The electrolyte composition of the soluble phase of cell homogenates

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Content -THE ELECTROLYTE COMPOSITION OF THE SOLUBLE PHASE OF CELL HOMOGENATES by- Deborah Boswell Goodell A Thesis Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF-SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE / (Biochemistry) August 1962 UMI Number: EP41349 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI EP41349 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 U N IV E R S ITY O F S O U TH E R N C A L IF O R N IA G R AD UA TE SCHOOL. U N IV E R S IT Y PARK LOS A NG ELES 7 . C A L IF O R N IA (^icr 6 3 G- 6^ t This thesis, written by . .............. Pe M ra h .. B e& w ell.. G Q Q . d e J L X .......... under the direction of hSX....r.T hesis Committee, and approved by all its members, has been pre sented to and accepted by the Dean of the Graduate School, in partial fulfillm ent of re quirements fo r the degree of .............JMaajtjar..-Q£...Spienae., ........... Dean Date... 1^.62 SI^COMMITTEE u y m J J L TABLE OP CONTENTS LIST OP TABLES ............................. ii |LIST OP FIGURES ........................... SChapter I ! I. HISTORICAL INTRODUCTION.............. | II. STATEMENT OP PROBLEM AND PLAN OP ATTACK III. MATERIALS AND METHODS............ ' . . Materials .......... ........ Animals Chemicals ........ Preparation of Solutions Methods Preparation of Rat Liver Homogenates. . Preparation of the Soluble Supernatant. Dialysis Method Measurement of Magnesium Concentration. Measurement of Potassium and Sodium Concentration ....... ........ Paper Electrophoresis ......... IV. RESULTS Dilution of the Homogenate before Centrifugation ........ The Effect of Dilution on Magnesium Concentration ............ The Effect of Dilution on Potassium Concentration The Effect of Time on the Concentration of Magnesium and Potassium......... Chapter Page Magnesium and Potassium Concentrations from Homogenate and Soluble Supernatant . k-2 State of Electrolytes in the Soluble | j Supernatant.............................. k-2 i i ' | Equilibrium Dialysis. .......... k-2 j j Electrophoresis . . b5 j V. DISCUSSION . . ............................ 53 j VI. SUMMARY........................... 65 I BIBLIOGRAPHY....... ............................... 69 iii LIST OF TABLES Table Page 1. Distribution of Metal Ions among Liver Cell Fractions ........... ........ . . . 13 2. A Comparison of .Cell Disruption Using Varying Numbers of Up-and-Down Strokes of the . . Homogenizer ............... 2b 3. Concentration of Potassium and Sodium in General Purpose Solutions Used for the Flame Photom eter. . 3b b. The Effect of Time on the Equilibration of Magnesium and Potassium between the Soluble Supernatant and the Particulates......... *+3 5. A Comparison of the Amounts of Magnesium and Potassium in the Homogenate and the Soluble Supernatant ¥+ 6. Distribution of Magnesium Inside and Outside a Membrane after Twenty-Four Hours of Dialysis. . *+6 7. Distribution of Potassium Inside and Outside a Membrane after Dialysis................... b7 8. The Effect of Time on the pH on the Soluble Supernatant and Dialysate .............. *+8 9. The Electrophoretic Movement of Protein of the Soluble Supernatant ....... •• 52 iv LIST OP FIGURES Figure Page 1. The Effect of Time on the Color Development of Titan Yellow . . ........................... . 30 2. Standard Curve for the Determination of Magnesium by Titan Yellow. .......... 31 3. The Effect of Dilution on the Magnesium Con centration of the Soluble Supernatant........ 32 *t. The Effect of Dilution on the.Potassium Con centration of the Soluble Supernatant. .... 36 5. The Effect of Dilution on the Sodium Concen tration of the Soluble Supernatant.......... 37 6. The Relationship between the -Concentration of Magnesium in the Soluble Supernatant and, the Per Cent Concentration of the Cytoplasm in the Uncentrifuged Homogenate ........... 39 7. The Relationship between the Concentration, of Potassium inr the Soluble Supernatant and' the Dilution of the Uncentrifuged Homogenate i . ‘ . . *+1 8. Analytrol Diagrams from Paper Electrophoresis of Soluble Supernatant Done at pH 6.60 • . . 5l v CHAPTER I HISTORICAL INTRODUCTION The use of tissue homogenates has proved quite valuable in many biochemical studies. It is then possible to separate and then study the various particulate struc tures of the cell, as well as the soluble cytoplasm. How ever, problems arise in trying to determine the optimum conditions for the preparation of breis. The ideal isola tion process is a method yielding the desired intracellular components unchanged, uneontaminated, and in quantitative amounts, as they actually exist in the cell (1). The investigation of the electrolyte composition of the soluble supernatant from rat liver homogenates was undertaken in an effort to formulate a more physiological medium for preparing breis. A neutral, isotonic sucrose solution, in general, is considered satisfactory as a homogenizing and suspending medium (2, 3)* However, media are still undergoing evaluation and the choice often depends on the intended use. This choice of media has remained a central problem in the preparation of breis and is far from being solved ( 1, *f, 5, 6). The use of an aqueous suspension medium has the 1 2 advantages of ease and rapidity of isolation of the cellu- j lar particulates and the soluble supernatant. Several | problems are introduced through the use of such a medium. ! First, there may be a loss of water soluble components i from the particulates, and secondly, there may be a trans- ; I fer of substances between the soluble phase and the par- 1 1 tieulates and their retention by absorption. However, ! i Schneider and Hogeboom (6) reported that there is no j experimental proof that water soluble materials are lost j from nuclei in aqueous media. Also, Poort (7) recently reported that beef pancreas nuclei must be completely dis rupted to cause an appreciable loss of soluble proteins in aqueous media. Elliott and Libet (8), in 19^2, recorded the first use of sucrose as a suspending medium for the preparation of brain breis. Hogeboom, et al. (9) were next to study the use of sucrose solutions for rat liver homogenates. They evaluated cytologieally water, salt solutions, and sucrose solutions of various molarities for their ability to free large granules, mitochondria and secretory granules, from the cells and to preserve the morphology of liver mitochondria. They also studied the effect of these media on intact liver cells. Their results indicated that water, while able to free large granules, caused visible swelling of these bodies, and the mitochondrial material was no ilonger morphologically intact. In isotonic saline solu- ! j tions at 0°C there was agglutination of the large grannies; ( j into lumps which "became more pronounced upon standing. i Similar results were obtained in the presence of other | electrolytes including KC1, KpSO^, and phosphate buffers. | Isotonic sucrose (0.25M) caused no agglutination and differ ential centrifugation was possible. However, liver cell mitochondria became spherical in this isolation medium. i In hypertonic sucrose solutions (0.88M) the mitochondria kept their rod-like appearance, the unbroken cells looked normal, and there was no agglutination. Also, free nuclei looked similar to those in normal liver cells. Despite the improved morphological condition of the mitochondria, the oxidative phosphorylative function was later shown to be impaired ( 1). Another difficulty in using a hypertonic sucrose solution is that the relatively high density and viscosity probably prevent complete sedimentation of the submicroscopic particles upon centrifueation. In later work, Hogeboom (10) indicated that the lower concentration (0.2^ 1) of sucrose was satisfactory for most purposes. The technique of homogenization has received much attention. Effective cell fractionation requires the rup ture of the cell "membrane" with minimum change to the particulates of the cell. It is generally thought that in aqueous media a coaxial homogenizer, which permits cell V | breakage in a liquid velocity gradient, is the best for ! i i gentle homogenization, The rate of shear depends on the j radii of the pestle and tube, and on the rate of rotation, i However, a hand (up-and-down) homogenizer is more effective) than a motor driven one in the same amount of time (1, 2, 5). A Teflon pestle has advantages over one of glass j i mainly because it does not alter the dimensions of the tubej i I by grinding as rapidly and therefore allows better repro- 1 ducibility. Also, there is no contamination with glass 1 fragments (1). The rapid post-mortem changes in a cell are a seri ous problem. These autolytic effects are minimized by working rapidly at low, but not freezing, temperatures. In an aqueous media even the pH is important and any change in pH can modify the results. Anderson (11) found that a fraction of the soluble phase was insoluble between pH 3*5 to 6. 3j and there was maximum precipitation of protein at pH 5. Also, the microsomes agglutinated at pH 5. In a neutral medium the cell "membraneM is broken first; then, as homogenization is continued, the nuclei become frag mented. At pH 3 and 5, in mammalian tissues, the cell "membrane" is broken but not the nuclear membrane. For this reason citric acid, 1-^+ per cent, has been added to sucrose media. However, many tissues contain cathepsins and nucleases with a pH optimum of 5 or lower so an acid suspension medium may introduce error. Alkaline solutions \ are just as harmful in preparing "breis. They cause swelling ' i land dissolving of the nuclei, along with clumping and > 1 agglut inat ion (1). Griswold and Pace (12) studied the distribution of ! t sodium, potassium, calcium, and magnesium among liver cell I fractions prepared by differential centrifugation. Approx-; i imately 50 per cent of the sodium and potassium were found | i in the supernatant while magnesium and calcium were chiefly, 1 in the particulate fractions. These results are broadly consistent with the known behavior of these metal ions with respect to their reactions with protein molecules (1 3, l*f, 15). Generally, magnesium ions are ionically ’ ’ bound'1 or complexed in a non-diffusible form by proteins when the protein carries a negative charge. Potassium and sodium ions are expected to form such compounds weakly, if at all. KLotz ( 1 3) states that experiments have demonstrated that sodium and potassium are bound by certain natural polymeric electrolytes. Gurd and Wilcox (16) say there is no present evidence that potassium and sodium ions are bound to pro teins but that these ions affect the ionic atmosphere of proteins, including their functions as counterions to the negatively charged macromolecules. Griswold and Pace ( 1 2) also found that the ions associated with the particulates are apparently non- | diffusible and are hindered in some manner from leaving 6’ the particulate. Thiers and Vallee (17) obtained similar iresults in their studies on metal distribution in subcellu-; i I lar fractions of rat liver. Amoore (18) investigated the 1 potassium ion concentration in mitochondria and suggests that there is no concentration difference between the ^ ! mitochondria and the soluble cytoplasm of the liver cells. Amoore believes that potassium is largely in the inaccessi ble phase of the mitochondrial water where the concentra- j I tion may be greater than 100 millimolal. Upon disruption 1 i of the mitochondria, only 10 per cent of the potassium remained associated with the fragments. He suggests that the millimolal ratio of potassium between the inaccessible and accessible space is controlled by a Gibbs-Donnan equi librium. Gamble (19) has shown that loosely bound potassium is not retained by the mitochondrial membrane during wash ings with potassium free solutions, but tightly bound potassium on acceptor sites located on the structural ele ments of the mitochondria, membrane or eristae, is retained. He has also shown that respiratory activity of the mito chondria enhanced the stability of the potassium binding sites and stimulated the rate of potassium exchange. Gamble fragmented mitochondria by treatment with digitonin and found that they still had the ability to bind and exchange potassium, were capable of oxidative phosphoryla tion, and still held the tightly bound potassium when washed with isotonic sucrose* Gamble’s evidence suggests |that potassium adheres directly to a substance on the mito- I chondrial membrane or cristae. Berger (20) also noted that' a certain amount of potassium can be removed from mito- i chondria by washing. She found that the uncoupling of oxidation from phosphorylation caused leaching of mito chondrial potassium. This same result was brought about by surface active agents and calcium salts. She found that mitochondria could maintain a concentration of potassium much greater than the surrounding medium with the aid of a metabolically available energy source. Scott and Gamble (21) observed the effects of various inhibitors upon potassium binding and oxidative phosphorylation of mito chondria. They found that low concentrations of inorganic i and organic mercury counpounds stimulated the rate of i potassium exchange between mitochondria and the medium. Also, these agents inhibited oxidative phosphorylation and diminished the total amount of bound potassium. Thiogly- colate and glutathione prevent this action on potassium binding and phosphate esterification, and reverse the / potassium exchange rate. Ethylenediaminetetraaeetie acid (EDTA) was found to prevent the toxic effect of the inhibi tors on potassium binding capacity but it did not change the exchange rate produced by the inorganic and organic mercury counpounds. 8 Another interesting aspect of mitochondrial behav ior is the close relationship between mitochondrial size and metabolism. Malamed and Recknagel (22) found that sucrose entered the mitochondria but only to a certain extent. Approximately 65 per cent of the water space of the mitochondria is permeable to sucrose solutions. This had been previously shown by Werkheiser and Bartley (23). Malamed and Recknagel also showed that the sucrose inacces- * • * sible space behaved as an ideal osmometer and obeys Boyle- van’t Hoff * s Law for an external sucrose concentration from < 0.05m to 0.30M. Packer (2*+) has shown the swelling and shrinking of mitochondria arise from different metabolic states under conditions favorable for oxidative phosphoryla tion. The regulation of the respiratory rate in mitochon dria is largely under control of adenosine diphosphate (ADP), and Packer (2*+) found that an increase in ADP caused mitochondrial shrinking while an absence of ADP and the presence of oxidizable substrates caused swelling. Bartley and Davis (25) found magnesium in higher concentrations in the mitochondria than in the surrounding medium, but this concentration was not affected by metabo lism. Later, however, Schneider and Hogeboom (6) stated that the mitochondria are able to concentrate, hydrogen, sodium, magnesium, and phosphate ions while undergoing active metabolism in vitro. A concentration gradient for 9 magnesium between the mitochondria and the supernatant fraction was also shown by Griswold and Pace (12). | ! Sanui and Pace (26) investigated sodium and potas- ‘ sium binding by rat liver microsomes. They found that i I sodium and potassium were adsorbed by the microsomes and I could be washed out only very slowly indicating that they were bound and not merely trapped in the membraneous sacs which are highly permeable to sodium and potassium. They ' i i found 16 to 18 milliequivalents bound per liter of micro- j somal pellet volume. The pH optimum for binding was a little greater than 7 and the two ions seemed bound to the same extent. Although potassium was bound slightly more strongly than sodium, there apparently is a competition for | binding sites. The fact that ion retention depends on con centration seems to indicate specific sites are present and retention is not osmotic. They found more than one species of sites for the hydrogen ions, so the binding capacity was much greater for this ion than for sodium or potassium. Sanui and Pace compare the microsomal binding of potassium, sodium, and hydrogen to a synthetic cation exchange resin and say the binding follows simple mass law. Recently there has been evidence for the binding of magnesium to ribonueleoproteins (RHP) and ribonucleic acid (RNA). The RHP from rat liver microsomes and similar RHP from other sourees has been shown to require some 10 I dialyzable substance for stability (27). Hamilton and Petermann (28) observed the importance of magnesium for the i structural integrity of rat liver REP. They have shown I that the structure of purified RNP in solution is dependent! upon the ionic environment and particularly the magnesium ! concentration. Changes in pH and ionic strength also ^ i I ! affect the association-dissociation reactions of RHP. The i ( presence of other ions greatly affects the dissociation of the principal component of RHP. Cations, such as sodium or potassium, compete with magnesium for sites on the RHP and can displace the magnesium if they are present in excessive amounts. However, unlike magnesium, these cations are not able to maintain the structural integrity of the RHP (27). It is evident that any agent which has an appreciable affinity for magnesium, such as phosphate, citrate, or EDTA, will have a destructive effect on micro- | somal RHP. Epp, et al. (29) give infrared evidence indi cating that the pyrophosphate portion and purine nucleus are involved in the complex formation with magnesium. This complexing may be intra- or intermolecular* Edelman, et al. (30) give evidence supporting the idea that magne sium is bound by RHA and RHP of rabbit reticulocyte micro somes but not by the protein. In fact, the protein in RHP blocks approximately 30 per cent of the sites. They infer that the magnesium binds to the negatively charged phosphate 11 groups, Zubay (3 1) found that the negatively charged phos-. phate group is not always the actual binding site. His 1 experiments suggest that magnesium may be bound by the ! i nitrogenous base groups. However, chelation may not J actually be possible because of the configuration of the j bases in native nucleic acids. The distribution of sodium and potassium was studied in isolated thymus nuclei by Itoh and Schwartz (32). They found that sodium was the more stable constituent of the j two. Potassium was lost easily after prolonged standing at low temperature, repeated washings, incubation in potassium free media, and particularly in the presence of pyridoxal, mercuric ion, phenol, and urea. Conversely, sodium was relatively stable under similar conditions. The potassium adhering to the nuclei increased when conditions favorable to metabolism existed. However, the potassium concentra tion decreased only slightly upon addition of metabolic jinhibitors. Again, the sodium content remained relatively stable. Haora, et al. (33) found that 29 per cent of the magnesium in isolated thymus nuclei was soluble in the isolation medium, 7 per cent was soluble in incubation media, and 6^ per cent was associated with deoxynucleie acid (DMA) and nucleotides. The retention of magnesium was entirely dependent on the presence of sucrose. Buffers, 12, I glycerol, and ethylene glycol at the same molar concentra- j tion showed a loss of magnesium. Since it had been found j that mononucleotide retention depends on the presence of ! sucrose, the magnesium loss could be due to the release of J l the mononucleotides. j i Table 1 gives a summary of the partition of common metal ions among the various liver cell fractions. Prom this table it is seen that approximately 50 per cent of the sodium and potassium is in the soluble supernatant, while calcium and magnesium are chiefly in the particulates. Sodium and calcium percentages are maximum figures because of possible contamination by extracellular regions. In the procedure of Griswold and Pace (12), each fraction was washed two additional times with no major effect on the concentration of any ion. Both moving boundary and paper-strip electrophore sis have been used to study the scalable supernatant pro teins obtained from centrifuged liver breis. Anderson and Swanson (35) used moving boundary electrophoresis with a phosphate buffer at pH 7.5 to study the supernatant from liver breis. They found that a definite part of the soluble protein of liver has an isoelectric point more basic than 7 .5 and therefore a net positive charge at physiological pH. Sorof and Cohen (36), using moving boundary electrophoresis with a veronal buffer of pH 8. 6, TABLE 1 . DISTRIBUTION OP METAL IONS AMONG LIVER CELL FRACTIONS Metal Ion Whole Homogenate (mg/gm tissue) i » . . i Per Cent of Homogenate Composition Pound in Cell Fraction Nuclei Mitochondria Microsome Supernatant Griswold and Race (12) K 13.6 1 8.i f ir.9 52.7 Na 12.5 6 .8 Ik.k 65.6 Ca 15.5 k7.7 23.7 26.6 ... Mg 13 A 21.8 k8.0 1 2 .8 Thiers and Vallee (17) K 2.900 29.0 7.8 2.8 58.0 Na 0.270 26.0 2.5 3.0 6 6 .0 Mg 0.160 *+7.8 17.*f 13.7 1 9 .2 Macfarlane and Spencer (3*+) K* *f .21 3.6 k.8 75.5 Berger (20) 6 .6 K 3.20 k .7 3.8 8 0 .0 * The fresh tissue was dried and ashed; not perfused. M Co n ~ iifi i J found nine components in the soluble phase of rat liver. ! Adjutantis (37) separated soluble rat liver-cell proteins j on filter paper. He used a series of buffers to find which) f produced the best resolution. Borate buffer at pH 8.6 was j found to give the best resolution, and he found five major components in the soluble supernatant. Veronal and phos phate buffers (pH 8.6) were less satisfactory. However, the soluble supernatant was prepared by lyophilization and suspension of the powdered material in isotonic sucrose. The original treatment of the liver cells makes it more difficult to apply his results to soluble supernatants prepared by perfusion and homogenization. 1 ' CHAPTER II STATEMENT OP PROBLEM AND PLAN OP ATTACK The purpose of these experiments was to investigate the magnesium and potassium concentrations of liver cell homogenates. The concentration of sodium is negligible in the liver eell, therefore sodium concentration was not measurable at all times. The study of these metals, • especially their state in the soluble supernatant, should help in formulating a more physiological medium for per fusion, homogenization, and suspension of cells. Much work has been done with various homogenizing media to determine their ability to keep the particulate structures morphologically and functionally intact. How ever, this approach to finding the optimum conditions for the preparation of homogenates is generally centered on the recovery and use of only one particulate structure. This takes into consideration only that particular structure and disregards the effects of the preparation on the rest of the cell. Another approach to the preparation of homogenates is possible. This method tries to reproduce as closely as possible the medium in which the particulates 15 16 are normally found. Ideally, this would mean simulating the soluble cytoplasm of the cell and therefore maintaining all the particulate structures in their true physiological condition. This second approach was kept in mind in these experiments. The perfusion technique and the method of prepara tion of the soluble phase used in these experiments were similar to those described by Anderson (11, 38). The effect of dilution with isotonic sucrose on the concentra tion of potassium and magnesium in the original homogenate was investigated. Dialysis experiments against an equal volume of water or NaOl solution and soluble phase were used to study the state, bound or free, of magnesium and potassium in the soluble supernatant. The state of binding of magnesium in the soluble supernatant was further studied by paper-strip electro phoresis experiments using phosphate buffers with and with out added magnesium. CHAPTER III MATERIALS AND METHODS Materials Animals ! ' i 1 l ; ! : ? Male albino rats of the Wistar strain from the j I stock of the University of Southern California, School of Medicine • were used. i j I Chemicals \ All chemicals were ACS Reagent Grade except the j | i j j followings | | Silicone Oil— Dow Chemical Co., Silicone Oil 0702. | ‘ !l Polwinvl Alcohol. 98$ hvdrol.vzed— Matheson Coleman ! and Bell, Division of the Matheson Co., Inc., Norwood, \ Ohio. Lot No. PXL295. Acid Fuchsin— Allied Chemical Corp., National } Aniline Division, University of Rochester Medical Center, ! | Rochester, New York. C.I. #*2685. j i j ! | Preparation of Solutions I j Deionized Water— Solutions for use in the deter- j | ! mination of sodium and potassium must be rigorously free | I of sodium and potassium. Por this purpose, distilled water 18 was passed through a.mixed "bed resin (Deemajet demineral- izer, Crystal Research Laboratories, Inc.). Alternately, distilled water was passed through a column of cation exchange resin (Dowex 50, Dow Chemical Co.) in the hydrogen form. Sucrose. 0.25 M.— 171.2 gm of sucrose were dissolved in deionized distilled water and diluted to 2 liters. The solution was stored at 5°0. Acid Fuchsin. 0.1 per cent— 25 mg of acid fuchsin were dissolved in distilled water and diluted to 250 ml. Sodium Tungstate. 10 per cent— 50 gm of Ifo^WO^. 2H2O were dissolved in distilled water and diluted to 500 ml. Sulfuric Acid. 2/3 N— 1.86 ml of concentrated t H2SOii .(35.6 R) were diluted to 200 ml with distilled water. Polyvinyl Alcohol (PVA), 0.5 per cent— 100 mg of PVA. were dispersed in 100 ml of distilled water. The dis persion was heated to 65°0 with stirring until dissolved. The solution was then diluted to 200 ml, filtered, and stored at 5°0. Sodium Hydroxide. * + N— -80 gm of HaOH were dissolved in distilled water and diluted to 500 ml. Titan Yellow. 0.5 per cent— 50 mg of titan yellow were dissolved in distilled water and diluted to 100 ml. This solution was stored in an amber bottle in the dark 19 and remade every week. Magnesium. Standard Solution— 5Q mg of magnesium turnings were dissolved in approximately V ml of 1 N HC1. Water was then added to make a volume of 100 ml. Potassium Stock Solution. 20 mEqA— KC1 crystals were dried overnight in an oven and cooled in a dessicator. Then 0.7b55 gm of KC1 were dissolved in deionized distilled water and diluted to 500 ml. This solution was stored in a polyethylene bottle. Sodium Stock Solution. 50 mEq/1— NaCl crystals were dried overnight in an oven and cooled in a dessicator. Then l.^l^ gm of NaCl were dissolved in deionized dis tilled water and diluted to 500 ml • This solution was stored in a polyethylene bottle. Lithium Stock Solution. 5000 ppm— Li2C0^ was dried overnight in an oven and cooled in a dessicator. Thirteen and three tenths gm of LigCO^ were added to 100 ml of deionized distilled water. Thirty ml of 12 N HC1 were slowly added until a clear solution resulted. . The solution was then diluted to 500 ml. • This solution was stored in a polyethylene bottle. Sodium Chloride Stock Solution. 0.2 M— Five and eighty-five hundredths gm of FaClwere dissolved in deionizec distilled water and diluted to 500 ml. Phosphate Buffer. 0.025 M— One and twenty-three 20 hundredths gm NaCI, O.M39 gm HagHPO^, and 0.155+ gm NaE^PO^.E^O were dissolved in distilled water and diluted to 1 liter. The total ionic strength was 0.031. The pE[ was adjusted to either 7.28 or 6.60 with 1 N NaOH or 1 N HOI. Phosphate Buffer. 0.025 M, plus 0.01 M MgCl?— This phosphate buffer was prepared as above with 2.0333 gm MgCl2» 6EU>0 added for every liter. The total molarity was 0.035 and the ionic strength was 0.061. The pH was adjusted as above. Methods Preparation of Rat Liver Homogenates Eat liver was chosen as the material for the homogenization study because its ready availability and because there is a great deal of information about liver cells and their use in homogenates. Also, the individual cells are large, easily disrupted, and have abundant cytoplasm (10). Rat liver contains approximately 20 ml of blood per 100 gm of tissue (38). The problem of contamina tion of the homogenate by red cells was eliminated by per fusion with 0.25 M sucrose. Of course, perfusion has an unknown effect on the whole cell and possibly on the elec trolyte composition. It has been observed that 0.15 M NaCl removes blood more completely than sucrose solutions (3 9)• 21 However, since some measurements of sodium concentration were to be made and since the electrolytes present were to j be studied, sucrose was used for perfusion. j i I The method used for the preparation of the liver homogenates was very similar to that described by Anderson 1 1 (11, 38). Young male rats weighing approximately 125 to j 175 gm were used. The rat was anesthetized or killed with ether, and decapitated. The rat was allowed to bleed for j a few seconds while suspended by the tail. The skin cover-*! ing the upper left side of the body was removed with curved scissors. An incision was made on the left side below the ribs, and continued around to the spine and along the spinal column to the top of the pleural cavity, severing all the ribs. The diaphragm was cut around to the midline, close to the chest wall. The aorta, lying along the spinal column, was partially cut and a #L5 gauge blunt needle with cold, 5°C, perfusion fluid flowing out, was inserted into the aorta. The needle was tied into the aorta with a piece of thread. The left lung was removed to allow free outflow of the blood. The perfusion fluid was 0.25 M sucrose. The pressure of the perfusion fluid was maintained at about 120 mm of water to simulate normal rat blood pressure (38). Perfusion was continued until the liver color lightened, or three to five minutes. The liver was then removed and placed in a previously weighed, chilled beaker. Three rat 22 i I livers were usually perfused in each experiment. The livers i were kept in the beaker in an ice bath and weighed together. i r I The livers were then cut into small pieces with a pair of j ! scissors and homogenized in several portions with a small I amount of the cold sucrose solution. The homogenizer was a r i glass tube, 12.5 cm by l.1 * cm (inside diameter), with a j i pestle of Teflon, 1.2 cm (diameter), mounted on a stainless; steel shaft. Thirty up-and-down strokes were used to homogenize the liver after the liver pieces were well broken up. The homogenizer was kept in an ice bath during this process. The brei was transferred to a graduated cylinder in an ice bath and diluted with 0.25M sucrose to a volume which was twice the weight of the liver. The brei was examined microscopically to determine the amount of cell disruption upon homogenization with varying numbers of up-and-down strokes. The maximum amount of cell breakage was desired with minimum damage to the particulates. Two methods were used in determining the optimum number of strokes with which to homogenize the rat liver. A small drop of brei was applied to a glass micro scope slide. An equal amount of 0.1 per cent acid fuchsin solution was added next and the slide was tilted until the dye was mixed into the brei. A cover slip was placed over the spot and the slide observed under a microscope before the spot dried. A lens of a Zeiss Standard GFL 23 microscope was used to view the slide. The results are shown in Table 2. A sample of brei homogenized with thirty | strokes was fixed on a slide, dried, and stained with \ | routine hematoxylin and eosin. Upon microscopic examina- j tion, many nuclei were visible along with a few liver cells j and some Kupffer cells. Approximately 95 per cent of the cells appeared to be ruptured and the nuclei looked normal, i Preparation of the ! Soluble Supernatant i To obtain the soluble supernatant from the liver brei, 12 ml portions of the brei were transferred to plas tic centrifuge tubes for the No, ^0 rotor of a Spinco Model L Preparative Ultracentrifuge. The No, *+0 rotor had been chilled overnight in a 3°C cold room. The brei was spun at 100,328 x g for one hour with refrigeration and the rotor was allowed to decelerate freely. The sediment in the plastic tubes after centrifugation was in five definite visible layers. The soluble supernatant was removed from the tubes either by using a syringe and long needle, or simply by decanting it from the sediment. In the latter case, some of the lipid layer came out with the soluble supernatant. Approximately 7 ml of supernatant were recovered from 12 ml of homogenate originally used. The soluble supernatant was then used directly or frozen in plastic bottles for future experiments. The color of the 1 ! 2k TABLE 2 A COMPARISON OP CELL DISRUPTION USING VARYING NUMBERS OP UP-AND-DOWN STROKES OP THE HOMOGENIZER Number of Extent of Strokes Disruption* 10 Masses of cells visible along with some nuclei 20 Approximately 92 per cent 30 Approximately 95 per cent 80 100 per cent; no whole cells visible * Breis stained with 0.1 per cent acid fuchsin as described in text. 25 soluble phase, in the various experiments, ranged from a straw color to a definite red. Varying dilutions of the brei were also centrifuged in the ultracentrifuge.' A series of dilutions of the origi nal homogenate was made . depending on the amount of the original homogenate available. The dilutions were made directly in the plastic centrifuge-tubes. A 10 ml pipette with an enlarged hole was used to pipette the brei. The brei was thoroughly mixed before each aliquot was removed as there was much settling. The dilutions,( made with cold 0.25 M sucrose,' were well mixed in the plastic tubes before centrifugation. When the diluted homogenate was less than the 12 ml needed to fill a plastic cell for the rotor, the difference was made up with Dow silicone* oil. The dilu tions were centrifuged as described above with the rotor decelerating freely. The soluble supernatant was then care fully removed and stored in plastic tubes in the freezer. Dialysis Method The materials used for the dialysis experiments consisted of a 20 cm piece of Tygon tubing (outside diame ter 1.2 cm, inside diameter 1.0 cm) with a piece of Visking dialysis tubing (0.95 cm diameter) placed inside. Pieces of the Visking tubing, approximately 20 cm long, were soaked in distilled deionized water for one half hour. The piece was then tied at one end and drawn into a piece of 2 6 Tygon tubing, A screw clamp was placed on the Tygon tube above the knot in the Visking tube. One and five tenths ml of the soluble supernatant was put inside of the Visking bag with a small syringe and a long needle, BD /20. Next, 1,5 • 1 f ml of deionized water or a sodium chloride solution was pipetted into the space between the Yisking bag and the Tygon tubing. Another screw clamp was placed above the liquid level of the tubes and tightened.’ in air space was allowed to remain in each compartment, There were two reasons for devising such an assem bly. First, the amount of soluble supernatant available for each dialysis bag was approximately 1.5 ml. The volume of the dialysate, either deionized water or a NaCl solution, was also to be 1.5 ml. This posed the problem of finding some way for the dialysate to cover • the supernatant in the Yisking bag. If a Isnot was tied in the tubing and the tub ing placed in a test tube, the volume of dialysate was not sufficient to cover the volume in the tubing. Ey using a piece of Tygon tubing, a knot could be tied in the Yisking tubing, facilitating the removal of the protein solution, i and a Hoffman screw clamp could be placed on the Tygon tub ing above the knot in the Yisking tubing. In this manner, the volume of the dialysate was sufficient to cover com pletely the supernatant in the Yisking bag. Secondly, it was preferable for the materials to be plastic or some 2 7 other inert material, such as the Tygon tubing, since sodium could be leached from glass. It was observed that deionized water stored in Pyrex glassware picked up detectable amounts of sodium and potassium in less than one week. Each dialysis.assembly was wired to a test tube rack. The rack was then attached to a rotating arm of a variable speed motor and rotated slowly. The dialysis was carried out in a cold room (5°C). Different time periods were used initially to determine the time needed for an equilibrium to be established on both sides of the mem brane • Twenty-four hours was found to be sufficient time for establishing an equilibrium* After twenty-four hours, the Tygon assemblies were removed. With the knotted end of the Visking tubing down and the Tygon tubing over a small plastic test tube, the screw clamps were earefully opened and the dialysate was allowed to run into the test tube. The Visking tube was then removed and emptied into a small graduated cylinder, the volume recorded, and the protein solution then poured into a plastic tube. The protein, solutions and dialysates were stored in a cold room. Measurement of Magnesium Concentration Magnesium was determined by a modification of the procedure of Weill and Neely (*f0). Polyvinyl alcohol (PVA) 28 was substituted for gum ghatti as a dispersing agent, and no calcium solution was added. Either a Beckman DU or Beckman B Spectrophotometer was used to measure the absorb ance instead of an EEJ Colorimeter with a number 62b filter. Finally, the standard magnesium solution was made directly from magnesium turnings instead of from MgCl2. 6H2 0. A blank was made with 3 ml of distilled water in a test tube, and the unknown solutions were made with varying concentrations of homogenate or soluble supernatant to a total volume of 3*0 ml. To each tube, 1 .0 ml of 10 per cent sodium tungstate then 1 .0 ml of 2 /3 N H^SOi*. were added, with mixing after each addition. The solutions containing protein were centrifuged at 1 ,0 0 0 rpm in a standard clini cal model desk centrifuge for ten minutes. The solutions were filtered and 2 .5 ml of each were transferred into a new tube. To each tube was added and mixed 1.0 ml of PYA solution; 0 .5 ml of titan yellow were next added and mixed. Finally, 1.0 ml of b E NaOH was slowly added to each tube and mixed gently. The absorbance of each solution was read against the blank at 5*+0 m/t on a Beckman DU or Beckman B Spectrophotometer using matched cells. The same pipette was used throughout the procedure for the addition of each reagent, being thoroughly washed out with distilled water before each new reagent, to enhance reproducibility. A time study was run on the color development with titan yellow. A magnesium solution containing 0.5 mg magnesium per ml of solution was used. The procedure followed was the same as given above. The results are [ i j I shown in Figure 1. From this study, it was decided to take’ i j the absorbance readings between ten and forty minutes after. I jthe addition of the sodium hydroxide solution. J ! A standard curve was made using solutions contain- j j ing known concentrations of magnesium. The results are j i ! | shown in Figure 2, which indicate that Beer’s Law is j I followed up to a concentration of approximately 8 /<gm per i j ; j ml or an absorbance of 0.250. All the unknown solutions i j were subsequently diluted so the absorbance would fall j below 0.250. The effect of dilution on the magnesium con centration of the soluble supernatant was determined and found to vary in a linear manner, as expected. The results are shown in Figure 3. Measurement of Potassium and Sodium Concentration A Baird Atomic Flame Photometer was used for the measurement of potassium and sodium in the homogenate, soluble supernatant, and their dilutions. All of the glassware used was rinsed four times with deionized dis tilled water, in addition to the regular rinsing procedure. A series of general purpose solutions was next prepared for the purpose of calibration of the photometer I scale. These solutions were made from the stock solutions *0.160 ■ 0 *150* 0*140 0.130 0.120* 10” 15 20 25 30~ Time (minutes) 35“ ^ 4*5 50 Fig. 1.-— The effect of time on the color development of titan yellow. The method described in the text was used for a solution containing 5.0 Agm per ml (.1:100 dilution of the standard mag nesium solution). Absorbance (5 * f 0 mju) 0 .3 0 0 0.200 '*+.006.00 8.00 10.00 12.00 Concentration C«gm Mg++/ml) 2.00 Fig. 2.— Standard curve for tlie determination of magnesium by titan yellow. 32 P S o •H -p 0 i —i o c a + + U0 100 -■ 80 . . A 6o ... 20 .. 10. . . 20 Concentration of Soluble Supernatant {%) 100 Pig. 3.-— The effect of dilution on the magnesium concentration of the soluble superna tant. The magnesium concentration was deter mined by the titan yellow method described. 33 i described previously. The final concentrations of the 1 ! general purpose solutions is shown in Table 3. [ The general purpose solutions, GP 1, GP 5, and GP 9 were stored in plastic bottles and were remade every three I or four weeks. The photometer was set according to the i directions given in the instruction manual so that "1" on j the scale was equivalent to 0.02 mEq per 1, "5“ was equiva-j lent to 0.10 mEq per 1, and ”9” was equivalent to 0.18 mEq , per 1, for both sodium and potassium. Each division on the scale equalled a change of 0.00*f mEq per 1. The homogenate, soluble supernatant, or their dilutions were diluted 1:100, or 1:500 if still too concentrated, before being tested. A known amount of lithium stock solution was added so the final concentration of lithium would be 250 ppm. If the sample range was 0 to 20 mEq per 1, the dilution used was 1:100. A 0.1 ml of sample and 0.5 ml of lithium stock solution were put into a 10 ml volumetric and deionized distilled water was added. If the sample range was 20 to 90 mEq per 1, the dilution used was 1:500. A 0.1 ml of the sample and 2.5 ml of lithium stock were put into a 50 ml volumetric and distilled deionized water was added. The air pressure for the photometer was maintained at 10 psi. The glass parts of the photometer were washed frequently and the atomizer was periodically cleaned with chromic acid. TABLE 3 CONCENTRATION OP POTASSIUM AND-SODIUM IN GENERAL PURPOSE SOLUTIONS USED FOR THE FLAME PHOTOMETER General Purpose Solution K mEq A Na mEq A Li ppm GP 1 0.02 0.02 250 GP 5 0.10 0.10 250 GP 9 0.18 0.18 250 t i The effect of dilution on the potassium and sodium ' I concentration of the soluble supernatant was determined and; i found to vary in a linear manner. The results for potas- j sium and sodium are shown in Figures b and 5, respectively.! !Paper Electrophoresis ! Paper electrophoretic studies were performed using I Spinco Burrum type cells with Spinco paper wicks and strips! of S&S paper. Two cells were used in each study. One eon- | | j tained a 0.025 M phosphate buffer, while the other con tained a similar phosphate buffer which was 0.01 M in MgC^. Electrophoresis was carried out at two pH*s, 7.28 and 6.60, at 125 volts, 18 to 21 miliamperes, for 15.1/2 hours at |room temperature. For the protein stain, strips were dyed ! j with Bromophenol Blue (^1). 36] bO • • 30.. 20 .. 100 20 Concentration of Soluble Supernatant (%) ------------------- t Pig. *+.— The effect of dilution on the potassium concentration of the soluble supernatant. The potassium concentration was measured by flame photometry. mEq Na+/1 20 . 20 ^0 60 80 100 Concentration of Soluble Supernatant (%) Pig. 5.--The effect of dilution on the sodium concentration of the soluble supernatant. The sodium concentration was measured by flame photometry. CHAPTER IV RESULTS Dilution of the Homogenate before Centrifugation The procedure for preparation of liver homogenates, dilution of the breis before centrifugation, centrifugation, and recovery of the resulting soluble supernatant, has been described in Chapter III. The Effect of Dilution on Magnesium Concentration Dilutions were made of the soluble supernatant so that the final magnesium concentration would be in the range following Beer's law for the titan yellow method described earlier. At least 0.5 ml of the soluble super natant was diluted for assay of magnesium as it was found that the concentration of magnesium determined in smaller amounts of supernatant could not be easily duplicated. As is shown in Figure 6, there is a linear relation ship between the concentration of magnesium in the soluble supernatant and the per cent concentration of the cytoplasm in the uncentrifuged homogenate, assuming the cytoplasm is 25 per cent of the cell (*f2) • 38 Concentration Mg+ + Cflrgm/nl) 20 l ! 20 $ Concentration Cytoplasm ) Pig, 6.— The relationship "between tration of magnesium in the soluble supernatant and the per cent concentration of the cytoplasm in the uncentri fuged homogenate, bO The Effect of Dilution on Potassium Concentration The procedure for measuring potassium concentration by flame photometry has been described previously. The machine was set with the standard solutions, GP 1, G-P 5, and GP 9» after every one or two readings. The samples of soluble supernatant were diluted either 1:500 or 1:100, with an appropriate amount of lithium stock solution. Generally, if the 12 ml centrifuge tube held up to 8 ml of the original homogenate (the remaining 12 ml volume made up with 0.25 M sucrose), a 1:500 dilution was used. For less than 8 ml of homogenate per 12 ml of solution, a 1:100 dilution was used. Figure 7 shows that there is a linear relationship between the concentration of potassium of the recovered soluble phase as plotted against the dilution of the homogenate before centrifugation. 4 * The Effect of Time on the Concentration of Magnesium and Potassium The effect of time ori the concentration of mag nesium and potassium in several dilutions of the homogenate * / ■ t was observed. Four centrifuge tubes, two containing 6 ml and two containing 8 ml of homogenate, each diluted to 12 ml with 0.25 M sucrose, were prepared immediately upon homogenization of the liver and well mixed. One tube of each dilution was then immediately centrifuged, as bl *fO- 30.- 20. •H 10. . o 10 12 ml HomogenateA2 ml Solution Fig. 7.— The relationship between the concen tration of potassium in the soluble supernatant and the dilution of the uneentrifuged homogenate. *KL of homogenate with enough 0.25 M. sucrose solution added to make the final volume 12 ml. previously described, for one hour at 100,328 x g. The | remaining two tubes were incubated for 90 minutes in an ice; bath before centrifugation. Table ^ shows the comparison 1 of the amounts of magnesium and potassium obtained under the two sets of conditions. Magnesium and Potassium Concentrations from Homogenate and Soluble Supernatant j Several measurements were made of the magnesium i and potassium concentration in the original homogenate and the soluble supernatant obtained from it. Table 5 shows this data. State of Electrolytes in the Soluble Supernatant Two methods were considered to study the state of magnesium and potassium in the soluble supernatant: equi librium dialysis and electrophoresis. Equilibrium Dialysis The dialysis method described previously was used in inve stigating the distribution of both magnesium and potassium in the dialysate and the soluble supernatant. A series of NaCl dilutions were made with deionized distilled water from the 0.20 M HaCl stock solution for each experi ment. The dilutions ranged from 0.01 M to 0.20 M ITaCl. Dialysis was also performed against deionized distilled water at the same time. Titan yellow was used to determine TABLE I f THE EPPECT OP TIME ON THE EQUILIBRATION OP MAGNESIUM AND POTASSIUM BETWEEN THE SOLUBLE SUPERNATANT AND THE PARTICULATES* Dilution Mg Concentration C^gm/ml) K Concentration (mEqA) Incubat i on T ime (Minute s) 0 90 0 90 6 ml homogenate per 12 ml solution 28.0 26.8 15.96 15.60 8 ml homogenate per 12 ml solution M)Jf 39.2 27.0 ■ 2*+.0 *Method described in text. Incubation at 0° C. -r OJ TABLE 5 A COMPARISON OP THE AMOUNTS OP MAGNESIUM AND'POTASSIUM IN THE HOMOGENATE AND THE SOLUBLE SUPERNATANT Electrolyte Homogenate Soluble Supernatant 30.5 35.8 ^Methods described in text. * 4 - 5 the magnesium concentration^ while' the flame photometer was used to measure potassium concentration. The effect of dialysis on magnesium distribution inside and outside of the membrane is given in Table 6. The original dialysates in these experiments were 0.01, 0.05, 0.10, and 0.20 M NaGl'solutions and deionized dis tilled water. Also, the final volume is shown in this table. Table 7 shows the effects of dialysis on potassium concentration of the soluble supernatant after treatment with the above NaCl solutions or deionized distilled water. Prom preliminary experiments it was found that sodium equilibrated rapidly across the dialysis membrane. However, magnesium and potassium required agitation and a twenty-four hour period to become equilibrated. The pH of the dialysates and the soluble super natants was measured after 6, 12, and 2b hours. This measurement was made at room temperature. The results are shown in Table 8. Electrophoresis Prom the results of the experiments involving equilibrium dialysis, it appeared that potassium was ubiquitously distributed in the soluble supernatant. How ever, magnesium appeared to be held or bound in some way in the soluble supernatant. The electrophoretic studies, TABLE 6 DISTRIBUTION OP MAGNESIUM INSIDE AND OUTSIDE„A MEMBRANE AFTER TWENTY-POUR HOURS OP-DIALYSIS Concentration of Nadi in Original Dialysate (Molar) Pinal Volume of Soluble Supernatant** (ml) Ms Concentration (Asm/ml) Soluble Supernatant Dialysate Experiment 1 0.00 1.90 ,57.6 17.1 0.01 1.90 58.0 17.3 0.05 1.90 58.8 18.8 0.10 1,80 61,2 18.2 0.20 1.80 60.0 18.2 Experiment 2 0.00 1.85 56.2 16.8 0.01 1.80 58.0 16.7 0.05 1.85 57.6 16.7 0.10 1.85 61.2 17.1 0.20 1.90 61.2 18.*+ Experiment 3 0.00 1.80 59.*+ 12.0 0.01 55.2 11.0 0.05 1.80 55.8 12.0 0.10 1.80 52.2 ~ - 13 .2 0.20 1.80 53.** 12.6 ■N* Methods described in text. Original volume of both the soluble supernatant and the dialysate was 1.5 ml. Dialysis for twenty-two hours. - F TABLE 7 DISTRIBUTION OP POTASSIUM INSIDE Ap OUTSIDE A MEMBRANE AFTER DIALYSIS Concentration of NaCI Final Volume of K Concentration (mEqA) in Original Dialysate (Molar) Soluble Supernatant** (ml) Soluble Supernatant Dialysate Experiment 1 0.00 1.90 16.5 16.5 0.01 1.80 1**.^ 13.6 0.05 1.90 17.5 17.0 0.10 1.80 16.5 17.5 0.20 1.90 16.8 16.0 Experiment 2 0.00 1.90 16.8 17.0 0.01 1.95 17.b 18.0 0.05 2.00 19.5 20.5 0.10 ---- 17.8 17.5 0.20 1.80 17.3 17.5 * Methods described in text. ** Original volume of both the soluble supernatant and the dialysate was 1.5 ml. *** Dialysis time **#* Dialysis time was sixty-six hours, was twenty-four hours. -r TABLE 8 THE EFFECT OF TIME OH THE pH ON THE SOLUBLE SUPERNATANT AND DIALYSATE Concentration of NaCI in Original Dialysate (Molar) ' pH 6 Time (hours) 12 27 1/2 Soluble Supernatant I Dialysate Soluble Supernatant i----------------- 1 " ■ ■ ■ ■ ......— I Dialysate i . . . . ! Soluble Supernatant i 1 I 1 Dialysate i 1 ' I 0.00 6.5*+ 6.71 6.53 6.73 6. 1 + 9 6.67 0.01 6.50 6.68 6.1+9 6.67 6. 1 + 8 6.61+ 0.05 6.35 6.66 6.38 6.63 6. 1 + 5 6.6 5 0.10 6.**1 6.55 6.1+3 6.56 6 M 6.56 0.20 6.37 6.52 6.3>+ 6. 1 + 9 6. 1 + 3 6.3 6 •r b9 therefore, involved only magnesium. In each case, two cells were used simultaneously under identical conditions of time and voltage. The buffer in one cell was 0 .0 2 5 M phosphate buffer, and in the other cell, 0.025 M phosphate buffer plus 0 .0 1 M MgClg. One electrophoretic experiment was carried out with the pH of the phosphate buffers at 7.28. In this experi ment, three samples of the soluble supernatant were dialyzec overnight in a cold room against distilled water, distilled water plus 0.01 M EDTA, and distilled water plus 0.01 M MgCl2* The pH of the EDTA solution was adjusted to the pH of the distilled water,used. The soluble supernatant dialyzed against distilled water was run in both cells. The soluble supernatant dialyzed against 0.01 M MgC^ was run in the 0.01 M MgC^ phosphate buffer cell, and the soluble supernatant dialyzed against the EDTA solution was run in the phosphate buffer cell. A sample of undialyzed soluble supernatant was also run in each cell. Duplicates of each sample were run. The strips were stained for pro^ tein with bromophenol blue and the color density was measured with a Spinco Analytrol, Model RA, with a 500 m// interference filter. A second pH, 6.60, closer to that of the original soluble supernatant, was ehosen for a second electrophoretic experiment. The experiment was carried out in the same 5o manner except that phosphate buffer was substituted for distilled water in the.overnight dialysis of the soluble supernatant. An example of two Analytrol diagrams made from the paper electrophoresis done at pH 6.60 is shown in Figure 8. The electroosmotie effect was determined in the two buffers using dextran as the reference material. Forty Stll of a 6 per cent dextran solution were applied to each strip. The two cells were run at the same voltage and for the same length of time as in the previous experiments. The strips were stained with a bromophenol blue saturated solution of methanol. It was found that the dextran moved 3 cm toward the cathode in the phosphate buffer and 2 .2 cm toward the cathode in the phosphate plus magnesium buffer. Dextran was checked for magnesium binding, which might have affected its movement in the phosphate plus magnesium buffer. A dextran solution with MgC^ added was applied to one half of the strips in the phosphate buffer cell. The movement of the dextran and the dextran plus magnesium was identical. The results of the electrophoretic experiment, corrected for the electroosmotie effect, are shown in Table 9. 5 i ; Origin Dextran Dextran Origin Anode Pig. 8.— Analytrol diagrams from paper electro phoresis of soluble supernatant done at pH 0. 6O. Condi tions described in text. I. Soluble supernatant dialyzed and run in 0.( pho s phate buffe r. II. Soluble supernatant dialyzed and run in 0.025M phosphate buffer plus 0.01M MgCl2* 52 TABLE 9 THE ELECTROPHORETIC MOVEMENT OP PROTEIN OP THE SOLUBLE SUPERNATANT Dialysate Used Buffer Used pH * Distance moved before Electrophoresis for Electrophoresis Anode (cm) Cathode (cm) ftft Phosphate Phosphate 6 .6 0 1 0 .2 1 .2 Phosphate plus 0.01 M EDTA Phosphate 6. 6o 1 0 .6 1.9 None Phosphate 6 .6 0 12.9 2 .8 Phosphate plus 0.01 M MgCl2 Phosphate plus 0.01 M MgCl2 6 .6 0 k-,5 1 .0 None Phosphate plus 0.01 M MgCl2 6 .6 0 k.7 1 .0 Water Phosphate 7.28 12.9 2.k Water plus 0.01 M EDTA Phosphate 7.28 13.0 2.5 None Phosphate 7.28 1 1 .1 1 .0 Water plus 0.01 M MgCl2 Phosphate plus 0.01 M MgCl2 7.28 4-.1 0.9 ft Distance moved corrected for the electroosmotie effect, ft. ft. Phosphate buffer, 0.025 M, ionic strength 0.031, throughout • CHAPTER V DISCUSSION The study of electrolytes in the soluble super natant of rat liver cells presupposes a method of obtaining the soluble supernatant without contamination of ions from the particulate structures and extracellular material. A method for recovering the soluble cytoplasm from cells without disturbing the delicate internal balance existing between the cytoplasm and the particulate structures seems, at this point, to be lacking. Even the death of the animal, by any means, has an unknown effect on all the cells, as well as on the distribution and state of the sub stances within the cells. Perfusion of a tissue, removal, and homogenization, in any medium, can easily, be expected to cause gross changes in the cytoplasm, as well as the particulates. A study by Anderson (^3) showed that a variety of structures form in tiie clear ultracentrifuged soluble supernatant from rat liver. He found six discrete types of structures. These may be formed during or after cell breakage and resemble components of liver cells. The idea is considered that cell components exist!in equilibrium 53 5*t with their constituent molecules, in solution, and a mechanism similar to that involved in the formation of structures in the soluble supernatant of rat liver cells may also occur in the living cell* Many investigators, mentioned earlier, have studied the electrolyte composition and state of binding in the microsomes, mitochondria, and nuclei of rat liver cells, and other cells in general. However, these studies have treated the particulate structure as an isolated body, without taking into account the irreversible changes that may have been produced in its isolation. To begin with, the homogenates in these previous studies were diluted at least one to four (weight liversvolume sucrose or medium). In most cases the dilution was even greater, usually one to nine. At such great dilutions, any equilibrium involving the electrolytes that might have existed between the cyto plasm and the particulate has most likely been destroyed. The ions may now be washed in or out of the particulates indiscriminately, or may'recombine in the particulates in some unphysiological manner. These distributions may then be mistaken for an in vivo condition. For these reasons, ■ - \ it was considered important to use1 as concentrated a brei as possible. A one to two (weight liversvolume sucrose) and a one to one dilution were prepared and both found satisfactory in regard to homogenization so a one to one 55 dilution was used throughout for the preparation of the homogenates. A more concentrated liver brei seemed mechanically impossible using the small homogenizer without producing great local heating effects and poor cell break age. After determining a suitable original dilution of the brei, the next step was to find out, if possible, just what effect further dilution of the brei might have on the electrolyte composition of the particulates. It was specu lated that if an equilibrium existed for any electrolyte between a particulate and the cytoplasm, a series of dilu tions of the brei, made before centrifugation, would • *' V release a certain amount of the electrolyte which would be related to., the equilibrium constant. The .soluble super natant obtained from the dilutions could then be checked for electrolyte composition. It would then be possible to * f ‘ - extrapolate the results back to the original cytoplasm and, in this manner, determine the true electrolyte composition of the soluble cytoplasm. Such a study was performed for both magnesium and potassium. The results showed a linear variance between the dilution of the soluble cytoplasm and the concentration of magnesium. The concentration of magnesium in the whole homogenate was always greater than the concentration in the soluble supernatant. This indi cates that the magnesium is bound in the particulates in a 56 manner which prevents its equilibration with the soluble supernatant. The binding may be relatively non-specific, as the electrostatic binding between magnesium and a non- diffusible anion. Of course, a more complex type of bind ing, as chelation, may also occur along with the electro static type. However, this is not necessarily a duplication of the in vivo conditions regarding magnesium binding in the particulates. Even a one to one dilution may start too far out on the dilution curve to show any relation but a linear one between dilution and magnesium concentration. If this is true, a general redistribution of magnesium ions may have taken place. A linear variance between brei dilution and potas sium content was found. The concentration of potassium in the soluble supernatant was approximately the same as that found in the homogenate. These results indicate that potassium is able to distribute itself freely between the particulates and soluble supernatant. As the homogenate is diluted with sucrose,-the potassium concentration of the t . 1 particulates equilibrates freely with the potassium of the soluble supernatant. Another variable considered was the time allowed for establishing an equilibrium between the electrolytes of the particulates and the soluble cytoplasm in the vari ous dilutions of the brei. The usual procedure was to 57 centrifuge the solution immediately upon dilution. However, after incubating for ninety minutes at 0°C, there was no additional release of either magnesium or potassium. This indicates that the equilibrium, or redistribution if any, takes place in a very short time. If anything, the results may indicate a slight readsorption. However, the amounts are so small no definite statement can be made. Preliminary experiments indicated that magnesium is in greater concentration in the particulates than in the soluble supernatant, and is probably bound in some manner. Dilution of the brei solutions did not seem to remove any more magnesium than already may have been removed by the original one to one dilution. The average per cent potassium found in the soluble supernatant was 58.^9. This value agrees well with that of Griswold and Pace, 52.7 per cent, and Thiers and Vallee, 58.0 per cent. However, the two groups do not agree about the distribution of potassium in the various particulates. The total amount of potassium found in the homogenate was 2.597 mg per gm of liver tissue. Thiers and Vallee found 2.90 mg potassium per gm of tissue. Other investigators have found much higher values for potassium content. Macfarlane and Spencer (31 *) found if. 21 mg per gm of tissue with 75*5 per cent in the soluble supernatant, and Berger (20) found 3.20 mg per gm of tissue with approximately 75 per cent in the soluble supernatant. Obviously, there is not good agreement between the various investigators. The difference may arise from the varied procedures used in perfusion. The isolation procedures of Griswold and Pace (12) and Thiers and Vallee (17) were very similar to those described in this paper. It must be realized that perfusion may have washed away a certain amount of the electrolytes, especially the potassium. Also, during homogenization both magnesium and potassium could be washed out of or into the particulates. Great care was taken to avoid contamination of the brei with electrolytes, especially those being measured, and there seems to be no significant contamination introduced in the procedure. The differences in the total magnesium found in several different experiments can be partially accounted for in the original dilution of the homogenate. Even though the livers were blotted after perfusion and < . \ r ’ *, removal, it is probable that different amounts of sucrose remained; also, the dilution of the homogenate was made in a graduated cylinder and was therefore not as accurate as desired. The state of the magnesium and potassium in the soluble supernatant was next studied, after finding that their existence in the soluble supernatant was probably not simply a dilution effect on the brei. It seemed of 59 interest to determine, if possible, if either magnesium or potassium were actually bound'to some cytoplasmic macro molecules or if they were present simply as free ions. Equilibrium dialysis against water and NaCI solutions was used as one method to determine the existence of any bind ing. The dialysis experiments were performed against NaCI solutions <?f different molarities to determine- the Donnan effect on the electrolyte distribution. Dialysis against water and the low concentrations of NaCI would show a Donnan effect on the electrolyte distribution if present, while the high concentrations of NaCI would virtually swamp any Donnan effect present. Surprisingly, there seemed to be no Donnan effect indicated by the potassium ions. The potassium was concentrated equally inside and outside of the membrane, regardless of the NaCI concentration used for the dialysate. This result would indicate that the non-diffusible macromoleeules inside of the membrane had no net charge. The measurement of magnesium concentration inside and outside of the membrane showed that the concentration of NaCI had no effect on the distribution of the magnesium. The concentration of the magnesium in the dialysate and in the protein solution remained constant, but the concentra tion of magnesium in the dialysis bag was approximately three times the concentration of magnesium in the dialysate. This indicates that the magnesium is bound in some manner to a non-diffusible macromolecule in the soluble super natant* It seems possible that the magnesium is neutraliz ing the charges on the bulk of the macromolecules and they are therefore nearly isoelectric. This would explain the lack of a Donnan effect on the distribution of potassium. From the dialysis experiments, the amount of mag nesium in the cytoplasm that is bound to maeromoleeules and the amount existing free can be calculated. An average of 0 .2 5 5 mg of magnesium are bound to non-diffusible maero moleeules, probably proteins, in every milliliter of cyto plasm, while 0.121 mg exist as free ion. This means that approximately 6 7 .8 per cent of the magnesium of the cyto plasm is in a bound form.. The separate dialysis experi ments of Table 6 .show a very constant amount of magnesium bound per milliliter of cytoplasm between the experiments. A variance in the magnesium concentration of the soluble cytoplasm is seen in the amounts of magnesium present as free ion. This could indicate that sucrose perfusion can remove and lose magnesium ion from the cytoplasm. From the dialysis experiments the amount of free potassium ion in the cytoplasm can also be calculated. From Tables 5 and 7 it can be calculated that there are 6.502 mg of free potas sium ion in every milliliter of cytoplasm. The change in pH as related to dialysis time was 61 measured. It was found that in each compartment, after 6, 12, and 27 1/2 hours, there was relatively no change in pH. It is interesting to note a difference in pH between the protein solution and the dialysate. The equilibrium between the protein solution and the dialysate was established at a temperature of 3°c ? while the pH measurements were made at room temperature. Since the two solutions contain different buffer systems and temperature has a definite effect on the pH of a buffer system, it seems logical that this is the cause of the pH difference measured. Paper electrophoresis was used to investigate the binding of magnesium to the proteins in the soluble super natant. The results, shown in Table 9> show that the pro teins are capable of binding magnesium in some manner which neutralized most of the negative charge present in a solu tion with the divalent ions removed. At a pH of 6.60, the protein moves only an average of 3 .8 8 cm toward the anode after dialysis and electrophoresis in a phosphate buffer that is 0.01 M in magnesium, while the protein moves 10.2 cm toward the anode after dialysis and electrophoresis in phos phate buffer alone• A great decrease.in the negative charge of the protein upon dialysis and electrophoresis in a com bination buffer of phosphate plus magnesium is obvious in ► comparing the two curves in Figure 8. In fact, there}is very little movement toward the anode. It is also 62 interesting to note that a dialysis against phosphate buffer seems almost as effective in removing magnesium from the soluble supernatant as is EDTA. The samples dialyzed against the phosphate plus magnesium buffer showed less movement toward the 'anode than the' soluble supernatant that was not dialyzed. However, the undialyzed soluble super natant appeared to have picked up magnesium fairly readily as the protein moved only b.75 cm toward the anode in com parison to the other undialyzed sample run in plain phos phate buffer which moved 1 3 .0 cm toward the anode. The electrophoretic studies run at a pH of 7.28 showed, in general, slightly more movement of protein toward the anode. As the pH increases, proteins, in general, become less positively-] charged. V * Again, at pH 7.28, there is a definite decrease in protein movement toward the anode between the sample dialyzed against water plus EDTA and run in phosphate buffer, and the sample dialyzed against water plus magnesium and run in phosphate plus magnesium. It can be seen that while much interesting informa tion has been obtained from these studies in electrolyte composition, some new questions have been raised. At this point it is difficult to determine to what extent the results found pertain to in vivo conditions in the liver cell. The fact that no measurable dissociation constant 63 was shown for either the magnesium or the potassium bound, in the particulates may be a good indication that this type of isolation procedure in a sucrose medium is not too damaging to the particulates, at least with respect to f their electrolyte composition* The fact that magnesium seems to be bound to some nondiffusible anions of the cyto plasm including proteins, raises the question of the type of binding and its- specificity. From the results of the electrophoresis experiments it would appear that magnesium is bound non-specifically to the cytoplasmic proteins. There are other macromoleeules in the soluble supernatant, such as soluble ribonucleic acid, which could exert a Donnan effect and are known to bind magnesium. As to formulating a new and better homogenizing medium, further information needs to be obtained as to the enzyme activity present in the isolated soluble super natant. If the true physiological condition of the cyto plasm is to be maintained, electrolyte composition and state are only tv/o variables out of many to be investigated, It has been shown that addition of small amounts of mag nesium to a sucrose medium causes agglutination of the mierosomes (11). Also, salts have been shown to cause agglutination of mitochondria. (9)* The simple addition of electrolytes to the medium does not seem to be the answer, even though certain electrolytes, such as potassium, seem j ' " ' ' ' " ' 6 ¥ j to be free in the soluble supernatant, The use of some large macromoleeular substance in the perfusing and homo genizing medium might also be investigated. If the macro molecule had a charge similar to that of the over-all charge of the macromolecules present in the soluble super natant, conditions very similar to that in the cytoplasm i might be created, j The problem of a "physiological homogenate” is still not solved,'but in the future more work on the composition I j and state of various substances and particulates of the cell may provide the answer. Until that time, every piece of information brings closer the solution of this problem. CHAPTER VI SUMMARY These experiments were performed for the purpose of investigating the electrolyte composition of cell homogen- ates. The composition and state of magnesium and potassium were studied in particular since these are the two metals which are found in cells to a greater extent than most • others. They are known to be important in certain enzymatic reactions in the cell, and have been found in the particu lates as well as the soluble cytoplasm. Previous studies have been made on the electrolyte composition in rat liver cells. However, these studies have centered around the recovery of an isolated particulate structure and the measurement of its metal composition. In order to perfect the technique of homogenization, it is important to study the relationship of the metal composition of the particulates to the metal composition of the soluble supernatant. The first step was to find ta>. homogenization pro cedure that was gentle enough not to damage the particulates yet would free them, and would not dilute the cytoplasm to 65 66 such an extent that the particulates would be in a com pletely unnatural environment. The final concentration of the homogenate used in these experiments was much greater than generally used— one to one versus the usual one to ten. The potassium concentration of the homogenate was the same as that found in the soluble supernatant. This indicates no particulate binding of potassium. A linear relationship was found between the concentration of potassium in the soluble supernatant and dilution of the homogenate. A linear relationship was found between the concentration of magnesium and the per cent concentration of the cytoplasm from the homogenate. The soluble supernatant was dialyzed against an equal volume of water or sodium chloride solution and the concentration of potassium and magnesium in the solutions was determined. The potassium was found to be distributed equally in the protein solution and dialysate, while the magnesium concentration was about three times higher in the protein solution. However, different concentrations of sodium chloride had no effect on the concentration of potas- * sium and magnesium: inside and outside of the. membrane indi cating that no Donnan effect was operating. It is postulatec that the magnesium is bound to the macromolecular anions of the soluble supernatant and they therefore have no net charge. 67 The bulk of the protein of the soluble supernatant, about 95 per cent, was found to migrate electrophoretieally toward the anode when dialyzed and run in phosphate buffer. The ability of the protein of the soluble supernatant to bind magnesium is shown by the great reduction in movement toward the anode when the soluble supernatant is dialyzed and run in a phosphate-magnesium buffer. Further work needs to be done on the type of mag nesium binding in the soluble supernatant and its signifi cance, before these results can be correlated to the in vivo condition of a liver cell. B I BLI OG RA PHY BIBLIOGRAPHY 1 . 2. 3. i f . 5. 6. 7. 8. 9. 10. 11. 12. Allfrey, Vincent. "The Isolation of Subcellular Components," The Cell. Vol. I. Edited by J. Bracket and A. E. Mirsky. New York: Academic Press, Inc., 1959. Potter, V. R. 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Creator Goodell, Deborah Boswell (author)

Core Title The electrolyte composition of the soluble phase of cell homogenates

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