................... 65 5. Ion-Exchange Separation of a Yeast Nucleic Acid Hydrolysate . • ................ 67 6. Ion-Exchange Separation of Yeast Nucleic Acid Hydrolysate ............ 70 7. Ion-Exchange Separation of Yeast Nucleic Acid Hydrolyzed with Snake Venom Diesterase . . . 73 8. Ion-Exchange Separation of the Cytoplasmic Acid- Soluble Constituents from Rat Liver Slices Incubated with Normal Cytidine ....... 75 9. Ion-Exchange Separation of the Cytoplasmic Acid- Soluble Constituents from Rat Liver Slices Incubated with 4-C^-Cytidine ........ 77 10, Ion-Exchange Isolation of Unidentified. Compound 80 11. Ion-Exchange Purification of Unidentified Compound , .................... 83 X FIGURE PAGE 12, Hypothetical Pathways of Cytidine Incorporation Into Nucleic Acids . . . . . . . . . . . . . 95 CHAPTER I INTRODUCTION It has become apparent in recent years that nucleic acids are intimately concerned with many of the most im portant and basic functions in biological systems. These functions which include cellular division, differentiation, growth and inheritance, may be better understood through a clearer comprehension of the metabolism of nucleic acids. In order to learn more about nucleic acid metabolism, experiments were designed to study the metabolism of cyti dine, presumably a nucleic acid precursor. It was estab lished by others that the free pyrimidines (1,2) and purines (1,3), with the exception of adenine (4), were neither incorporated into nucleic acids nor were they found free in nature. The nucleotides, which were considered as chemical degradation products of nucleic acids seemed to be a poor choice since some question as to their absorption had been raised. The corresponding nucleosides were known to support growth of certain micro-organisms (5) and were incorporated into nucleic acids In vivo (6,7,8). For these reasons and since synthetic methods were developed for the pyrimidine nucleosides it was decided to investi gate the metabolism of cytidine. Cytidine was chosen rather than uridine because the latter was less effective as a precursor of nucleic acid pyrimidines in vivo (7), Most of the studies reported prior to this investigation by other workers concerned the incorporation of nucleic acid precursors in vivo. However, it was felt that incorporation studies of systems in vitro offer greater possibilities for investigations of the metabolism in different cell particu lates and for subsequent isolation of the enzyme systems involved. Although both biological systems can be sub jected to analysis of degradation products and anabolic studies of precursor, incorporation, in vitro studies re quire smaller amounts, of substrate, and, therefore, experi ments with a greater number of replicates are possible. In addition, an in vitro system may be used for the determina tion of environmental effects of nucleic acid metabolism such as anaerobiosis, pH change, effect of cofactors, etc,, much more readily than intact.animal studies. Accordingly, experiments were designed to investigate the incorporation of labeled cytidine into the pyrimidine nucleotides of rat liver slices. Earlier studies of simple nucleic acid precursors, such as labeled phosphate (9), and glycine (10), indicated that there was a heterogeneity with respect to the incor poration of these precursors into the nucleic acids of different cellular components. These results and those re ported by Caspersson (11), in which the ultraviolet ab sorption of different cellular organelles and their changes under different metabolic conditions were studied, gave rise to the suggestion that nucleic acids or their pre cursors were synthesized within the nucleus and then mi grated to the cytoplasm. It seemed of interest, therefore, in this study of cytidine incorporation to distinguish be tween the nucleic acids of nuclei and cytoplasm. The early investigations of the glycolytic pathway and the tricarboxylic acid cycle were greatly facilitated by the presence of the intermediates derived from these systems in the acid-soluble or water-soluble fractions of tissues. Studies of these soluble fractions, so important in the carbohydrate work, had not been investigated by those studying nucleic acid biosynthesis until recently (13-19), These fractions now have been shown to contain many cofactors such as coenzymes I and II, adenosine tri phosphate and flavine-adenine dinueleotide, the more re cently identified group of uridine diphosphate coenzymes containing glucose, galactose and acetylglucose amine, and many of the 5'-purine and 5•-pyrimidine nucleotides. Pre liminary results of the present investigation showed a pref erentially greater incorporation of cytidine into nucleic acid uridylic acid than into cytidylic acid. It appeared pos sible, therefore, that cytidine is converted to some other form prior to nucleic acid incorporation, which by analogy might be expected to be contained in the acid-soluble frac tion. Study of this fraction was facilitated by the fact that it was routinely obtained in the course of preparation of the nucleic acid fractions. Ion-exchange chromatographic methods for the analy sis of tissue acid-solubles previously developed by Potter et al. (19) were modified for the analysis of the acid- soluble extracts obtained from these incorporation studies. This approach has.proved to be.fruitful in the. isolation of a new pyrimidine nucleotide biologically derived from 4-C1^- cytidine. CHAPTER II HISTORICAL At the present time there is very little information concerning the intermediary metabolism of pyrimidines and their derivatives.despite rapid advances in the field of purine metabolism. It can be stated, categorically, how ever, that there are differences in the catabolic and ana bolic pathways of purines and pyrimidines. In addition to the fact that purine precursors, such as glycine and for mate, do not play a role In pyrimidine biosynthesis, the catabolism of these nitrogenous compounds differs markedly. In higher animals, the purines are catabolized to uric acid and allantoin whereas the pyrimidines.are degraded to urea and carbon dioxide or to beta-amino acids. Pyrimidine catabolism. The available evidence concerning pyrimidine degra dation indicates that there may be at least two pathways for pyrimidine catabolism, one involving reduction and the other oxidation. Much of the original work on pyrimidine catabolism, performed by Cerecedo et al. (20-27),. depended on the measurement of urea levels in the urine of dogs to which products of pyrimidine oxidation in vitro had been pre viously administered. To account for the results of these experiments, Cerecedo suggested the following scheme for pyrimidine degradation. OH 1 OH I 1 / K W CH 1 1 ! 1 /'X W G-OH V 1 I I 1 II--- > —CL GH --- > 1 I I ------ H0~Cv CH V ' Uracil Is©barbit uric acid h h». I i=o I nh2 Urea 0 HIT N0=0 ■» I i 0=0 13-OH QH Ha- ) HN COOH c=o NH / Isodialuric Pormyloxal- aeid uric acid COQH I GOOH Oxalic ac id Half i GOOH 0=C 0=0 NH Oxaluric acid Subsequent investigations of pyrimidine catabolism, by Wang and Lampen (28), in a cell-free extract of an aerobic soil bacterium revealed the presence of a uracil oxidase which formed .barbituric acid from.uracil. An en zyme further oxidizing barbituric acid to maIonic acid was isolated by Hayaishi and Kornberg (29) from uracil-adapted cells. Pyrimidine degradation according, to this information may be summarized as follows. 7 I 0=C GH I V CH I I H H Uracil j H N* NCH2 I I HO-U CH-GH OH I .Cv N' CH ■ * I I I - HO-C C-OH / Barbituric -acid H V° °\ CH 2 C-OH W o NH. 2 -f G=C. \ ■NH* Malonic acid Urea pink et al. (30,31) have recently demonstrated the existence of a pathway of pyrimidine catabolism through an initial reduction. The system studied involves the sub sequent formation of beta-aminoisobutyric acid (BA.IB) from thymine, dihydrothymine or desoxyribonucleic acid (DNA) and of beta-alanine from uracil or dihydrouracil. Sub sequent investigations by Pink and McGaughey (32) have re vealed some of the intermediate steps that occur both in vitro and in vivo. This information may be summarized as follows* 8 OH OH HO 0 I* A H*N CH-CHg HO 0 V/ 0. N C-CHa I I ch-ch3 0*=C GH HslT' CH* H Thymine Dihydrothymine Beta-ureido Beta-aminoiso- isobutyric acid butyric acid The enzymes responsible for the conversion of the dlhydro- pyrimidine to the beta-amino acids have been located in the supernatant of rat liver homogenates. These apparent dissimilarities in pyrimidine degra dation may be explained by one or more of the following possibilitiesi (a) the mode of pyrimidine degradation may be species specific; (b) two alternate pathways may exist within the organism for pyrimidine degradation; (c) these systems may not represent normal catabolic pathways of polynucleotide pyrimidine, but might represent the action of nonspecific enzymes. The latter possibility seems quite tenable since free pyrimidines are neither incorporated in to nucleic acid nor are they found free in nature. Biosynthesis of pyrimidines. The availability of isotopic compounds has provided a means of studying the incorporation of small molecular weight precursors into a complex ring structure. The information provided from such studies has aided in the elucidation of metabolic pathways of biosynthesis. The extent of incorporation of N^®-ammonium citrate into the pyrimidines has been found to be comparable to that of the purines in both the rat and the pigeon (33). C14-bicarbonate is equally effective as a precursor of position 2 of uracil as it is for carbon atom 6 of the purines (34,35,36). The carbon.atoms of glycine, however, are not utilized to any significant extent in the formation of RNA and D M pyrimidines in the rat (37), In contrast to its incorporation into the purines, the carbon of c^- formate is not extensively incorporated into the uracil or cytosine component of nucleic acids of the rat (34) and yeast (38), Although foraate does not contribute its car bon atoms to the synthesis of the pyrimidine ring, G^- formate, beta-C^4-aerine and alpha-C^-glycine (39) are in volved in the formation of the 5-methyl group of thymine. Investigations concerning the source of carbon atoms,. 4, 5 and 6 have received much impetus from studies of aliphatic compounds capable of replacing uridine in a uridine-requiring Neurospora mutant (40). Aliphatic com pounds in addition to orotic acid which were effective in replacing uridine are aminofumarie acid, aminofumarie acid diamide and oxalacetic acid. Amino acids, including aspar- 10 tie acid and compounds of the tricarboxylic acid eycle, were much less effective in meeting the growth requirements of the organism. The work of Wright and co-workers (41) demonstrated that ureldosuccinic acid was 10 to 20 per cent more effec tive as a precursor of nucleic acid pyrimidines than orotic acid for L. bulgaricus 09. These workers proposed that ureldosuccinic acid was converted to ureidofumaric acid, or some derivative thereof. Wright postulated that ureido- succinic acid may be formed by a transcarbamylation reac tion on the amino group of aspartic acid in a fashion simi lar to the formation of citrulline from ornithine (42). The conversion of ureldosuccinic acid to orotic acid would require at least two steps; ring closure and the introduc tion of a double bond. . Depending on which of these reac tions occurs first, either dihydroorotic acid or ureido fumaric acid would be the. intermediate. Lieberman and Kornberg (43) have recently demonstrated that in an extract from an aerobic bacterium the synthesis of orotic acid from ureldosuccinic acid proceeds through dihydroorotic acid. As further evidence for this metabolic sequence, it has recently been reported that ureidosuccinate is utilized via dihydroorotic acid in rat liver homogenates (44). A summary of the formation of orotic acid may be 11 represented as follows. HO 'e< CHS I NH, * V \ H2N CHa CH COg HgN vCOOH Aspartic acid OH I .0. J CHg ose ch \ /\ NH COOH Ureidosuccinie acid 0=C ,CH nNH COOH D ihydroorotie acid I OH A N CH f i XNH ^COOH Orotic acid Additions of N15-aspartic acid, l,4-C13-L-aspartic acid or 2,5-C14-L-aspartic acid with unlabeled orotic acid to liver slices and reisolation of the orotic acid at the end of the experiment indicated that the N^, 04, Cg, Cg, and Cy atoms of orotic acid were derived from aspartic acid (45). The means by which carbon dioxide can enter the pyrimidine ring has been proposed from some recent work by Ratner and co-workers (46,47) who isolated arginosuccinic acid from a reaction mixture of citrulline and aspartic ' acid in the presence of an isolated enzyme system. This intermediate when in aqueous solution is transformed into the anhydride, which does not produce arginine and fumaric acid in the presence of the proper enzyme systems* However, the anhydride will react enzymatically to form these prod ucts when previously treated with alkali, A hypothetical scheme, which has been made more tenable by recent work (48) in which eitrulline labeled in the ureide carbon acted as a fairly specific precursor.of the number 2 carbon (ureide) of nucleic acid.pyrimidines, can be summarized as follows# yCOOH HG— -NHo NH I G=0 + I NHa Citrulline GOGH HC(— NHa (CHa)s R H i /■ A ? Kt-nV >'1 N-G H COOH HaNiH I CHa - I COOH Aspartic acid NH c k i NHa N GOOH COOH H CHS A [ CH, 'cx I I 0 H 1 ' C-COOH I Arginosuc cinic acid 0=0' HG-C00H I I HN. CHa I I o Dihydroorotic acid The role of orotic acid. The occurrence of orotic acid in natural sources was 13 t first demonstrated by Biscaro and Belloni (49) who isolated this substance from cow*s milk in 1905. The finding of Loring and Pierce (50) and Rogers (51) that uracil can be replaced by orotic acid as a growth stimulant for certain bacteria suggested a connection between the acid and nu cleic acid biosynthesis. Mitchell and co-workers (40) ex tensively studied the function of orotic acid in the pyrim idine metabolism of different strains.of Neurospora mutants. Prom genetic evidence, Mitchell, Houlahan and Hyc (52) came to the conclusion that orotic acid is formed as a by-product in,a side reaction during pyrimidine synthesis Neurospora and thus is not a normal intermediate in pyrimidine biosynthesis.. Instead it was proposed that pyrimidines are formed from oxalacetic acid, via amino- fumaric acid, and that ribosidation of an acyclic pyrimi dine precursor precedes ring closure. Subsequent to this work Michelson, Drell and Mitchell (53) isolated from Neurospora a riboside of orotic acid, orotidine, which was rather labile to acid. The acid lability of orotidine may explain the existence of orotic acid as a by-product rather than an intermediate in pyrimidine biogenesis. Interest in the metabolism of orotic acid was greatly stimulated by the finding of Arvidson et al. (54) that N*5-orotic acid was utilized by the rat for the 14 synthesis of RHA pyrimidines, confirmation of these re sults has come from different laboratories where it has been demonstrated that orotic acids labeled in either the 2 or 4 positions, are equally well utilized for pyrimidine biosynthesis by a great variety of different organisms (55, 56). The incorporation of orotic acid into the polynucleo tide pyrimidines in vitro has been demonstrated by Weed and Wilson (57) and Reichard (158) . Reichard has shown that the presence of orotic acid greatly diminishes the incor poration of isotope from N^H^Cl into uridine of rat liver slices (58). At the end of the incubation, the orotic acid isolated from the medium contained a high level of N*5. Reichard concluded that orotic acid is.a direct inter mediate in pyrimidine biosynthesis. The question of whether or not orotic acid is an intermediate is rather academic at this stage of our know ledge of pyrimidine biosynthesis; however, some recent work of Lieberman and Kornberg (59) sheds some light on the sub sequent fates of orotic acid. An enzyme from pigeon liver was shown to be capable of converting ribose-5-phosphate (R-5-P) and adenosine triphosphate (ATP) to 5-phospho-l- pyrophosphoribose (FRPP) and adenylic acid (AMP). The pyro- phosphorylated pentose in the presence of a yeast, enzyme 15 was capable of converting orotic acid to orotidine-5- phosphate (OMP). The latter nucleotide was converted to uridylic acid (UMP). These transformations are summarized as follows. pigeon liver ATP + R-5-P .... .... ....■ > PRPP + AMP yeast extract PRPP + orotic acid .j :>• OMP + P-P OMP ----> UMP + C02 Nucleosides and nucleotides as nucleic acid precursors. While it has been shown that preformed purines and pyrMidines are not dietary essentials, and that most organisms synthesize them from simple metabolic precursors, there, nevertheless, has been a continuing interest in the metabolic reactions of the breakdown products of nucleic acids, the nucleotides, nucleosides and free bases. The apparent uniformity of biosynthesis of nucleic acid pyrimidines ffom aliphatic precursors in widely dif ferent species is in contrast to the manner in which organ isms react to preformed pyrimidines and their derivatives. Plentl and Schoenheimer (1) fed N^-labeled guanine, thymine and uracil to rats and isolated the purines and pyrimidines of the nucleic acids. Although the compounds were absorbed and catabolized, they were not incorporated into nucleic acids to any appreciable extent. Subsequently, Brown and co-workers (3,60,61) administered labeled adenine and cytosine to rats. Cytosine, like guanine, thymine and uracil, was not incorporated into nucleic acids. Adenine, on the other hand, was extensively incorporated into nu cleic acid adenine and guanine. None of the pyrimidine constituents of nucleic acids, therefore, is utilized for nucleic acid synthesis in rats. The only pyrimidine known to participate is orotic acid, which has already been dis cussed. It is perhaps significant that orotic acid, unlike other pyrimidines, is known to occur in the free form, namely in milk. Brown and his group (62,63) in an effort to detect larger and perhaps more immediate precursors of nucleic acids, administered a mixture of N15-labeled pyrimidine and purine nucleotides to rats. There was a significant incorporation into the pyrimidine components of ribonucleic acid but not into the purines, since the free pyrimidines are not utilized for nucleic acid synthesis the implication was that the pyrimidine moieties were taken up in the nucleoside or nucleotide form. Confirmatory results were obtained by Hammarsten and his group (7). They 17 administered labeled cytidine and uridine to rats, Cytidine was a very effective precursor of both RNA and DNA pyrimidines, It appears from these results that ribo- cytidine is convertible to desoxyeytidine and that ribo- cytidine and uridine are interconvertible. Reichard and Estborn (64) studied the incorporation of desoxyeytidine into the pyrimidines of both RNA and DNA of the rat. The desoxynucleosides were incorporated only into the DNA. pyrimidines but not into those of RNA, Recently Rose and Schweigert (65) studied the incorporation of cytidine labeled with in both the pentose and pyrim idine moieties of the nucleoside. They stated that in the rat both the base and the pentose were incorporated into the RNA cytidine and uridine to the same.extent. It is also of interest that the desoxyribose moiety was as highly labeled as the pyrimidine to which it was attached, in dicative of an irreversible reduction of the number 2 car bon of the ribose. Since many of the original investigations of pyrimi dine nucleotide incorporation into nucleic acids depended on deriving the labeled nucleotide from an alkaline hydroly- sate of labeled RNA (66) it was believed that the 2', S'" nucleotides, which are the isomeric forms resulting from such hydrolytic methods, were the isomeric form of the nucleotides involved in nucleic acid biosynthesis. More recent studies would tend to implicate, rather, the 5'~ isomers, Hurlbert (15) showed that G^-orotic acid was in corporated into the acid-soluble uridine-5’-phosphate of rat liver with little dilution of the radioactivity. Ion- exchange chromatographic analyses, from the same laboratory (19), revealed that only the 5'-isomers of the purine and pyrimidine nucleotides are present in the acid-soluble fractions of mammalian tissue. It was found, by Rose and Carter (67), that uridine-5'-phosphate was almost as ef fective in meeting the growth requirements. of. L. bulgaricus 09 as was orotic acid; however, under similar conditions uridine-2», 3'-phosphate, uridine and uracil, in agreement with previous studies (68), did not support growth. The mechanisms whereby nucleosides or nucleotides may be incorporated into nucleic acid have been partially elucidated through some recent investigations by Rose and Carter (67). They observed that p32«labeled cyclic uridyUc acid or guanylic acid was incorporated into the RHA. nucleo tides of L. bulgaricus 09 in a random fashion in a manner precluding an equilibration with the inorganic phosphate of the growth medium. However, an analogous study with E. coli indicated complete equilibration of isotopic phosphate of the medium. Merrifield and Woolley studied the microbiological activities of some dinueleotides (69,70). The organism L. helveticus 335 was shown (71) to have a very specific re quirement for uracil, and to be unable to replace this need with uridine or uridylic acid or with any of the natural pyrimidines, purines, nucleosides, nucleotides, B- vitamins, or folic acid. However, certain pyrimidine-con taining dinucleotides and dephosphorylated dinucleotides could completely replace the uracil requirement of this organism (69,70). Prom their data these authors concluded that uracil is utilized.in L. helveticus 535 not by way of the nucleosides or nucleotides, but by direct incorporation at the dinucleoside phosphate level. They also postulated that transamination occurred at this level since cytosine was completely inactive. Roberts and Visser (72) found that 3-methyluridine or 5-chlorouridine inhibited the growth of Heurospora 1298 in the presence of uridine or cytidine. However, these antimetabolites were inactive when added to the medium of the. . “wild-type1 1 strain. The authors concluded that cytidine and uridine were not normal intermediates in the major biosynthetic pathway to RHA. pyrimidines in Neurospora. Although considerable verification is necessary, the following scheme summarizes the interrelationships of pyrimidines, nucleosides and nucleotides based on the above evidence: Small molecular weight precursors OH .0. N' CH. OH I . C v s 0-CL „CH I 0»C OH I a CH s i i i • o=c OH Jp\ N nCH "^NH^COOH ” ~xNHCvC00H % Dihydroorotic Orotic acid acid R-P Or ot idine- phosphate OH - A W CCHa I I I <£- 0=0. GH ' >N/ I dR-P NH2 I I S 0=C. CH xw dR-P NH2 ■ I A W CH 0-C CH 0=c CH 00H I R-P Uridylie acid I t NHo A W CH -I II - 0-C CH dR-P R-P Thymidylic Desoxy-5- Desoxycytidylic Cytidylic acid methylcyti- acid acid dylic acid OH N CH ♦ I I I 0=C CH • vn' I R Uridine NHS ,cx W CH ■ I I I 0=C CH \N/ I R Cytidine CHAPTER III EXPERIMENTAL Preparation of 4-C^-cytidine (Pig. 1) g-Thio-A-C-^-uracil, (pig, 1, I) A modification of Heidelberger and Hurlbert*s method (73) for the synthesis of orotic acid and oxalacetic acid was employed, sodium hydride (5.16 gm.) was added to 30 gm. of dry ethyl formate in a three-necked flask. The.flask was placed in an ice- bath and 20 gm. of carboxy1-C ^ -ethy1 acetate was slowly added to the reaction mixture with constant stirring. The mixture was stirred at this temperature until a gel formed. The flask was then removed from the ice-bath and permitted to come to room temperature. The sodium enol salt of the ethyl formylacetate formed a solid when kept overnight at this temperature. Dry thiourea.(5 gm.) was added to the solidified salt and the solids mixed thoroughly, then 10 ml, of dry xylene was added. The mixture was refluxed for 6 hours with constant stirring using all precautions to ex clude moisture from the reaction mixture. After removal of the xylene by lyophilization, an aqueous solution of the residue was decolorized with activated charcoal and acidified with acetic acid. The white crystalline product, having the same ultraviolet absorption spectrum as CHa-C1400Et + HCOOEt NaH o^ost s m CH NHs-C-NHg AcO- C 0 I I AcO— C C-CHaOAe i H i H NH- aH NaO OH M N CH I H HS-C. CH N V 22 EtO-C NaOEt CH < H0-C NHS I N' CH I i i HQ-C. CH H i JSL HO-C 0 HO-C C-CHBOH ixi H- w H0-C/Wn0 H #3CCl OH HA HO-C 0 ACoO HO-C C-OH k°\ H OH HO-C-— A-CHgOCjZfa f H VI Cl-CHs-COOH V OH I c-» Ii' XCH i ii HO-C. .CH r ii Br H c^H ' Ax Aco-O 0 t r I H AcO- IX t OAc H A^H C-CHaOAc HBr AeO-C AcO-C- ?/ C-CHaQAe gVIII A xa' Ac20 AcBr OAc H A-- H f A AcO-C 0 AcO-C ^-CHgOC^a I H H VII FIGURE 1 SYNTHESIS: OF 4-Cl4-CYTH>INE 23 recrystallized commercial thiouracil, started melting at 340°* Recrystallization from water yielded 19,0 gm. (65 per cent), 4-C-^-uracil, (Pig. 1, II (74)> Monochloracetic acid (17 gm.) in 350 ml, of water was mixed with 19 gm. of I and brought to 135° in an oven. A flask, placed on the outside of the oven was attached to the reaction flask to , receive the distillate. At the end. of 18 hours, the dried product was brought to room temperature.and enough 99 per cent ethanol added to immerse the dried product completely. The mixture was heated on a steam bath for 5 minutes, fil tered, and dried, in vacuo. The yield from I was 12 gins, (93 per cent). 2,4-Dichloro-2-C1^-pyrimldine. (Pig, 1, III (75))i Phosphorus pentachloride (12 gm,) and. phosphorus oxy- chloride (48 ml.) were mixed with II (12 gm). and the mix ture refluxed for 5 hours with constant stirring. At the end of the reaction,, the excess phosphorus.oxychloride was removed from the dark brown homogeneous mixture by distil lation at 60° under aspirator vacuum. The viseousr. residue was dissolved in 35 ml, of diethyl ether, and crushed ice (100 gm.) was added slowly to the mixture at first so that the temperature of the mixture, remained below 20° at all 24 times. The ether layer was removed and the strongly acid solution was repeatedly extracted with ether. The combined ether extracts were washed with a sodium bicarbonate solu tion and dried over calcium chloride. The ether was re moved and the residue distilled at 85° and 6-7 mm. pressure to yield 8,5 gm, (65 per cent) of a white crystalline solid which started to melt at 61°, 2,4-Diethoxy-4-C14-pyr.imidlne. (Pig. 1, IV (76)X Sodium metal (2.7 gm.) dissolved in 40 ml. of dry ethanol was added to a mixture of III and 42.5 ml. of dry ethanol. The sodium chloride which precipitated was filtered im mediately, The excess alcohol was removed by distillation and the residue was treated with 23 ml. of a 30 per cent sodium hydroxide solution. The alkaline solution was re peatedly extracted with ether. The combined extracts were dried.over, anhydrous sodium sulfate. The ether was removed under aspirator vacuum, and the product distilled at 40° at a pressure of 0.1 mm. Hg. The product which was a viscous liquid at room temperature had a freezing point of 19-20°, The yield was 7.8 gm. (88 per cent). Tetraaceto-D-ribofuranose synthesis, 5-Trityl-D-ribofuranose. (Pig. 1, VI (77))^ To an hydrous pyridine (140 ml.) which had been cooled to 0°, 25 were added D^ribose (40 gm.) and triphenylchloromethane (75 gm*). The solution which was obtained after shaking was allowed to stand at room temperature for 4 days* When the solution was poured into 5 liters of ice water a pre cipitate formed which settled rapidly. The supernatant was then decanted and discarded* The precipitate was washed with one liter of ice water, dissolved in 500 ml. of chlo roform, placed in a separatory funnel, and the solution washed with 50 ml. portions of a 4 per cent potassium acid sulfate solution. The solution was then washed three times with 50 ml. portions of water and dried over anhydrous sodium sulfate overnight. The sodium sulfate was removed by filtration and the solution concentrated at reduced pressure (water aspirator) to a thick syrup. The syrup was dissolved in 100 ml. of carbon tetrachloride, and again concentrated to a thick syrup in order to remove the resid ual chloroform. Hot carbon tetrachloride was added until a thin syrup was obtained and 0.5 gm. of activated charcoal was added. The filtered carbon tetrachloride solution was cooled in the refrigerator overnight. The crystals were filtered and washed with carbon tetrachloride. The yield was 55 gm. (52 per cent) of VI, which started melting at 125°, 1,2,3-Triaceto-5-trityl-D-ribofuranose. (Pig. 1, 26 VII (*77). Forty gm. of VI was added to a mixture of acetic anhydride (70 ml.) and pyridine (140 ml.) which had been cooled to 0°. The mixture was stirred at room temperature until most of the solid had dissolved and allowed to remain at room temperature overnight. The solution was added to a mixture of ice and water (total volume, S.5 liters) and stirred for one-half hour. The precipitate was dissolved in 300 ml. of chloroform and washed twice with 50 ml. por tions of a 4 per cent potassium acid sulfate solution. The chloroform solution was washed twiee with 50 ml. portions of a saturated sodium bicarbonate solution. The chloroform solution was then washed three times with 50 ml. portions of water and dried over anhydrous sodium sulfate. After removal of the sodium sulfate, the solution was concentrated at reduced pressure (water aspirator) to a thick syrup on a water bath (50°). The syrup, was dissolved in 350 ml. of 95 per cent ethanol and again concentrated. Hot 95 per cent ethanol (350. ml.) was added and the mixture stirred until the syrup dissolved. The solution was added, with stirring, to a mixture of ice and water (total volume, 3.5 liters). The resulting precipitate was filtered by gravity through cheese, cloth and air dried. The yield was 42 gm, (81 per cent) of VII. Since the compound is amorphous, purity was determined by detritylation, which consisted of dissolving the tritylated compound (200 mg.) in 2 ml. of concentrated sulfuric acid. About 50 ml. of water was added and the solution was filtered through a weighed Gooch crucible. The precipitate was washed until the filtrate was neutral and then dried at 110° to a constant weight. Calculation of the purity of VII based upon the weight of the precipitated triphenylmethylcarbinol indicated that VII was approximately 100 per cent pure, 1,2,3,5yTetraaceto-D-ribofuranose. (Pig. 1, VIII (78)). Fifty gm, of VII was dissolved in 125 ml, of aeetie anhydride and cooled to 0°. Freshly distilled acetyl bromide (14,5 ml,) was added and the solution was warmed to room temperature and allowed to stand for 30 minutes. Crystals of triphenylbromomethane formed and were removed by filtration. The filtrate was added to an ice-water mix ture (total volume 2.5 liters) and stirred for 4 hours. The supernatant was decanted and the precipitate washed twice with 50 ml. portions of water. The washings and the supernatant were combined and extracted six times with 200 ml, portions of chloroform. The chloroform extracts were combined and washed twice with 50 ml. portions of saturated sodium bicarbonate, and then twice with 50 ml. portions of water. The chloroform solution was dried over anhydrous sodium sulfate, filtered, and concentrated at reduced 28 pressure (water aspirator) to a thick syrup. Hot 95 per cent ethanol (200 ml.) and 0.25 gm. of charcoal were added and the mixture was filtered while hot. The solution was concentrated again to a thick syrup. Isopropyl alcohol (100 ml.) was added and the solution reconcentrated. Crystals formed after alternate cooling in a dry-ice ace tone bath and warming to room temperature. . The flask was placed in the refrigerator overnight and the crystals filtered. The yield was 10 gm. (55 per cent) of VIII which started melting at 59°. l-Bromo-2,5,5,-triaceto-D-ribofuranose. (Pig. 1, IX (79)) A bomb tube containing 2.2 gm. of VIII was immersed in liquid nitrogen. Anhydrous hydrogen bromide, washed successively by passage through phenol, carbon tetra chloride and chloroform, was condensed in the tube until about 8 ml. wes-i obtained. The tube was sealed and brought to a temperature of 20° by immersing in water. After a period of 10 minutes the tube was cooled in a dry-ice ace tone bath, opened, and the hydrogen bromide allowed to boil off. Dry benzene (50 ml.) was added and the mixture lyo- philized. This was repeated three t imes in order to re move all the hydrogen bromide without allowing the mixture to come to room temperature. 4-g^-Cytidine, (Pig, 1, X (79». Fifty ml, of dry benzene containing 7,8 gm, of IV was added to IX and the mixture lyophilized to remove the benzene. The mixture was heated overnight at a temperature of 65° and 10“^ mm, of Hg in order to condense the sugar and the pyrimidine. The unreacted IV (6,2 gm,) was removed by distillation in a molecular still at a pressure of 10“^ mm, of Hg and at 100°. The residue was dissolved in 15 ml, of benzene and transferred to a bomb tube. The benzene was removed by lyophilization, 10 ml, of ethanol was added to the residue and the mixture was saturated with ammonia at -20®, The tube was then sealed and heated at 50° for a period of 5 days. The resulting light brown solution was distilled in vacuo (50°) using a water aspirator. Methanol (50 ml.) was added and the.distillation repeated. The dark brown resi due was taken up in a minimum amount of water, brought to a pH of 3,4 with HC1 and placed on an ion^exehange column (Araberlite IR 120 A, H* form) 25 cm. long and 2 cm. in diameter. The column was percolated with water until the ultraviolet absorption of the effluent at 260 mji was re duced to a negligible value. The material on the column was then eluted with 2,0 N HCl. Samples of 25 ml, were collected and analyzed in the Beckman DU spectrophotometer at 260 and 280 mu. Those samples having a 260:280 mp. ratio of 0.5 were pooled and dried by lyophilization. A small portion of the residue was dissolved in a sodium acetate buffer (pH 5.7) and submitted to filter paper ionophoresis (80). At the end of 4 hours at 20 v/em. and 5° the filter paper strip was removed from the apparatus and dried. The dried paper strip viewed with a Mineralight lamp revealed the presence of two ultraviolet-absorbing components. One component migrated 9 cm, while the faster component mi grated approximately 18 cm. from the origin.' The ultra- violet-absorbing areas were eluted from the paper strips by capillary action with water as the eluent. The samples were placed on aluminum planchettes and dried after neu tralization to phenolphthalein with a few drops of an NaOH solution. These samples were counted in a windowless flow counter. The radioactivity and mobility data seemed to in dicate that the more rapidly migrating component was cyto sine and the slower component was cytidine. Commercial cytidine and cytosine were subjected to filter paper iono phoresis both as a mixture and as single components with the radioactive mixture as the control. The results showed that cytosine was the contaminant. Cytidine purification. Preparation of Dowex-50, hydrogen-form. One pound of Dowex-50 was prepared according to the methods of Moore and Stein (81). The crude Dowex-50 was first washed with 4 N HCl on a Buchner funnel with very gentle suction. After washing with 4 to 8 liters of acid, the filtrate which was initially yellow, was nearly colorless. After two washes with distilled water (colloidal material-ap peared in the filtrate), the resin was washed with 2 N NaOH until the filtrate became alkaline. The resulting sodium salt of the resin was suspended in about three times its volume of normal NaOH and heated over a steam bath for about 3 hours with occasional stirring. The supernatant fluid was decanted or siphoned off after allowing approx imately 30 minutes for settling and replaced with fresh hot N NaOH. This procedure was repeated four times. The initial supernatants appeared very cloudy; the final washes were almost clear. The resin was filtered, washed free of alkali and passed through a 120 mesh screen with 6 to 8 liters of distilled water. By this procedure, a few large particles were removed. The resin was filtered, washed with 2 N HGl until the filtrate was acid, washed with water, and either stored or used directly. Ion-exchange resolution of the cytidine mixture. Resolution of 150 mg. of the impure cytidine preparation was accomplished by the use of an ion-exchange column 32 (Dowex-50, H* form), 15 cm. in length and 1.0 cm. in diame ter. The mixture, in a total volume of 10 ml., was placed on the column at pH 3.0, and the resin was washed succes sively with 100 ml, of water and one liter of 0.2 N HCl. The labeled nucleoside then was eluted with 100 ml. of 2.0 N HCl. An additional 300 ml. of acid was required to elute 1 the labeled cytosine. The results of this separation are given in Fig. 2. Peterminations of the purity of cytidine. The labeled nucleoside in.the first 100 ml..of eluent from the resin column was subjected to filter paper ionophoresis at ' pH 3.7. The only visible component had the same mobility as commercial cytidine. The paper strip was cut into one cm, strips and eluted with water. The eluents were placed on aluminum planchettes and counted. The only radioactiv ity present was that associated with the cytidine spot. - Determinations of purity by the use of reported molar extinction coefficients (82) showed that the 4-C^- cytidine was at least 95 per cent pure. Tissue incubations. Buffer. A Krebs-Ringer-Bicarbonate buffer (83) was used in all the incubations (see Table I). The buffer was aerated with a gas mixture consisting of 95 per cent 0S OPTICAL DENSITY A T 260 M JJl 33 360 280 200 120 80 CYTIDINE 8 0 0 1000 0 200 o.tn VOLUME ( i n CYTOSINE 4 0 0 6 0 0 NCL-------- > MLS.) FIGURE 2 ION-EXCHANGE PURIFICATION OF 4-C14-CYTIDINE 34 TABLE I COMPOSITION OP KREBS-RINGER-BICARBONATE BUFFER Compound Stock sol,27 Vol. used per (gm./liter) 100 ml. buffer Final cone, * (gm./liter) Nad 90.0 7.70 ml. 6.94 KGl 57.g 0.62 0.356 CaCl2 61.0 0.46 0.231 KJfePO* 105.5 0.16 0.169 MgS0«-7Ha0 191.0 0.16 0.306 NaHC03 65.0 3.20 0.416 Glucose 100 mg. 1.00 Hs0 to 100 ml. —^ These solutions were stored in the refrigerator and were kept no longer than 2 months* and 5 per cent C02 for 50 minutes prior to the experiment. The pH of the buffer was determined after aeration was completes*. Preparation of tissues. The animals used in this study were male rats, weighing between 250 and 350 gm,, and were of the University of Southern California strain. The experimental animal was sacrificed by decapitation and the liver was removed immediately and placed in an ice- cold buffer bath. The tissues were then sliced into 0,5 mm, slices with a Stadie-Riggs (84) tissue slicer. The slices were placed in.a tared 100 ml. beaker containing 6,5 ml, of cold buffer. When 5 gm. of slices had been ob tained the contents of the beaker were poured into a 125 ml, Erlenmeyer flask containing 5 mg, of 4-C1^-cytidine in 1.0 ml, of buffer. The flask was stoppered with a two-way stopper and attached to a special adapter to fit the War burg bath. One end of the flask was attached to the source of 95 per cent 02 and 5 per cent C02 and the other opening led to a flask of concentrated sodium hydroxide which served to absorb any radioactive C02 arising from the incubation flask. After 5 minutes equilibration period in which the gas mixture was flushed through the flask at the rate of 5 cu, ft. per hour, the gas rate was reduced to one bubble per 2 seconds. The incubations were carried 56 out with constant shaking and at a temperature of 38° for 4 hours. Tissue fractionation. Cellular fractionation. (85) At the end of the in cubation period, the stopper was removed from the flask and the contents filtered through cheese cloth. The tissue was washed once with 10 ml, of. cold 0.25 M sucrose solution containing 0,0018 M CaCls (Solution A) (85). All subse quent operations were carried out at 0-5°, The liver slices were placed into a Potter-Elve£$em all glass homog- enizer tube (86) and homogenized for 2 minutes in nine vol umes of Solution A. The homogenates were then filtered through one layer of single napped flannelet. The nuclei were isolated from the filtered homoge- nate according to the following procedure: A 10 ml. aliquot of the homogenate was layered over 20 ml, of 0.34 M sucrose- 0.0018 M Cacla (Solution B) and centrifuged at 2000 r.p.m. for 10 minutes (International refrigerated centrifuge, hor izontal yoke No, 269), The centrifuge was accelerated and decelerated slowly to avoid mixing of the two layers. The entire supernatant, which represented the cytoplasm, was withdrawn and immediately refrigerated. Five ml. of Solution A was added to the nuclear pellet which was re suspended by homogenization for 15 seconds. Ten ml. of 37 Solution B was then slowly introduced beneath the suspen sion of nuclei and the mixture was centrifuged for IQ minutes. This procedure of homogenization of the pellet, layering, and centrifugation was repeated twice more. The nuclear fraction was examined histologically with an alcoholic solution of a methyl green pyronine staiiir; and found to be free of cytoplasmic debris. Preparation of the protein residue. (87) To the cytoplasmic extract trichloracetic acid (TCA) was added to a final concentration of 5 per cent. The nuclear fraction was resuspended in the homogenizing tube with three volumes of 5 per cent TCA. Both fractions were permitted to react with the acid at room temperature for one-half hour. The tubes were shaken frequently during the 30 minute ex traction period, after which they were centrifuged. The supernatants in each case .were collected and frozen im mediately and represented the "acid-soluble" extracts. The. precipitates were suspended in 5 per cent TCA, extracted for 15 minutes, and centrifuged. Preparation of the acid-soluble fraction. The tri chloracetic acid present in the §cid-soluble fraction was removed by washing the solution with equal volumes of ethyl ether. The washing was discontinued when the pH of the extract was raised to S. The extract was then brought to pH 8 with 5 N NH^OH. The "acid-soluble" was then sub jected tb ion-exchange chromatography, RemovaX of Xlpids from the protein residue, (87), The precipitates from the TCA washings were suspended in XO mX. of an ethanoX-ether soXution (3:1) and were shaken for 2 hours at room temperature. After centrifugation the residue was suspended in XO mX, of a chXoroform-methanoX soXution (X:I) and refiuxed for 30 minutes at 70°, Follow ing centrifugation the residue was washed three times with XO ml, portions of ethyi ether and dried £n vacuo. This residue, termed the I'protein residue", contains protein and nucieic acid in addition to other undefined components. Fractionation of the protein residue. (80). The protein residue was homogenized in a Potter-EXve^jem homogenizer with 5.0 mi. of ice coid 0.2 N NaOH per X00 mg. of residue and extracted for 30 minutes at 0°. After centrifuging, the supernatant was decanted into a test tube and the coid extraction repeated twice more. The pooled supernatants were adjusted to pH 8 with acetic acid and the volume measured. To the solution was added one and one-half volumes of cold ethanol and one-half volume of 2 per cent lanthanum acetate solution. After standing in the refrigerator overnight, the precipitate of lanthanum nucleate was removed by centrifugation and the supernatant discarded. The precipitate was washed once more with a cold 50 per cent ethanol solution containing 0,1 per cent lanthanum acetate, and centrifuged. The residue was shaken mechanically for 2 hours with 5 ml, of 0,5 N Na2€0a solution.per 100 mg. of original protein residue, centri- fuged, and the supernatant decanted. The precipitate was washed once more with a small volume of water, recentri fuged, and the supernatants pooled. Solid NaOH was then added to the pooled supernatants to a final concentration of 0,3 N and the solution incu bated for 15 to 24 hours at 37°. During this period ribo nucleic acid was hydrolyzed to ribonucleotides. Following the incubation, a cation resin (Dowex-50, H* form) was added to the incubation mixture in small portions until the pH of the solution was reduced to a value between pH 3,7 and 4,1, The resin was removed by decantation and washed several times with.small aliquots of water, The solution was centrifuged to remove small amounts of DNA and protein in suspension. The supernatant was evaporated to dryness with an air jet and dissolved in exactly 5 ml, of water, A small amount of this solution was diluted and the absorption at 40 260 mp. determined, in the spectrophotometer. An average value of 300 (80) was assumed for the calculation of 1 cm. v ' the total amount of mixed nucleotides. The remainder of the solution was transferred to a smaller vessel, evap orated to dryness and water added to a final concentration of 5 mg. of mixed nucleotides per ml. Aliquots of this solution containing 500 to 800 micrograms of mixed nucleo tides were then submitted to ionophoresis. Isolation of pyrimidine nucleotides. Buffers for ionOphoresis of nucleotides. Sodium acetate buffers (80) were replaced by ammonium formate, which offered distinct advantages as a buffer salt for ionophoresis of C14-labeled compounds, since it was completely removed at 60° in 7 hours, thereby reducing the self-absorption corrections for radioactive determin ations. Ammonium formate buffers were prepared according to the direction in Table II, Filter paper ionophoresis at pH 6.0. Ionophoresis for 10 hours at 450 volts and pH 6.0 resulted in one ultra violet -absorbing area 12 to 16 cm. in length and approxi mately 30 cm. from the origin. Although the nucleotides were not separated from each other, they were isolated from many contaminants present in the crude mixture. The area 41 TABIE II PREPARATION OP AMMONIUM FORMATE BUFFERS*/ pH Ml. of 23 N formic acid Ml. NHiOH of conc. (S.G. 0.90) 5.70 6.0 4.3 6,0 3.0 4.3 1/ The indicated volumes of acid and alkali were adjusted to the desired pH and diluted to a total volume of 1 liter with water. The buffers had an ionic strength of 0,07. 42 containing the nucleotides was cut from the filter paper strip and eluted with water onto watch crystals treated with Dri-Filmi^, The water was evaporated from the sample and the material dissolved in a small amount of water and submitted to ionophoresis at pH 3,7. Filter paper ionophoresis at pH 3,7. After iono phoresis at pH 6,0, the mixed nucleotides were eluted from the filter paper, concentrated and submitted to ionophor esis at pH 3,7 for 12 hours and 450 volts. The results of ionophoresis at this pH are tabulated in Table III, The spots were cut from the filter paper strip and eluted with water into siliconed watch crystals for isotopic and spectrophotometrie analysis. Ion-exchange purification of labeled nucleotides. Each pyrimidine ribonucleotide was further purified on an ion-exchange resin (Dowex-1, formate form, 1.5 cm, long and 1.0 cm. in diameter). Preparation of the resin is given below. The samples were placed on the column in slightly alkaline solution. The column containing the uridylie acid was first washed with 10,0 ml. of water and 10,0 ml. of 1.5 N formic acid and then eluted with 30,0 ml, of 4,0 H General Electric Ho, 4487, 43 TABLE III IONOPHORETIC SEPARATION OP RIBONUCLEOTIDES AT PH 3,7 Nucleotides Relative mobility.^/ Uridylic acid - 0*53 / Guanylic acid - 0.43 Adenylic acid - 0.20 Cytidylic acid - 0.08 1/ "Relative mobility” is defined ag: migration of.nucleotide in centimeters migration of iodate ion in centimeters formic acid. The column containing the cytidylic acid was washed with 10.0 ml. of water and 10.0 ml. of 0.1 N formic acid and eluted with 30 ml. of 0.5 N formic acid. The samples were evaporated to dryness by an air jet, dissolved in a small amount of water, and placed on watch crystals treated with Dri^film for counting. Isotopic analysis of labeled nucleotides. The material eluted onto the tared watch crystals was evap orated to dryness at 60° for 7 hours. The material dried into a circular spot whose thickness varied with the weight of the material on the crystal. After drying, the diameter of the spot was measured to the nearest 0.5 mm., the weight on the crystal determined and the thickness in mg, per 2 cm. calculated. In some cases it was necessary to add inert material to increase the weight on the watch crystal, A solution of inositol (1.0 mg. per 0.1 ml.) was used for this purpose since this compound does not absorb in the ultraviolet at 260 or 280 mju, and does not migrate in an electrical field in the pH range employed. The material on the watch crystals was eounted in a Nuclear gas flow counter with a Technical Associates scaler. The slide of the gas counter was machined to accommodate the one inch,watch crystals. Self-absorption curve. All samples were corrected to 2 mg. per cm.2 with the inositol self-absorption curve (88). This curve was obtained by adding inositol in vary ing amounts to the watch crystals containing a constant amount of c-^-glucose. Methods of reporting radioactive data. Specific activity. Specific activities were cal culated as observed counts per micromole of substrate. Relative specific activity. The relative specific activity of a fraction was determined as a ratio of the specific activity of the fraction to the specific activity of the 4-C^-cytidine times 100. Spectrophotometrie assay of nucleotides. The loca tion of the nucleotide bands on filter paper strips after ionophoresis was accomplished by means of a Mineralight lamp 2/, Following elution of the nucleotides from the filter paper strips, a quantitative assay of the. nucleotides was accomplished by the use of a Beckman Model DU quartz 2/ —' Ultraviolet Products, inc., South Pasadena, California, spectrophotometer with standard one cm. matched quartz cuvettes. Bluates from blank strips were carried also through the same procedure in order to correct for the ultraviolet absorbing material extracted from the paper. The optical density of the samples at 260 and 280 rap was used as a criteria of purity. The quantity of nucleotides present was calculated from published molar extinction co efficients at 260 mp. The extinction coefficients used in the calculations and the ratio of the optical density at ; 260 and 280 rap. are given in Table IV. Ion-exchange chromatography. Preparation of the resin. The crude Dowex-2 was treated in the same manner as described above for Dowex-50, with the exception that the resin was percolated with 2 N ammonium formate in 2 H formic acid instead of hydrochloric acid. The eluates were collected periodically in order to determine the extent of the ultraviolet absorption at 260 mp* The washings were terminated when the optical density did not exceed 0.005. The resin then was washed with water until the eluates were neutral in pH and no longer gave a positive Nessler»s test for nitrogen. Uniform particle size was obtained through the use of a 120 mesh screen as described. The resin was used in this form for all 47 TABDS IV EXTINCT I OH COEFFICIENTS AND RATIOS OF OPTICAL DENSITY AT 260 AND 280 ftp FOR RIBONUCLEOTIDES Compound M.W. Em X 10-3 1/ 260/280 1 cm. Range Avg. 1/ Cytidylic acid 323 6,800 210 0.504-0.536 0,52 Adenylic acid 347 13,900 400 4.14-4.38 4.26 G-uanylic acid 363 11,800 325 1,49-1.52 1.51 Uridylic acid 325 . 10,000. 309 2.80-3.42 3.02 —^ Measured at 260 ap and pH 2.0 48 chromatographic analyses. Column preparation. The ion-exchange resin was con tained in a 25 ml, burette, having an inside diameter of 1,0 cm, and equipped with a stopcock, A small ball of glass wool, previously washed with water, was inserted into the burette and pressed securely against the constriction leading to the stopcock. The glass wool was washed once again with 20 ml, of water and the burette completely filled with.distilled water. Powdered glass (mesh 20) was added at the top of the column to a level of 5 mm, above the glass wool. The sur face of the powdered glass was adjusted so that it was level. Powdered glass (mesh.70) was added to the heavier glass bringing the.powdered glass bed to a level of 10 mm, above the. glass wool. The side of the burette was tapped to cause the smaller.particles, of glass to fill the spaces below them. When the surface of the powdered glass was level a small amount of resin, in a slurry with water, was added to the column. As the particles of resin settled to the bottom, the stopcock was opened and more resin slurry poured into the burette. The resin was poured to a height of 20 cm, above the powdered glass and water added to 2 cm, below the burette opening. The column of resin was washed with water from a carboy 90 cm, above the column. The 49 hydrostatic pressure aided in packing the resin securely. The water source, which was introduced through an airtight connection, was disconnected when 500 ml. of eluate had been collected. The column was filled to the top with water and powdered glass (mesh 70) added until a 5 mm, layer was obtained. The larger particles of powdered glass (mesh 20) were added above this to provide a total layer height of 10 mm. This layer served to maintain a level surface of resin which could, not be disturbed when a sample was applied to the resin. Sample introduction. The level of liquid above the resin was removed prior to the introduction of the sample to the resin. The sample, which was introduced with care so as not to disturb the resin surface, was applied to a level of 2 cm. below the column opening. When larger volumes were to be applied, a glass funnel was placed over the column and attached to it with an airtight connection. When the sample had been absorbed by the resin, water was added to 2 cm. below the burette opening as a wash. When the wash was absorbed, a 4 ml, level of water was maintained over the resin. ion-exchange chromatography by gradient elution, (19) The column was eluted with formic acid whose 50 concentration increased continuously (gradient elution). Elution was begun with water in the mixing flask, about 7 ml. of water above the resin column and formic acid in the reservoir. The elution was continued until the uridylic acid came off the column. Since the mixer was not changed, the chromatogram was not subjected to sudden increases in concentration of the influent. The rise in formic acid concentration may be calcu- 3 / lated from the following derivation^'. CR * Reservoir concentration CE = Eluent concentration x = Throughput volume V - Mixer volume 1. 30*, = % ^ ^ V Let u = Cjj-Cg; then, since Cr is a CR“CE V constant, -du = dCE 3. -du = dx u V 4. -ln(CR-CE) = | * K. When CE - 0, x = 0; then K = In Cg 5. -ln(cR-CE ) - 2 - lnCCR 3/ —' Kindly provided by Dr. W, C. Werkheiser, 51 6. la CR - In (CR-CE) - | 7, In CR _ x Or “Os v 8 » lo g _C r _ = X CR”CE 2.5 V 9« Or = -573? Cr-CE ® E2_ « antilog x Cr-Ce 2.3 v 11. Cr-C^ _ cr 12, 1 - Or n antilog 13. CE * CR antil°g ~^73~V “ 1 2.3 V X 2.3 7 X 2.3 V Plotting C j j , against V from equation 8 offered the possibility of estimating the pattern of elution (Pig. 3). Curve A represents the elution pattern of a system with a 250 ml, mixer and 4 M formic acid in the reservoir. Utilizing such a set of conditions provided for an initial rapid elution (0-500 ml.) followed by a relatively slow increase in acid concentration. Such a system provides less discrimination of compounds eluted initially and the M X « C » O M L X • 4 MMOOOM .V.'tOOOML. X«SMHC*OH M.%.« XOOOMC X » S M HOOOH 40 0 VOLUME ( ) FIGURE 3 GRADIENT ELUTION PATTERN I 55 latter part of the range is sufficiently slow to cause broader peaks of compounds held more tenaciously to the resin* The ideal set of conditions would provide a con stant change. The system most appropriate for providing constant elution pattern was found to include a 2000 ml. mixer with 9 M formic acid in the reservoir (curve B)« Resin regeneration. At the end of each chromato graphic analysis, a slight.positive air pressure was ap plied through the bottom of the column. . As the resin emerged from the column, the first cm. of resin was dis carded and the remainder pooled with other ''exhausted* 1 resin. Regeneration of the "exhausted" resin was accom plished by washing with 2 N HaOH until the first alkaline eluates no longer absorbed in the ultraviolet at 260 rap. The resin was then washed with 2 N ammonium formate. Ex change of hydroxyl ion by formate ion was determined by a rise in pH. Washing with ammonium formate was continued until 100 ml. of eluent was collected (100 ml. per column volume 20 cm, x 1 cm.). The resin then was washed with water until the eluents did not react positively with Nessler's reagent. Preparation of 5*-cytidyllc aeid (CMP-51), The location of the "acid-soluble" CMP-5* from 54 tissue extracts subjected to ion-exchange chromatography in which gradient elution techniques were employed was facil itated by the addition of pure CMP-5* to the extracts. Since CMP-5* is not commercially available, this component was isolated from yeast nucleic acid hydrolyzed with snake venom diesterase, A hydrolysate and some crude cobra venom were kindly supplied by Dr, A, Deutsch of the California Biochemical Foundation, Los Angeles, California, The isolation procedure of CMP-5* was essentially ! that of Cohn and ifoikin (89) for fractionation of a nucleic acid hydrolysate, with the exception that the hydrolysate was applied to the column. (Dowex-2, formate form, 20 cm, long and 1*0 cm, in diameter) at pH 8,0 rather than pH 10.0. At the latter pH* guanosine is eluted in the same region as I CMP-5* thereby limiting the purity of the desired nucleo tide. since the pK* for guanosine is 9,5, it will not ad sorb onto the resin at pH 8.0. The column was washed with 250 ml. of water and placed over the Technieon Automatic Fraction Collector (drop counter). The initial influent used was 0,02 M formic acid. The flow rate was maintained at 10 ml. per hour and the collector adjusted to collect 3 ml, fractions. After 500 ml. of eluate had been collected the next eluent (0,15 M formic acid) was begun. This strength of acid 55 elutes the 2‘ and 3*-isomers of cytidylic acid whereas the former acid concentration elutes only the 5’-isomer. Each fraction was assayed spectrophotometrically at 260 and 280 mp. The peak representing CMP-5* was pooled, lyophilized and subjected to enzymatic analysis. Of the enzymes present in snake venom only two will act upon ribonucleic acid and its derivatives. One enzyme, phosphodiesterase, acts on polymerized ribonucleic acid, and the other enzyme, phosphomonoesterase, acts only on 5 *-ribonucleotides, One hundred microliters of the enzyme solution (10 mg, of venom per ml,) were added to 250 micrograms each of commercially available 5‘-adenylic acid and 2‘-adenylic acid in 7,2 x lO"^ moles of NaOH in.a total volume of 1,0 ml. In addition, control samples containing the substrate in the absence of enzyme were analyzed. The beakers were placed in a constant temperature bath at 37° for 4 hours. At the end of the hydrolysis period the samples were placed in 10 ml, test tubes and evaporated to dryness in an air jet, taken up in 0.5 ml, of water, placed on siliconed watch crystals and the samples again evaporated to dryness. The samples were dissolved in 50 microliters of water, placed in the center of filter paper strips and submitted to ionophoresis at pH 4.0 for 4 hours. The resulting 56 strips indicated that the enzymatic hydrolysis was specific for 5*-nucleotides. The same hydrolysis procedure was applied to 250 micrograms of the.isolated CMP-51• The ionophoretic re sults showed that the CMP-5* treated with the crude enzyme preparation was converted to cytidine,. which had a positive mobility, whereas the ■untreated CMP-5* had a negative mobility indicating that the isolated material was, indeed, the 5*-isomer of cytidylic acid. Chemical testa. Known chemical tests for biological products of pyrimidines and their derivatives were applied to a com pound biologically derived, from cytidine and isolated from , liver slices in minute quantities. The tests described be low were found useful in the identification of the unknown compound and were found to be sufficiently specific and sensitive for the detection of less than 5 micrograms of material. The tests utilized are as follows* Organically-bound phosphorus, (90) The reagent described by Hanes and Isherwood (91), which was utilized for the determination of organically-bound phosphorus con sisted of: 57 5 ml. of a 60 per cent perchloric acid solution 10 ml. of 1.0 N HGl 25 ml. of a 4 per cent ammonium molybdate solution water to 100 ml, A 5 microliter sample {5 mg. per ml.) was placed on a small square of filter paper (Whatman Mo. 1) with a micropipette and dried at room temperature. The dried paper was sprayed with the above reagent and exposed to a current of warm air to remove the water from the paper, then heated at 85° for 7 minutes. Inorganic phosphorus ap pears as a yellow spot at this stage whereas organically- bound phosphorus appears as a blue spot when exposed to ultraviolet light for 10 minutes. The standards employed were uridylic acid, cytidylic acid and dihydrocytidylie acid—' Pentose determination. (92) The Brody modification of the Dische pentose test (95) which was used consisted of placing an equal volume of 3 per cent cysteine-HCl in 3 M HaS04 and a 3 per cent unknown solution on a porcelain spot plate and heating at 110° for 8 minutes. Color de velops upon heating. The nature of the eolor depends on the nature of the carbohydrate employed. D-ribose and ribose-containing uridylic and cytidylic appeared as 1 7 ------------- Kindly supplied by Dr. R. E. Cline. 58 cherry-red spots, desoxycytidylic acid turned brown and sucrose appeared as a blue spot. Saturated pyrimidine ring and carbamyl compound de termination. The color determination developed by Pink et al. (9sl) for saturated pyrimidines and carbamyl compounds was modified for a dipping rather than.a spraying technique. The reagent consisted of 50 mg. of p-dimethylaminobenzalde- hyde in 100 ml. of 70 per cent ethanol containing 10 ml, of concentrated HCl. The reagent for hydrolysis was 1 N UaOH in 70 per cent ethanol. A 5 microliter sample (5 mg. per ml.) was placed on a square of filter paper and dipped into the color reagent and heated at 85° for 3. minutes. Carbamyl compounds ap peared as a.yellow spot, saturated pyrimidines, which re quired prior alkaline hydrolysis, were dipped into the alkaline reagent and permitted to dry at room temperature. The dried paper strip then was dipped into the color re agent and heated in the same manner to produce the yellow color. The standards employed for this color test were urea, tryptophane, dihydrouridine^/, arginine, acetamide and ureidOsuccinic acid. Urea and ureidosuceinic acid appeared * “ 57 — Kindly supplied by Miss S'. K. Fukuhara. as yellow spots without alkaline hydrolysis, tryptophane appeared as a purple spot without prior hydrolysis, and the dihydrouridine appeared yellow only if it had been hydro lyzed previously* Arginine and acetamide did not react with the reagent under any condition. The yellow color with the reagent appears to be absolutely specific for compounds containing, or capable of forming after hydrolysis, a carbamyl group. Hydrolysis of saturated pyrimidines, which is required for color, development, evidently causes ring opening between the 3 and 4 positions. Prom investigations of the specificity of this re agent with dihydrocytidylic acid it appears that an amino group attached to position 4 of the pyrimidine ring is suf ficient to prevent color development. Thus, deamination overnight at room temperature with nitrous acid, prior to alkaline hydrolysis, is necessary for color development with this reagent. OH 0 OH N* N3He UaOH H2H ch2 Y 0=0 J 3 H a ' NH > Dihydrouracil A -ureidopropionic acid 60 Dihydrocytidylic acid prepared by electrolytic re duction®/, reacted in exactly the same manner as that pre pared by catalytic reduction^/. ------- — Kindly supplied by Miss T. K. Fukuhara, 7/ — Kindly supplied by Dr, R. E. Cline. CHAPTER IV RESULTS All of the investigations of pyrimidine nucleoside incorporation to date have involved the study of biosyn- thetically prepared ribosides isolated from the nucleic acids of microorganisms grown in the presence of simple labeled precursors (66,95). Ribosides isolated in such a manner, although more easily prepared than through chemical synthesis, are labeled randomly in both the ribose and pyrimidine moiety of the nucleoside. The advantage of a synthetically prepared nucleoside is that it can be labeled in a specific carbon atom of the ring. Therefore, it is certain that any radioactivity isolated in a fraction from tissue Incubated with pyrimidine-labeled cytidine can be derived only from the pyrimidine ring. Although synthetic methods for the preparation of pyrimidine rings have been known (96,9V,98), the yields ob tained by the author were too low, involving loss of labeled starting compounds, A revised procedure was de veloped which not only increased the yields considerably, but is also applicable to the synthesis of other bio logically significant pyrimidines. 62 ■ Incorporation studies. Cytidine, which has been shown to be utilized to a significant extent for the synthesis of ribonucleic acid pyrimidines in vivo, is also readily incorporated into the RNA of rat liver slices. Prom Table V it can be seen in the nuclear RNA that after a 4 hour incubation period of 4-C-1 - 4-cytidine with liver slices, 9.3 per cent of the radio- . activity was recovered in the uridylic acid and 3.6 per cent in the cytidylic acid, whereas in the cytoplasmic RNA. only 1.23 per cent was recovered in the uridylic acid and 0.87 per cent in the cytidylic acid. Some of the 260:280 mu ratios following the filter paper ionophoresis at pH 3,7 were not within the acceptable range of purity (see Tables IV and V). Each pyrimidine i nucleotide was, therefore, purified on anion exchange resin (materials, methods page 42). The 260:280 mp. ratios after the final purification had an acceptable degree of purity, while the specific activities were essentially unchanged, indicating that the actual extent of contamination by radioactive or ultra- violet-absorbing materials following filter paper ionophor esis was slight. Ion-exchange chromatographic analysis. 65 TABLE V INCORPORATION OP 4-C14-CYTIDINE INTO RAT LIVER NUCLEAR AND CYTOPLASMIC RNA PYRIMIDINE NUCLEOTIDES .IN VITRO Optical density Relative Ratios atw260: C.p.m./ specific , 280 mp* pM activitiesi/ Cytoplasmic fraction Uridylic acid, Experiment I 4.70 3.30 12.80 15,00 1,06 1,25 Experiment II 3,15 3.00 14.60 14,60 1.21 1.21 Cytidylic acid, Experiment I 0.51 0,52 10.30 10,30 0.86 0.86 Experiment II 0.60 0,53 10.50 10.90 0.87 0.90 Nuclear fraction Uridylic acid, Experiment II 3.60 3.18 109.00 112.00 9.10 9.30 Cytidylic acid, Experiment II 0.62 0.51 42.00 43.00 3.50 3.60 (a) - Separation of ribonucleotides by filter paper ionophoresis at pH 6.0 followed by ionophoresis at pH 3.7, (b) - Further purification of (a) by anion exchange resin (Dowex-1). * Used as a criterion of purity (80): Uridylic acid 2,8 - 3.42, mean - 3.02 Cytidylic acid 0.514 - 0.536, mean - 0.520 —^ - (Counts per minute per micromole of nucleotide) / (Counts per minute per micromole of 4-C-cytidine) x 100. 64 During the routine isolation of nucleic acids from the liver slices an acid-soluble fraction was obtained after the initial precipitation of nucleic acids and pro tein* Determination of the level of radioactivity present in this fraction was sufficiently high to warrant further investigation. An ion-exchange chromatographic technique was recently developed by Schmitz et al, (19) at Wisconsin for the analysis of the acid-soluble fractions of mammalian organs* The method, which employs a gradient elution technique, was applied to an acid-soluble extract from rat liver slices incubated with non-labeled cytidine and pre pared in exactly the same manner as required for the radio active experimental extract (see page 37), The results of this method for the separation of the acid-solubles, given in Pig, 4, did not appear adequate for the identification of constituents. The 2f -and 3*-isomers of the purine and 0 pyrimidine nucleotides derived from an alkaline hydrolysate of yeast nucleic acid, likewise, were not separated by this method (Pig. 5). To obtain a more complete separation of the compon ents of the acid-soluble fraction, it seemed advisable to revise some of the conditions of this method. One of the steps in the derivation of a mathematical expression o 2 0 40 60 80 100 120 TUBE NUMBER (** “L *""T U B f ) FIGURE 4 ION-EXCHANGE SEPARATION OF COMPONENTS OF THE ACID-SOLUBLE CONSTITUENTS FROM THE CYTOPLASM OF RAT LIVER SLICES INCUBATED IN THE PRESENCE OF NON-LABELED CYTIDINE LEGEM) FOR FIG. 4 The peaks represent the ultraviolet absorption of the acid-soluble components at 260 and 280 mji. The separ ations depicted here were carried out on an anion exchange column (Dowex-1, format e-form) 20 cm. long and 1 cm. in diameter. The flow rate was maintained at 1 drop per 15 seconds with a reservoir concentration of 4 N formic acid and a mixer volume of 250 ml. OPTICAL DENSITY A T 260 M J4 I 125 IOC 7! 5C 25 CMP AMP OMP (IMP 5 4 3 2 I 0 4 0 50 6 0 70 8 0 90 TUBE NUMBER ( w m l k * n » r ) FIGURE 5 ION-EXCHANGE SEPARATION OF A YEAST NUCLEIC ACID HYDROLYSATE Ol P.P. A T 2 60 MJJ " " " 2 8 0 MJJ LEGEMD FOR FIG. 5 The peaks shown in the lower graph represent the ultraviolet absorption at 260 mp of a 5 mg. sample of yeast nucleic acid hydrolysate placed on the column at pH 8,0, The line graph represents uncorrected 260:280 mju ratios. The ranges of values for the ratios of the nucleo tides are given on page 47, The separation was accomplished with an anion exchange column (Dowex-1, formate form) 20 cm, in length and 1 cm, in diameter. The flow rate was maintained at 1 drop per 15 seconds with a reservoir con centration of 4 N formic acid and a mixer volume of 250 ml. useful for the gradient elution technique is to determine the change in eluent concentration (99) at a given volume, which also is applicable to determining the reverse situa tion, namely, the determination of the volume of eluent necessary to. pass through the system in order to attain a given concentration of eluent. Using equation 8 in the derivation (page 51) to plot the concentration of the acid against the volume of eluent through the column when the reservoir concentration (4 N formic acid) and the mixer volume (250 ml.) are those utilized for the previous separ ation, results in a curve given in Figure 3 (page 52), curve A. It appeared from this calculated curve and the actual separation resulting under such conditions that the rise in eluent concentration was too rapid to provide an adequate separation of components eluted initially. From the re sults it appeared that a desirable pattern of elution should provide for a gradual, but constant, rise in acid concentration more closely resembling the straight line of curve B in Figure 3. The condition required for such an elution pattern included 9,0 N foraic acid in the reservoir and a 2,000 ml. mixer volume. These were the conditions employed for the separation of nucleotides from the yeast nucleic acid hydrolysate given in Fig. 6. The separation of components, particularly during the initial stages of OPTICAL DENSITY X l<T* A T 260 MJi 1000 AMP 900 8 00 CMP 7 0 0 6 0 0 500 SUP 4 0 0 UMP 3 0 0 200 100 190 I5 0 » I7 0 20 4 0 90 110 130 TUBE NUMBER (» * “-P E R T U B E ) FIGURE 6 ION-EXCHANGE SEPARATION OF YEAST NUCLEIC ACID HYDROLYSATE 0. D . A T 260 M JJ 71 LEGEND FOB FIGUBE 6 The peaks represent the ultraviolet absorption at 260 mp of a 5 mg. sample of yeast nucleic acid hydrolyzed with alkali and placed on the column at pH 8*0. The lines represent uncorrected 260:280 mp ratios* The ranges, of values for the ratios of the pure nucleotides are given on $i[ge 47. The separation was accomplished with an anion ex change column (Dowex-1, formate-form) 20 cm. in length and 1 cm. in diameter. The flow rate was maintained at 1 drop per 15 seconds with a reservoir concentration of 9 N formic acid and a 2 liter mixer volume. elution, though adequate, was subsequently improved con siderably by reducing the volume of the collected samples from 3,5 ml, to 0,5 ml. The results of this modification when applied to an enzymatic (snake venom diesterase) hy- 8 / drolysate of yeast nucleic acid—' are given in Figure 7, The separation provided by a system capable of resolving the three isomers of each of the nucleotides, with the ex ception of uridylie acids 2* and 3* (separated in other experiments), seemed sufficient for further analysis of the acid-soluble fraction of liver slices which had been in cubated in the presence of the non-labeled cytidine. The results of this separation are given in Figure 8, Isolation and separation of labeled nucleotides. The aforementioned methods of separation were ap plied to the acid-soluble extract of rat liver incubated in the presence of 4-C^-cytidine. The eluents from each of the collected fractions were placed on watch crystals, after spectrophotometric assay, for isotopic analysis. The results cf this separation and isotopic analysis are given in Figure 9, The solid peaks represent the ultraviolet absorption at 260 mp and the striped peaks indicate the --------q 7----- —' Kindly provided by Dr, A. Deutsch of the California Biochemical Foundation, OPTICAL DEN8ITY A T 260 MU CMP UMP 5’ 2', 3' 40 8 0 120 160 2 0 0 2 2 0 260 3 0 0 TUBE NUMBER (3 -5 m l.p e r t u b e ) FIGURE 7 ‘ ION-EXCHANGE SEPARATION OF YEAST NUCLEIC ACID HYDROLYZED WITH SNAKE VENOM DIESTERASE " 2 8 0 M JJ 74 LEGEND FOR FIGURE 7 The peaks represent the ultraviolet absorption at 260 mp of a 10 mg, sample of yeast nucleic acid hydrolyzed with snake venom diesterase and placed on the column at pH 8*0, The 6'-isomers were determined through the use of crude snake venom monoesterase. The lines represent un corrected 260:280 mp ratios. The range of values for the ratios of the pure nucleotides are given on page 47, The separation was accomplished with an anion exchange column (Dowex-1, formate form) 20 cm, in length and 1 cm. in diameter. The flow rate was maintained at 1 drop per 16 seconds with a reservoir concentration of 9 N formic acid and a 2 liter mixer volume. One-half ml. fractions were collected in the initial elution stages. After 100 ml. had been collected the tube volume was increased to 3.5 ml. =1 2 O * 2 0 0 0 t - < 1750 IO • 9 O 1500 X > » 1250 L 5 z 1000 ui o -1 7 5 0 < F 500 & 250 6 0 8 0 140 VOLUME (IN MLS.) FIGURE 8 TacT "555 5 4 3 2 I 0 ION-EXCHANGE SEPARATION OF THE CYTOPIASMIC ACID-SOLUBLE CONSTITUENTS FROM RAT LIVER SLICES INCUBATED WITH NORMAL CYTIDINE P.P. A T 260mm I I 76 LEGESD FOR FIGURE 8 The peaks represent the ultraviolet absorption at 260 mp of an acid-soluble extract of the cytoplasm from 5 gm, of rat liver slices incubated with unlabeled eytidine for 4 hours. The lines represent the uncorrected 260:280 mp ratios. The range of values for the ratios of the pure nuclebtddes are given on page 47. The separation was ac complished with an anion exchange column (Dowex-1, formate form) 20 cm. in length and 1 cm. in diameter. The flow rate was maintained at 1 drop per 15 seconds with a reservoir concentration of 9 If formic acid and a 2 liter mixer volume. One-half ml. fractions were collected during the initial elution stages. After 100 ml, had been col lected the tube volume was increased to 4,0 ml. OPTICAL DENSITY X I0'8 A T 260 M L I 1500 CMP-5* ^ - AMP-5 6 0 8 0 DO 140 180 2 20 VOLUME ( IN ML ) FIGURE 9 600 500 400 300 200 100 ION-EXCHANGE SEPARATION OF THE CYTOPLASMIC ACID-SOLUBLE CONSTITUENTS FROM RAT LIVER SLICES INCUBATED WITH 4-C14-CYTIDINE -a C R M P E R SAMPLE 78 LEGEM) FOR FIGURE 9 The solid peaks represent the ultraviolet absorption at 260 mji of an acid-soluble extract of the cytoplasm from 5 gm. of rat liver slices incubated with 4-C^-cytidine* The striped peaks represent the radioactivity in counts per minute per sample over background. See Legend for Figure 7 for explanation of symbols and conditions of separation. location and extent of labeling. The peaks of radioactiv ity represent approximately 99 per cent of the total iso tope of the anionic material of the cytoplasmic acid- solubles, whereas the nuclear extract did not contain significant amounts of radioactivity. It can be seen from this figure that the radioactivity in this extract is not associated with any ultraviolet absorption at wave lengths usually associated with absorption of pyrimidines and t;heir derivatives. However, the radioactive peak, by virtue of the location of isotope in the ring, certainly must have been derived from the pyrimidine moiety of 4-C^- cytidine. Larger quantities of this radioactive material, which were necessary for the positive identification, were obtained by pooling the acid-soluble extracts from ten in cubations of 5,0 gm. each of liver slices with non-labeled cytidine. In order to locate this material one-fifth of the radioactive peak was added to the large scale extracts for ion-exchange separation. The results of this separation are given in Figure 10. Purification of this peak was ac complished by using the same mixer volume but reducing the reservoir acid concentration to 2.0 N formic acid. These conditions were selected by finding that concentration of reservoir formic acid necessary to provide an eluent OPTICAL DENSITY X I0‘ A T 260 MU topoor 9.000 8,00C 7.000 6.000 5p00 4j00C 3.00C 2,0 O C I.O O C 2 0 4 0 6 0 80 100 VOLUME ( IN MLS.) FIGURE 10 ION-EXCHANGE ISOLATION OF UNIDENTIFIED COMPOUND CD o 12,000 50 40 30 20 I 0 ) C.RM. P E R SAMPLE 81 LEGEND FOR FIGURE 10 The solid peaks represent the ultraviolet absorption at 260 mp of an acid-soluble extract of the cytoplasm from 50 gm. of rat liver slices incubated with unlabeled cyti- dine and pooled with one-fifth of the radioactive peak from the separation given in Figure 8, The striped peaks represent the radioactivity in counts per minute per sample over background. See Legend for Figure 7 for ex planation of symbols and conditions of the separation. 82 concentration sufficient to elute cytidylic acid in a 100 ml. volume, instead of 50 ml„ as obtained by equation 8 on page 51, These conditions are represented as curve C in Figure 5 (page 52). The results of this separation given in Figure 11 indicate that purification was effected satis factorily. It might be assumed from Figures 9, 10 and 11 that the radioactive material bears some resemblance to CMP-5* in its behavior on the ion-exchange column,for the unknown compound consistently precedes CMP-5’, just as the latter nucleotide precedes CMP-2* and 5f• In addition, the re sults of recent studies of pyrimidine biosynthesis and deg radation suggest a common pathway for both synthesis and degradation of pyrimidines. If these mechanisms were to include those of the riboside and ribotide derivatives of pyrimidines, then the radioactivity might be associated with either dihydrocytidylic acid, beta-ureidopropionic acid ribotide or with the ribotide of beta-alanine, On the basis of this hypothesis, qualitative tests were chosen to distinguish between.the possibilities sug gested. Qualitative analysis of the unidentified radioactive peak. The qualitative determinations of the nature of the O P TIC A L D E N S IT Y X 1 0 * * A T 2 6 0 MU 6 0 2500 50 2000 40 CMP-8 * 30 1500 IOOC 20 50C 80 60 VOLUME (in ml ) 120 100 40 20 FIGURE 11 ION-EXCHANGE PURIFICATION OF UNIDENTIFIED COMPOUND CD w C P -M . O P . A T 2 6 0 P E P SAM PLE "" " 280 MM 84 LEGEND FOR FIGURE 11 An ion-exchange purification of the pooled, radio- ■ active samples from the separation given in Figure 9. The I j separation was accomplished on an anion exchange column ; (Dowex-1, formate form) 20 cm, in length and -1 cm, in ' diameter. The flow rate was maintained at 1 drop per 15 I ! seconds with a reservoir concentration of 2 N formic acid i I | and a 2 liter mixer volume. One-half ml, fractions were i ' collected. See legend in Figure 8 for explanation of the ; symbols, 85 radioactive peak, were confined to spot tests. Since the quantity of the isolated material was approximately 1 mg., the choice of tests was very limited for it was of utmost importance that the tests be specific and extremely sensi tive as well. Organically-bound phosphorus. Determinations des cribed by Bandurski et al, (90) and Hanes and Isherwood (91) were used to distinguish between inorganic phosphorus, which appears as a yellow spot prior to treatment with ultraviolet light, and the organic phosphorus which appears blue after irradiation with ultraviolet light. The test was found tq be sensitive to 5 micrograms of nucleotide or to one microgram of organically-bound phosphorus. The utilization of this test, which is described on page 56, with a 25 microliter sample of the unknown (1 mg. in 1.8 ml, of water) resulted in a spot which turned blue in the presence of ultraviolet light. Pentose determination. The original Dische test (93) was designed to determine desoxyribose; Brody (92), however, recently modified the test so that ribose could be detected. Modification of this latter method for a ribose test resulted in a method which reacted positively with both ribose, desoxyribose and sucrose. However, the three 86 different carbohydrates could be distinguished from each other by color difference, Ribose turned pink to cherry red, desoxyribose appeared as a brown spot and sucrose turned blue in the presence of the reagent. Twenty-five micro liters of the unknown sample, in the presence of this re agent, turned red when heated. Prom this and the foregoing evidence, it was concluded that the unknown was a ribotide. Nitrogenous base determination. The. color test de veloped by pink et al, (94) for the detection of carbamyl groups was found to be sensitive for detection of as little as 5 micrograms of carbamyl aspartic acid and specific for carbamyl groups only. Since dihydropyrimidines, which hydrolyze quite readily in the presence of 1,0 N alkali, also react positively with this reagent, it appeared to be a particularly suitable reagent for testing the presence of compounds conceivably resulting from pyrimidine degradation in biological systems. This procedure, which was reported to be responsive to the presence of dihydropyrimidines, was found to be limited to the detection of dihydrouracil and dihydrothymine and their derivatives. The reagent did not react positively with dihydrocytosine and its derivatives. It was presumed that an amino group on carbon atom number 4 of the pyrimidine ring either prevented hydrolysis or interfered with the color test. Therefore, since the 87 unknown did not react with the reagent, a sample of it was deaminated with nitrous acid, and treated with the reagent after alkaline hydrolysis. The compound reacted positively; it was, therefore, concluded from these data and the ion- exchange behavior that the compound was probably dihydro- 1 cytidylic acid. CHAPTER V DISCUSSION Incorporation studies. Although pyrimidine nucleosides are incorporated into nucleic acids,, subsequent studies have indicated that they are not intermediates, per se, in the major biosyn thetic pathways to nucleic acid pyrimidines. However, the data concerning the mechanism of cytidine. incorporation into nucleic acid pyrimidines in vitro does provide some insight into the intermediate steps involved in nucleic acid metabolism. Cytidine, which has been shown to be utilized to a significant extent for the synthesis of ribonucleic acid in vivo (6,7,8), also is incorporated readily into the RHA of rat liver slices. After a 4 hour incubation period of 4_Ql4_eytidine with liver slices a significant amount of the label was recovered in the RHA pyrimidine nucleotides. The incorporation data in Table V show that the nuclear pyrimidine ribonucleotides have a greater specific activity than those isolated from the cytoplasm. Jeener and Szafarz (9) from similar observations with P^04 pro posed that nuclear R1A is a precursor of cytoplasmic RHA fractions. On the other hand, Barnum and Huseby (100) 89 made extensive calculations which led them to believe that nuclear RNA is not a precursor of cytoplasmic RNA, but rather that they both have a common precursor. The heter ogeneous incorporation of labeled glycine (10) and orotic acid (14) into the nucleic acids of different cellular fractions also has been observed. The data in Table V show that the uridylic acid of both cellular fractions has a greater specific activity than the cytidylie acid. These results are similar to those obtained with c14-orotic acid in vitro (57) during a 4 hour incubation period, Hurlbert and Potter (14) found that the degree of C14—orotic acid incorporation in vivo into uridylic and cytidylie acids of nuclear and cytoplasmic RHA, at the end of 4 hours, is similar to that shown in Table V. Similar results were obtained by Anderson and Aqvist (101) 2 hours after the administration of N^-orotie acid. Hurlbert and Potter (14), from time studies of G^- orotie acid incorporation into the nuclear and cytoplasmic pyrimidine ribonucleotides, showed that the preferential incorporation of orotic acid into the uridylic acid rather than into cytidylie acid, which was evident in the early stages of exposure to isotopic orotic acid, diminishes and reverses with increasing time after administration. 90 Similar results were obtained by Hammarsten at al. (7) and Reichard (;„58) in which both N^®-orotic acid and cytidine are preferentially incorporated into Rffi eytidylic acid rather than uridylic acid 24 hours after administration. It is of interest that in the extended experimental time periods also orotic acid and cytidine are incorporated in to the two pyrimidines of RHA in a parallel fashion. The similarity in the degree of incorporation of these pre cursors into the nucleic acid uracil and cytosine in all cases is suggestive of the possibility that cytidine and orotic acid are converted to a common precursor. More over, the studies of Hurlbert and Potter (14) and Ham marsten et al. (7) involving the reversal of labeling in nucleic acid uracil and cytosine from cytidine and orotic acid ( -.58) indicate that the amination of uridylic acid to form eytidylic acid occurs at the polynucleotide level. A similar conclusion was reached by Merrifield and Woolley (69) from investigations of the growth requirements of L. helveticus 335. Lieberman and Kornberg (59) provided evidence, from isolated enzyme studies, that orotic acid in the presence of 5-orthophosphoribose-l-pyrophosphate and the appropriate enzymes is converted to orotidine-5-phosphate which is then transformed to uridylic acid. Hurlbert (15) showed 91 that acid-soluble uridylic acid is labeled from C^-orotic acid with little or no dilution. This evidence in con junction with the results of the present investigation and the results of studies in which uridylic and not eytidylic acid was able to meet the growth requirements of several microorganisms (67,69), would tend to implicate uridylic acid as the common pyrimidine precursor of orotic acid and cytidine. The finding that cytidine is incorporated prefer entially into the uridylic rather than the eytidylic acid of RHA. cannot be explained logically by assuming that cytidine is deaminated to uridine initially, Hammarsten et al. (7), when comparing the incorporation of N^-cyti- dine and uridine, showed that the former nucleoside was utilized effectively for RHA pyrimidine biosynthesis, whereas the latter was a very poor precursor. This is supported by Greenstein et al, (103), who failed to demon strate the presence of a cytidine deaminase in rat liver. Tissue extract investigations. Although the investigations of labeled cytidine in corporation into the RHA pyrimidines provide some evidence concerning the intermediate steps, the identity of pos sible intermediates cannot be ascertained by such an o 92 indirect approach. Therefore, application of a suitable method for the isolation and identification of intermedi ates formed from cytidine in liver slices was desirable. The method reported recently by Schmitz afc al, (19), for the separation of a number of nucleotides by the gradient eiLution technique was successfully modified to ac comodate ion-exchange chromatographic analysis of the acid- soluble constituents of liver slices incubated in the presence of labeled cytidine. The use of the gradient elution technique in conjunction with ion-exchange chro matography has the potential.of being a very useful method particularly for the isolation and identification of inter mediates in nucleic acid metabolism. However, the means for predicting how substances behave in the presence of ion-exchange resins are still uncertain and present many difficulties in the resolution of complex mixtures present in a tissue extract. Certainly through further investiga tion of this behavior there will be devised more effective separations and identifications of the many substances present in the acid-soluble fractions of tissue. Moreover, present in the acid-soluble fraction of liver are a large number of compounds associated with dif ferent metabolic mechanisms such as the urea cycle, the tricarboxylic acid cycle, the glycolytic pathway, nucleic acid intermediates and many coenzymes. It should be emphasized that problems of purity of an isolated compound become rather important. In addition, the selection of each labeled precursor should be considered in terms of its contribution of labeled atoms to compounds other than those under investigation. This problem becomes evident when i aliphatic intermediates of nucleic acid pyrimidines and ; purines, which do not absorb in the ultraviolet, are to be isolated and Identified, As discussed in detail in earlier sections of the dissertation, two separate and distinct pathways have been suggested for the participation of dihydropyrimidines in nucleic acid metabolism. One of the catabolic schemes in volving an initial reduction (page 7), which was postulated by pink and McGaughey (32), entails the conversion of a pyrimidine to a dihydropyrimidine which is then hydrolyzed to the corresponding ureido-derivative. However, current concepts of pyrimidine biosynthesis entail the same mecha nism, namely, that ureidosuccinic acid is converted to di- hydroorotic acid which is desaturated to orotic acid. It is entirely conceivable that the mechanisms for the bio synthesis and degradation are identical and that the rate and direction of the reactions are governed by the meta bolic needs and environment of the organism. Such a 94- metabolic pathway is in accord with our present concepts of carbohydrate and lipid metabolism. Hitherto, the evidence for these pathways of synthesis and degradation has rested on the demonstration that orotic acid participates in both the synthetic and degradative mechanisms. Since orotic acid is the only free pyrimidine reacting in this manner, not only would orotic acid be an obligate intermediate in synthetic pathways from simple precursors, but it also would be involved in the catabolic path from nucleic acid pyrimidines. Such a scheme though not excluded by the evidence now available seems somewhat implausible. The discovery of dihydrocytidylie acid along the pyrimidine metabolic pathway suggests the existence of saturation and desaturation at the nucleotide level. The original hypoth esis becomes more attractive if the mechanisms involved can be shown to operate at the nucleoside or nucleotide level thus relieving orotic acid from its central role in the metabolic scheme. The conceivable mechanisms for cytidine incorpor ation into nucleic acid pyrimidines correlating the data from this investigation with those of others are repre sented in Figure 11. The initial stages of cytidine incorporation into nucleic acid pyrimidines might proceed along three possible NHS c N CHS 0 C CHS N R NHg C N 0 C CH CH Dihydro- eytidine 9 OH C N CHg 0 C CHS N R Dihydro- uridine Ribotide of -ureido- propionamide N R NH 17 Cytidine L 2 C N CH 0 C CH N R-P 11 10 95 NUCLEIC ACID CMP 6 NUCLEIC ACID UMP 5 OH C N CH 0 C CH N R-P Cytidylic acid Uridylic NHg acid 4 C H CHa 5 OH 0 C CHg C N 4-4 N - C H s . Dihydrocyti- R p dylic acid N 15 8 R-P 12 0 NH HSN 0 C 14 ' 2 CHg CHS N R-P 16 -alanine+ribose-5-P Dibydro- ■uridylic acid 0 OH C HgN CHg 0 C CHg N Ribotide 15 R_p of - P-R-HN-CHgCHgCOOH ureido- Ribotide of -alanine propionic acid FIGURE 12 HYPOTHETICAL PATHWAYS OF CYTIDINE INCORPORATION INTO NUCLEIC.ACIDS . f ch8 0-Q^ ^CH, <- Y' 'CH * N > Dihydro- cytidine 0=C CHa r R Dihydro uridine N 17 Cytidine 11 NUCLEIC ACID CMP 6 NUCLEIC ACID UMP t 5 r 10 Cytidylic acid F r 0=CX CH [-P 8 4 Uridylic acid OH A Dihydrocyti^ F fH* dylic acid 0=G CH, N I I < yn. Dihydro- uridylic acid Ribotide of -ureido- propionamide HS|J 0=C Ha Ha: 16 x P 3 c\ h2n chs 0=6 CHg IT ^ t Ribotide R-P T* / R~p Of p - 14 ureido- -alanine+ribose-5-P « P-R-HN-CHgCHs©0OH propionic - - - ' acid Ribotide of (f-alanine FIGURE 12 HYPOTHETICAL PATHWAYS OF CYTIDINE INCORPORATION INTO NUCLEIC ACIDS 96 metabolic pathways (steps 1, 9 and 17), Cytidine might be phosphorylated directly to eytidylic acid which may be in corporated directly into nucleic acid (step 11), However, cytidine, from the results of the present investigation, is preferentially incorporated into RHA' uridylic acid. Moreover, uridylic acid, but not eytidylic acid, has been shown to be capable of supporting the growth of certain microorganisms (67,69) and acid soluble uridylic acid is labeled from orotic acid with little or no dilution (15), Prom these data it would seem likely that eytidylic acid is not an intermediate in nucleic acid biosynthesis. This assumption also would exclude step 10 which involves the deamination of eytidylic acid to uridylic acid. The direct incorporation of cytidine into nucleic acid pyrimi dines through a mechanism in which the pyrimidine moiety of the nucleoside would be exchanged with the nucleic acid pyrimidine (step 17) would seem to be unlikely, since Rose and Schweigert (65) found that the pyrimidine and ribose moieties of randomly-labeled cytidine were incorporated in tact, Moreover, such a mechanism could not account for the preferential labeling of RHA uridylic acid from cytidine nor is it in accord with the results obtained by Roberts and Visser from inhibition studies. It, also, seems un likely that cytidine is deaminated to uridine and then 97 phosphorylated to uridylic acid, since cytidine deaminase is not present in rat liver (103). In addition, cytidine has been shown to be more effective than uridine as a nucleic acid pyrimidine precursor in rats (7), Cytidine may, however, b;e reduced to dihydrocytidine (step 1). Subsequent phosphorylation (step 2) followed by oxidative deamination (step 3) or the same process in reverse order (steps 7 and 8) then.would give rise to di- kydrouridylic acid. These hypothetical steps are in accord with the results of previous investigations which implicate uridylic acid in the biosynthesis of nucleic acid pyrimidines. The preferential labeling of RNA -uridylic acid from labeled phosphate (9), orotic acid (14), and cytidine (12), the specificity of uridylic acid in promoting the growth of certain microorganisms (67,69), the apparent participation of uridylic acid in the synthesis of pyrimidine-containing coenzymes and the extent of labeling in the acid-soluble uridylic acid from orotic acid (15) would seem to suggest uridylic acid is a key intermediate in nucleic acid pyrimidine biosynthesis. » For these reasons the pathway of cytidine incorpor ation into nucleic acid would seemingly be directed towards uridylic acid, and conceivably involve the intermediate 98 formation of dihydroeytidylic acid (step 2) and dihydro- uridylic acid (step 3). Step 15 was included in this scheme as a possible pathway for dihydroeytidylic acid degradation, although It is open to objection on the basis.of prior chemical evi dence of the resistance of dihydrocytosine derivatives to alkaline hydrolysis. It is possible that hydrolytic cleav-i age occurs after conversion to dihydrouridylic acid as has i k been demonstrated for the degradation of uracil (30,31,32) (steps 12, 13 and 14), CBAPTER VI 'SUMMARY 4-C^-Cytidine, synthetically prepared from labeled ethyl acetate, was incubated with rat liver slices. An acid-soluble fraction and the pyrimidine ribonucleotides were isolated from both the nucleus and cytoplasm of the liver slices. Radioactivity determinations indicated that the pyrimidine ribonucleotides of the nucleus were more highly labeled than those from the cytoplasm. Moreover, the uridylic acid of both cellular fractions contained higher levels of radioactivity than the eytidylic acid. Ion-exchange methods employing a gradient elution technique were developed for the analysis of the acid- soluble components of the liver slices. Application of these methods resulted in the isolation of a hitherto un known nucleotide biologically derived from cytidine. 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Creator Grossman, Lawrence (author)

Core Title The metabolism in vitro of 4-C¹⁴-cytidine

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1954-10

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