1. A seasonal change was observed in the total level of pyruvate kinase [EC ] in rats fed on laboratory chow or a high carbohydrate

Transcription

1 The Journal Biochemistry, Vol. 62, No. l, 1967 Crystallization, Characterization Metabolic Regulation Two Types Pyruvate Kinase Isolated from Rat Tissues* By TAKEHIKO TANAKA,** YUTAKA HARANO, FUMIAKI SUE HIROKO MORIMURA (From Division Protein Metabolism, Institute for Protein Research, Osaka University, Osaka) (Received for publication, January 16, 1967) 1. A seasonal change was observed in total level kinase [EC ] in rats fed on laboratory chow or a high carbohydrate diet. 2. Two s kinase were identified by electrophoresis an immunological procedure, named L M. Both s were purified extensively were isolated as crystals. The molecular weights M L were 250, ,300 respectively. 3. No cross immunological reaction was observed between two s. The antibody for M was used in estimating levels two s enzyme in tissue extracts. 4. The level L varied greatly under various physiological conditions, whereas that M changed only slightly. In alloxan diabetic animals those fed on a high protein diet or fasted for 48 hours, total level kinase as well as ratio L to M decreased greatly, on subsequent insulin administration administration a normal diet, respectively, level returned to normal. 5. The level M increased markedly in regenerating liver was only found in ascites hepatoma cells. 6. Type M was present in muscle, brain, heart, liver, kidney, fat pads leucocytes, L was only present in liver erythrocytes. 7. The kinetic properties M L1 were quite different. The apparent Km L1 for phosphoenolpyruvic acid was about 10 times that M, with L, substrate seemed to act as an activator. The sensitivities L1 to inhibition by ATP acid were also very much higher than that M. 8. The physiological significances two s kinase were discussed. work were reported at Annual Meetings Japanese Biochemical Society, in 1964, ** Present address : Department Physiological Chemistry Nutrition, Osaka University Medical School, Osaka. 71

2 72 T. TANAKA, Y. HARANO, F. SUE H. MORIMURA In gluconeogenetic state, it has been proposed that newly synsized which is formed from lactate, alanine serine is converted to phosphoenolpyruvic acid via oxaloacetic acid, reaction being catalyzed by carboxylase [EC ] phospho carboxykinase [EC ] (1). However, this phosphoenolpyruvic acid which is formed at expense two high energy phosphate bonds would simply be reconverted to pyruvic acid again if kinase level was maintained. Thus to diminish a wasteful cycling such as this, re must be some mechanism controlling kinase level in gluconeogenetic organs, such as liver kidney. Recently, it was reported from three laboratories (Krebs Eggleston (2), Weber et al. (3) Tanaka et al. (4)), that kinase [EC ] level in liver was under hormonal dietary regulation, like levels or key enzymes. In contrast, enzyme level in skeletal muscle was not found to change under various conditions. This difference between responses liver muscle to hormonal dietary conditions has stimulated authors to undertake study which showed presence two s kinase, named M (muscle) L (liver) (5). Pyruvate kinase M is an usual muscle enzyme, its characteristics have already been described in detail (Boyer (6)). Pyruvate kinase L is not neutralized with antibody against kinase M, fluctuates in activity under various physiological conditions. Various experiments on se enzymes were reported previously (5), crystallization characterization two s kinase are described in present paper. METHODS AND MATERIALS Animals-Male Sprague-Dawley albino rats, weighing 150 g. to 200 g., were used throughout experiments. Animals were fed on laboratory chow water ad libitum, for at least 10 days after transfer from breeding farm to animal room in Institute, n were used for experiments. Syntic diets contained 4 g. inorganic salt mixture, 1 g. vitamin mixture, 2 g. cellulose powder, 0.01 g. choline chloride indicated amounts casein. The diets were made up to 100 g. by addition appropriate amounts dextrin. The animals were fed on indicated diets for 3 days before sacrifice, unless orwise mentioned. Alloxan Diabetic Animals-Seven mg. per 100 g. body weight alloxan monohydrate was injected into animals intravenously. Rats, with blood sugar levels more than 300 mg. per dl., were used for experiments 5 days after injection. Enzyme Assay-Two methods were used for assay kinase. During purification kinase studies its metabolic control in liver, 2, 4-dinitrophenylhydrazone method was routinely employed. Animals were sacrificed by a blow on head were exsanguinated. The livers were rapidly isolated chilled, 3 g. liver tissue were homogenized with 10 ml. cold KCl solution (0.15 M) containing MgSO4 (0.005 M) EDTA (0.001 M) using a Potter- glass homogenizer. The homogenates were centrifuged at 105,000 ~ g for 60 minutes. The supernatants were used for assay kinase activity protein estimation. At same time 1.5 g. tissue from left femoral muscle were homogenized with 13.5 ml. same solution as used with liver. The homogenates were centrifuged supernatants were employed for experiments as with liver material. Pyruvate kinase was assayed by measurement formed from phosphoenol in presence ADP with 2, 4- dinitrophenylhydrazine, according to a slight modification method Kimberg Yielding( 7). The composition assay mixture was as follows ; tris (hydroxymethyl)-aminomethane 50 ƒêmoles, phosphoenol 2 ƒêmoles, ADP 2 ƒêmoles, MgSO4 5 ƒêmoles, KCl 100 ƒêmoles, total volume 1.0 ml., ph 7.5. The reaction was started by addition 0.1 ml. suitably diluted enzyme solution, (containing about 1.0 unit per ml. enzyme) to 1.0 ml. assay mixture. After 3 minutes incubation at 37 Ž, 0.2 ml. reaction mixture was added to 0.5 ml. 2, 4- dinitrophenylhydrazine ( % in 2 N HCl) mixture was allowed to st for 10 minutes at 37 Ž. Then 1.2 ml. 2.4 N NaOH solution containing M EDTA was added to mixture optical density was measured at 510 mƒê, after full color development (after about 10 minutes). The blank run was carried out in exactly same way, except that ADP was omitted from assay mixture. The stard curve 2, 4-dinitrophenylhydrazone was obtained in exactly same way using known amounts lithium instead phosphoenol. One unit enzyme is expressed as amount enzyme which catalyzes formation 1 ƒêmole per minute under conditions described above. The specific activity

3 Two Types Pyruvate Kinase 73 enzyme is expressed as units enzyme per mg. protein. In kinetic studies experiments on inhibition by ATP p-chloromercuribenzoic acid, kinase was assayed by following decrease absorption at 340 mƒê in coupled reaction lactate dehydrogenase system with kinase using a recording spectrophotometer (Shimazu Automatic Recording Spectrophotometer or Gilford Multichannel Spectrophotometer). The composition stard assay mixture in latter method was as follows ; Tris (hydroxymethyl)-aminomethane 50 ƒêmoles, MgSO4 5 ƒêmoles, KCl 100 ƒêmoles, NADH 0.2 ƒêoles, lactate dehydrogenase [EC ] 2 units, amounts phosphoenol ADP as indicated in text in a total volume 1.0 ml. The reaction was started by addition ADP solution after termination small initial decrease absorption due to contamination phosphoenol solution with. The enzyme assay was carried out at 25 Ž. The above reaction mixture was incubated, with water instead ADP solution for blank. Protein Estimation-Protein contents were estimated by Biuret method (8). When a sample contained much hemoglobin, two kinds blank were taken, that is, enzyme plus alkaline solution water plus Biuret reagents. The optical densities all samples were measured against distilled water sum optical densities two blanks was substracted from value sample. Electrophoresis-Zone electrophoresis crude extracts organs was carried out on potato starch blocks (5 ~ 20 ~ 350 mm.), with 0.05 M potassium phosphate buffer (ph 8.0). After electrophoresis for 13 hours in a cold room (350 V), starch block was cut into 10 mm. lengths, each piece was throughly mixed with 1 ml. 0.15M KCl solution to extract enzyme. The starch was allowed to sediment an aliquot supernatant was used for assay kinase by 2, 4-dinitrophenylhydrazone method described above. Preparation Rabbit Anti-Enzyme Sera-One ml. a solution crystalline kinase L or M, (10 mg./ml.) was completely emulsified with 1 ml. Freund's complete adjuvant in a Potter glass homogenizer. Two ml. emulsion were injected subcutaneously into foot pads once every two weeks (9). Two or three injections emulsion were carried out. Three weeks after last injection emulsion, 0.2 or 0.3 ml. same enzyme solution was injected as a booster. A week after this booster injection, blood was taken from an ear vein. Antibodies were partially purified by ammonium sulfate fractionation. One unit antibody was expressed as amount antibody which neutralized one unit enzyme. One ml. best antiserum obtained against M enzyme neutralized 44 units M enzyme, one ml. best antiserum obtained against L enzyme neutralized 22 units L enzyme. Both s enzyme were neutralized almost immediately after mixing m with ir antibody. Differential Assay Type L Type M Pyruvate Kinase-No immunological cross reaction between L M kinase was observed. The antibody against kinase M neutralized only M but not L enzyme vice versa. Fig. 11 shows results neutralization experiments on M enzymes, isolated from muscle liver, with antibody against M enzyme. The two M enzymes isolated from muscle liver were both neutralized similarly with antibody against M enzyme isolated from muscle, whereas activity L enzyme was not neutralized at all with this antibody. Since neutralization curves two M enzymes were parallel, it was concluded that neutralization titers two M enzymes were same. As described later, precipitation lines two M enzymes also fused completely in gel diffusion test. To measure amounts each kinase, enzyme was assayed in presence absence antibody against M enzyme. The enzyme activity found without antibody was taken as total activity. The activity remaining after neutralization with antibody was taken as L activity. The M activity was calculated as difference between total L activity. Usually an amount antibody 5 times excess was used in se experiments. Gel Diffusion Analysis-Agar medium contained 1 g. agar (Special Agar-Noble), 5 ml. 0.2 M phosphate buffer, ph 7.8, 1 ml. 1% merthiolate solution in saline, was made up to 100 ml. with saline. This agar medium was dissolved by heating a microslide was coated with a 2-3 m.m. thick layer it. After solidification agar, wells were made by sucking agar into a square-tipped tube (diameter 2.5 mm.) ( 10 ). In center well were placed 30 pl. antibody against muscle kinase in or wells were put enzyme samples containing units activity. The slide was placed in a petri-dish, which was sealed tightly with a tape to prevent drying agar at room temperature. After precipitation line had developed, petri-dish was filled with 0.15 M KCl solution (ph 7.4) slide was washed by changing solution several times. Then it was covered with a wet filter paper dried with a fan. It was stained with 0.5% amidoblack solution containing 1% glacial acetic acid for 5 minutes. The

4 74 T. TANAKA, Y. HARANO, F. SUE H. MORIMURA excess stain was washed out by placing slide in a petri-dish filled with rinsing solution (methanol 5, glacial acetic acid 1, distilled water 4). Immunoelectrophoresis-A glass plate (0.1 ~ 7.6 ~ 5.5 cm.) was coated with a 1.7% solution Special Agar Noble in barbital buffer, ph 8.6, u =0.05. A trough holes were cut with blade a safety razor a needle. Then 1.5 units enzyme sample was placed in hole both ends slide were connected with filter paper to buffer vessels filled with barbital buffer, ph 8.6, u =0.05. A current 4 volts per 1 cm. was applied for 2 hours (11). Then trough was filled with antibody against muscle kinase slide was placed in a sealed petri-dish at room temperature. After development precipitation line, slide was washed stained as described above. Materials-The sodium salts ATP ADP, barium salt phosphoenolpyruvic acid lithium salt were purchased from Sigma time. It does not seem conceivable that temperature is an essential factor in this seasonal change in enzyme level. The decreased level kinase in liver during summer time did not increase on breeding in complete darkness for a month or on pinealectomy (cf. (12, 13)). It should be added that inducibility liver kinase by a high carbohydrate diet was also depressed during summer. The mechanism this seasonal change remains to be elucidated in detail. Effect Starvation on Pyruvate Kinase Levels in Liver-Table I shows decrease in kinase level in liver starved rats. All animals were fed on 10% casein diet ad libitum for three days before experiment Chemical Co., U.S.A. Laboratory chow, for rats was purchased from Oriental Yeast Co., Ltd., Osaka. The salt mixture, vitamin mixture, casein dextrin used for animal diets, were obtained from Tanabe Pharmaceutical Co., Ltd., Osaka. Insulin was purchased from Novo Industry. A/S, Denmark. Lactate dehydrogenase was purchased from C.F. Boehringer Co., Germany. Clel's reagent was obtained from Calbiochem., U.S.A., polyvinyl pyrrolidone (molecular weight, 700,000) was purchased from Nakarai Chemicals, Kyoto. RESULTS Seasonal Change in Pyruvate Kinase Level in Liver Rats Fed on Laboratory Chow 10% Casein Diet-The temperatures animal rooms on breeding farm in Institute are well controlled at 23 Ž, all year round. It takes one or two days to transfer experimental animals from breeding farm to Institute. The animals are exposed to atmospheric temperature only during this transportation period. As described in methods, to minimize variations due to stress during transportation, animals were maintained in breeding room Institute for at least 10 days before use in experiments. As seen in Fig. 1, kinase levels livers rats fed on laboratory chow 10% casein diet showed a seasonal fluctuation. The highest levels kinase were observed during winter time, lowest levels during summer FIG. 1. Seasonal change in kinase levels in livers rats fed on laboratory chow 10% casein diet. Each point is mean values more than four animals. The enzyme was assayed by hydrazone method described in text. Diet : Laboratory chow œ 10% casein TABLE I Effect Starvation on Pyruvate Kinase Level in Liver Rats were fed on a 10% casein diet ad libitum for 3 days before experiments. After fasting for period indicated, animals were sacrificed. The enzyme was assayed by hydrazone method as described in text. The activity is expressed as mean value specific activities } stard error.

5 Two Types Pyruvate Kinase 75 n starved. Fasting animals were sacrificed at intervals normal animals after three days on 10% casein diet. The level kinase in liver decreased gradually, became about one third that normal animals after 96 hours starvation. On contrary, level in muscle remained unchanged even after 96 hours starvation. Effect Concentration Carbohydrate (Casein) in Syntic Diets on Pyruvate Kinase Level in Liver-Fig. 2 shows level kinase in liver rats which were fed on syntic diets containing various amounts carbohydrate (casein). As described in methods, 100 g. syntic diet contained 4 g. inorganic salt mixture, 1 g. vitamin mixture, 2 g. cellulose powder, indicated amounts casein, weight was made up to 100 g. by addition an appropriate amount dextrin. Therefore, a 0% casein diet is a 93% carbohydrate diet a 93% casein diet is a 0% carbo- hydrate diet. The animals were fed on syntic diets for 3 days before sacrifice for enzyme assay. As shown in Fig. 2, highest level kinase was observed in liver animals fed on a 10% casein diet (high carbohydrate diet) lowest level was found in liver animals fed on a 93% casein diet (0% carbohydrate). The enzyme level in muscle did not show any response to diets containing various amounts casein. Effect Diabetic State on Pyruvate Kinase Level in Liver-The facts that kinase level decreases during starvation increases when animals are fed on a high carbohydrate diet, suggest that kinase level in liver is controlled by various hormones. Table II shows change in kinase level in liver alloxan diabetic animals. Seven mg. alloxan per 100 g. body weight were injected intravenously into rats, 7 days after this injection, animals were fed on a 10% casein diet for 4 days before sacrifice. The enzyme level in liver diabetic animals decreased to 28% that normal animals. No response to composition diet was observed in alloxan diabetic animals. As seen in Table II, enzyme level in muscle did not change in diabetic condition. Recovery Pyruvate Kinase Level in Liver Diabetic Animals on Insulin Administration- Fig. 3 shows time course recovery kinase level in liver TABLE Pyruvate Kinase Levels in Liver Muscle Diabetic Normal Rats II FIG. 2. Effect concentration carbohydrate (casein) in diet on kinase level in liver. Each point is mean values four animals. The enzyme was assayed by hydrazone method as described in text. A 0% casein diet is a 93% carbohydrate diet a 93% casein diet is a 0% carbohydrate diet. The composition diet is given in text. The animals were fed on indicated diets for 3 days before sacrifice. Seven mg. alloxan per 100 g. body weight were injected intravenously into rats, 7 days later, blood sugar levels were measured animals with levels over 300 mg. percent were sacrificed. The enzyme was assayed by hydrazone method as described in text.

6 76 T. TANAKA, Y. HARANO, F. SUE H. MORIMURA diabetic animals on insulin administration. The animals were used for experiments 7 days after subcutaneous injection alloxan. The rats were fed on laboratory chow ad libitum. Five units insulin per 100 g. body weight were injected subcutaneously at 0,3 5 hours animals were sacrificed for enzyme assay 0,3,5,8 10 hours after first injection insulin. One group animals which were sacrificed after 14 hours, was given 6 units zinc protamine insulin per 100 g. body weight as a single injection FIG. 3. Time course recovery liver kinase level in alloxan diabetic rats on administration insulin. Animals with blood sugar levels between mg. per dl. 7 days after intravenous injection alloxan were used for experiments. Details experiment have been described in text. Each point shows mean value specific activities four animals. Enzyme was assayed by hydrazone method described in text. at beginning experiment. The decreased levels in livers diabetic rats started to increase slightly 3 hours after first injection insulin, were elevated to normal levels after 8 hours. Zone Electrophoretic Patterns Pyruvate Kinase in Various Organs-As described above, re was a clear difference in response kinase levels in liver muscle to changes in diet to hormones. This suggested that enzymes in se two organs were different from each or. To test this, crude extracts various organs were subjected to starch zone electrophoresis. As seen in Fig. 4, electrophoretic patterns crude extracts various organs could be roughly divided into two groups. Crude extracts muscle, brain heart showed only a single peak kinase activity, whereas liver kidney showed two or more peaks on starch block zone electrophoresis. Peak M liver enzymes had same mobility at that muscle enzyme three or peaks were tentatively named L1, L2 L3 as shown in Fig. 4. Peaks L2 L3 were not always clear in extracts, sometimes only peaks M L1 were detectable. Type M L were named after organs, i.e., muscle liver, in which y occurred at highest concentrations. The differences in natures L group enzymes have not yet been elucidated, except ir mobilities on starch zone electrophoresis ir elution patterns from a DEAE-cellulose column. FIG. 4. Zone electrophoresis kinase crude extracts various organs. Crude extracts organs were employed for starch zone electrophoresis. Details experiment have been described in text. The enzyme was assayed by hydrazone method as described in text.

7 Two Types Pyruvate Kinase 77 Purification Type M Enzyme from Muscle freshly isolated muscles rats were homogenized in a Waring Blendor with 7 liters ice cold 0.15 M KCl solution containing M MgSO M EDTA, ph 7.4. All procedures were carried out in a cold room, unless orwise mentioned. The homogenate was centrifuged at 10,000 ~ g for 30 minutes. To 6,830 ml. supernatant were slowly added 320 g./litre solid ammonium sulfate. After 30 minutes stirring, precipitate was collected by centrifugation at 10,000 ~ g for 30 minutes was discarded. Then a furr 83.5 g./litre ammonium sulfate was added to supernatant precipitate was collected as previously. Up to this first ammonium sulfate fractionation, procedure was that T i e t z Ochoa (14). The precipitate was dissolved in a minimal volume same KCl solution. This enzyme solution was dialyzed overnight in a cold room against M Tris (hydroxymethyl)-aminomethane buffer containing M MgSO M EDTA, ph 7.4. Then 410 ml. dialyzed enzyme solution was slowly mixed with saturated ammonium sulfate solution (adjusted to ph 7.4 with concentrated ammonium hydroxide solution) in a proportion 1.23 litre/litre preparation. After stirring for 30 minutes, precipitate was collected by centrifugation at 10,000 ~ g for 30 minutes was discarded. The supernatant was slowly mixed with 0.8 litre/litre same saturated solution. After stirring for 30 minutes, precipitate was collected as above, dissolved in a minimal volume same KCl solution. The enzyme solution was dialyzed, overnight in same way, dialyzed solution was passed through a Sephadex G-25 column equilibrated with 0.05 M maleate buffer, ph 5.0. Then it was chromatographed on a phosphocellulose column which had been washed with 2 N KOH distilled water equilibrated with 0.05 M maleate buffer (ph 5.0). One ml. bed volume phospho- cellulose was used per 20 mg. protein. The column was washed with two volumes maleate buffer (ph 5.0) after enzyme had been adsorbed. The enzyme was eluted with a linear gradient maleate buffer from 0.1 M, ph 5.0, to 0.1 M, ph 6.8. Fig. 5 shows elution pattern enzyme. The enzyme began to be eluted at ph 5.4. Fractions with specific activities more than 300 were combined dialyzed in cellophane tubings against saturated ammonium sulfate solution described above. The solution was concentrated to one tenth its original volume by this dialysis. The enzyme precipitated inside tubings was collected by centrifugation at 10,000 ~ g for 30 minutes, dissolved in a minimal volume 0.05 M maleate buffer. The enzyme was rechromatographed on a phosphocellulose column in same way as before. Eluted fractions with specific activities more than 500 were combined. The enzyme again precipitated. inside cellophane tubing, was col- lected by centrifugation as before. It was dissolved in a minimal volume 0.05 M maleate buffer, ph 6.8, dialyzed against same buffer. The dialyzed enzyme solution was chromatographed on a Sephadex equilibrated with same buffer. Fractions with specific activities more than 600 were combined. A summary purification muscle kinase is shown in Table III. The concentration purified enzyme at last step, was FIG. 5. Chromatography ot kinase M isolated from muscle on a phosphocellulose column. Elution by linear gradient maleate buffer from 0.1 M, ph 5.0 to 0.1 M, ph 6.8 was performed as described in text. Fraction volume, 19 ml. : Absorbancy at 510 mĐ hydrazone (enzyme assay) : Absorbancy at 550 mĐ Biuret (protein assay)

8 78 T. TANAKA, Y. HARANO, F. SUE H. MORIMURA TABLE Purification Pyruvate Kinase Type M from Muscle III adjusted to 30 mg. protein per ml. This solution was slowly mixed with saturated ammonium sulfate solution until it became slightly turbid. The precipitate which formed was removed by centrifugation, solution was allowed to st in a cold room. A double refraction pattern was observed after a few days. The growth crystals was stimulated by placing solution alternately in a 0 Ž a -15 Ž bath. Thin needle-shaped crystals were seen under a microscope at a magnification x 80 (Fig. 8A). The crystals was collected by centrifugation recrystallized in same way. The specific activity crystalline muscle kinase was 780. The crystalline preparation showed a single peak on ultracentrifugal analysis (Fig. 9) cellulose acetate paper electrophoresis. Stabilization Type L Pyruvate Kinase by ADP, Phosphoenolpyruvic Acid Sulfhydryl Reagent-During purification L1 enzyme, it was found that enzyme was very labile, especially at a low protein concentration. This instability made enzyme purification very difficult. As shown in Fig. 6, a L preparation with a specific activity 31.8, lost almost all its activity after 36 hours storage in cold room. It was, however, found that inactivation enzyme was prevented by addition 0.5 mm ADP, 0.5 mm phosphoenolpyruvic acid 1 mm Clel's reagent. The best protection was observed in presence se three chemicals ir FIG. 6. Stabilization kinase L by substrates sulfhydryl protecting agent. A preparation L specific activity 31.8, protein concentration 5.66 mg. per ml., was stored in a cold room with without 0.5 mm ADP phosphoenolpyruvic acid, 1 mm Clel's reagent. At intervals aliquots were taken for enzyme assay by hydrazone method. presence facilitated purification enzyme to a great extent. Purification Type Li Pyruvate Kinase-Two hundreds rats were fed on a high carbohydrate diet (68% dextrin, 15% sucrose, 10% casein, vitamin mixture, salt mixture cellulose powder) for 2 or 3 days before sacrifice. Their livers (1,600 g.) were isolated immediately homogenized with 2.4 litres cold 0.15 M KCl solution containing M MgSO M EDTA for 1 minute at a low speed in a juice mixer. All purification

9 Two Types Pyruvate Kinase 79 steps were carried out at 0 Ž, unless orwise mentioned. The homogenate was centrifuged at 8,000 ~g for 20 minutes. The supernatant was filtered through a funnel packed with a small amount glass filter slowly brought to 28% saturation with saturated ammonium sulfate solution (adjusted to ph 7.4, with concentrated ammonium hydroxide solution) using a magnetic stirrer. After sting mixture for 20 minutes, supernatant was collected by centrifugation at 8,000 ~ g for 20 minutes. Saturated ammonium sulfate solution was added to supernatant to 45% saturation. The mixture was allowed to st for 20 minutes n centrifuged. The precipitate was dissolved in a minimal volume Tris (hydroxymethyl )-aminomethane buffer, ph 7.4, containing M MgSO4, MEDTA, solution was dialyzed against 20 volumes same buffer for 16 hours in cold room. The dialyzed enzyme solution from previous step was mixed with final concentrations 0.2 mm ADP, 0.2 mm phosphoenolpyruvic acid 1 mm Clel's reagent to protect it from inactivation during acetone fractionation. Redistilled acetone at Ž was slowly added to 50 ml. enzyme solution (0 Ž) to a concentration 20%, (v/v) using a magnetic stirrer. The mixture was n immediately centrifuged in a refrigerated centrifuge ( -5.0 Ž) at 4,000 ~g for 10 minutes. To supernatant more cold acetone was added to a concentration 45% (v/v) with constant stirring in a acetone-dry ice bat!i ( Ž), mixture was immediately centrifuged at 4,000 ~ g for 5 minutes. The precipitate was dried in a vacuum desiccator over P2O5 for 2 hours to remove acetone enzyme Was obtained by two extractions precipitate with small volumes 0.15 M KCl solution. The supernatant this extract was brought to 45% saturation ammonium sulfate by slow addition saturated ammonium sulfate solution using a magnetic stirrer. After sting mixture for 20 minutes, precipitate was recovered by centrifugation. The precipitate was as dissolved in a minimal volume 0.15 M KCl solution clear supernatant was recovered by centrifugation. This enzyme solution (13.4 ml.) was dialyzed overnight with constant stirring against 250 ml M Tris buffer, ph 7.4, containing M MgSO4, M EDTA, 0.2 mm ADP, 0.2 mm phosphoenolpyruvic acid 1 mm Clel's reagent. The dialyzed enzyme solution containing about 700 mg. protein was applied to a DEAE- Sephadex column (35 ml. packed volume) previously equilibrated with buffer used in dialysis. The column was eluted with twice bed volume M Tris buffer containing 0.1 M KCl stabilizers contaminating inactive protein was eluted. The activity kinase L1 ap- peared after second elution with M Tris buffer containing 0.2 M KCl stabilizers (Fig. 7). Fractions with specific activities more than 46, were combined. The enzyme solution was placed in a cellophane dialysis tubing concentrated by immersing tubing in polyvinylpyrrolidone powder, 1.5 g. powder was used per 1 ml. enzyme solution. This procedure resulted in reduction volume from 26 ml. to 4.3 ml., without substantial loss total activity. The concentrated solution was passed through a Sephadex G-25 column (30 ml. bed volume) equilibrated with 0.02 M acetate buffer, ph 5.0, containing stabilizers, n immediately applied to a phosphocellulose column. Phosphocellulose, prepared as described in purification M enzyme, was equilibrated with 0.02 M acetate buffer, ph 5.0, containing stabilizers. One ml. bed volume phosphocellulose was used per 25 mg. enzyme protein. After enzyme adsorption, column was washed with two bed volumes same buffer enzyme was eluted with a linear gradient from 0.02 M acetate buffer, ph 5.0 to 0.05 M maleate buffer, ph 7.0. The enzyme was eluted at above ph 5.4, fractions with sepcific activities more than 250, were collected in a dialysis tubing (Fig. 7). The combined solution was concentrated by means polyvinylpyrrolidone as previously, to reduce volume from 12.9 ml. to less than 1 ml. The enzyme solution was taken out from tubing tubing was washed with 0.15 M KCl solution

10 80 T. FIG. 7. TANAKA, Chromatography Y. HARANO, pyruvatc kinase F. Sta.: L, from H. livers MORIMURA rats on a on (A) a DEAE-Sephadex column (B) a phosphoccllulose column. The DEAE-Sephadex column was eluted with stepwise increase in concentration to 0.2 M (A). The phosphocellulose column was eluted with a linear gradient from ph 5.0 to 0.05 M malcate ---- buffer, ph 7.0 (B). : Absorbancy protein (A by Biuret reaction at 550 mu. B by Folin-Ciocalteau Crystals M 510 A : Crystals kinase from B : Crystals kinase L, mĐ (enzyme L, muscle from liver diet KCl to 0.1 M 0.02 M acetate buffer, size : (A) 4.5 ml. (B) 2.4 ml. 8. at Fraction : Absorbancy Fin. hydrazone high carbohydrate assay; (magnification ~ (magnification ~ kinase. 80) 100) reaction at 500 ma;

11 Two to make washing up total procedure temperature The solution turbid dialysis tubing volume was to carried solution was placed Types 1 ml. This at room out became again in into room Kinase 81 dried with a fan. After several hours, turbid solution was examined under microscope, rhombohedral crystals were observed (Fig. 8B). After centrifugation, one third total activity was collected as turbid. put a cold Pyruvate a TABLE IV Purifccation Pyruvate Kinase Type Li from Liver Rats Fed on a High Carbohydrate Diet A B FIG. A : Type 10 B : 9. L, minute Type 10 Ultracentrifugal (7.4 intervals, M minute enzyme patterns enzyme intervals, mg./mi.) 12 (8.9 minutes mg./mi.) 15 minutes speed 55,100 after attaining speed 42,040 after attaining M r.p.m., full r.p.m speed., full L Ž. Photographs kinase. from right to left taken at taken at Ž. speed Photographs. from right to left

12 82 T. TANAKA, Y. HARANO, F. SUE H. MORIMURA crystals, specific activity was 388. This crystalline preparation contained only L1 kinase showed only a single peak on ultracentrifugal analysis (Fig. 9). The results purification L1 enzyme are summarized in Table IV. Molecular Weights Type M Type Li Pyruvate Kinase-The molecular weights two s enzyme were determined by sedimentation equilibrium method (15). E- quilibrium centrifugation was carried out at several different concentrations enzyme protein plot results was extrapolated to value at zero concentration enzyme protein, as shown in Fig. 10. The molecular weight M enzyme at zero concentration was 250,000 that L1 was 208,300. Immunological Studies on Type M Type la Pyruvate Kinase-As described in methods, crystalline M L1 enzymes were in- jected into rabbits antibodies against se enzymes were prepared. No immunological cross reaction was observed between M I enzymes. The antibody against M enzyme neutralized muscle kinase also M enzyme isolated from liver, but not L1 enzyme (Fig. 11), similarly, antibody against L1 enzyme neutralized only L enzyme. In previous experiments described in this paper, kinases were d by ir mobility on starch block zone electrophoresis. However, one peak in kidney extract showed same mobility as L1 enzyme, but was neutralized with antibody against M enzyme rar than against L1. Thus kinases must be d on bases ir mobilities on electrophoresis also ir neutralization FiG. 11. Neutralization curves M kinase muscle liver with antibody against muscle kinase. The same amount each enzyme was mixed with various volumes antibody. A few minutes after mixing, enzyme was assayed by hydrazone method as described in text. The solid line shows results with muscle M enzyme FIG. 10. Determination fo molecular weights M Li kinase. Type M was centrifuged in 0.05 M Tris buffer, ph 7.4, containing 0.1 M KCl, at 5,563 r.p.m. (at Ž). Type L1 was centrifuged in 0.05 M Tris buffer, ph 7.4, containing 0.1 M KCl, 0.4 mm ADP, 0.4 mm phosphoenolpyruvic acid 1 mm Clel's reagent (at 11.1 Ž) at 5,563 r.p.m. broken line shows those with M enzyme isolated from regenerating liver. Type NT regenerating liver was partially purified by ammonium sulfate fractionation (50% to 60% saturation), heat treatment at 55 Ž for 3 minutes phosphocellulose chromatography in way used for purification muscle kinase, as described in text.

13 13 Two with antibodies. The enzyme in peak observed with kidney tentatively called I.M. Fig. which was 121 shows placed gel against in precipitation antibody pletely antibody a antibody line enzyme from between in Only One liver. No antibody line was precipitation lines which Formed fused completely with each or. It was concluded from se results that kinase in brain, heart, fat pad muscle, M liver are immunologically indistinguishable. com- formed between purified NI observed crystalline I., (13 (A Fin. The 12. Gel diffusion antibody against M-P.K. is partially is crystalline L-P.K. from muscle, method are analysis kinases extracted muscle kinase was placed purified crude described M from kinase L1 from liver, extracts brain, in M-P.K. liver. In heart from various in center is crysialline picture (B), fat M-P.K. pads were put organs. well. Onc in sample antibody are described method (B) units L serum upper were trough, crystalline in in crystalline placed against Immunoelectrophoresis Two wells. Details (B) units electrophoresis (A), muscle, kinase text.. (A) Immunoelectrophoresis half In picture kinase from is crystalline (A) FIG. was placed in center well, antigen crude extracts brain, heart fat pad were placed in sample wells. All enzyme between it found 83 Kinase enzyme (Fig. 121). In experiment shown in Fig. 12B, antibody against muscle kinase is test muscle well. Pyruvate curious diffusion center preparation was Formed antigen, with line partially Types lower L was put crystalline muscle crystalline muscle muscle kinase kinase. were kinase placed was put in into sample holes. troughs. two units After Details text. crystalline L well. into L1 enzyme After were lower placed electrophoresis trough. kinase. in antibody upper well against L was put anti into

14 84 T. TANAKA, Y. HARANO, F. SUE H. MORIMURA Fig. 13A shows results immunoelectrophoresis crystalline muscle kinase. Only one precipitation line formed with antibody against muscle kinase on electrophoresis crystalline muscle kinase. Fig. 13B shows results immunoelectrophoresis crystalline L1 kinase. Only one precipitation line formed with antibody against L1 enzyme on electrophoresis crystalline L1 enzyme. Neutralization Liver Pyruvate Kinase after Electrophoresis with Antibodies against Type M Type L1 Pyruvate Kinase-As shown in Fig. 4, crude liver extracts contained at least four electrophoretically separable kinases, including two major components (M L1) two minor components (L2 L3). In experiments shown in Fig. 14, crude liver extracts rats fed on laboratory chow on 10% casein diet were subjected to zone electrophoresis, enzyme activity extract from each starch segment was assayed in presence absence antibody. Only peak M, which moved furrmost towards cathode, was neutralized with antibody against muscle kinase, or three peaks (L1, 1,2 L3) were neutralized with antibody against L1 enzyme. This result also confirmed that crude liver extracts contained two groups kinase (L M). It should be noted, that M was predominant in liver extracts rats fed on laboratory chow L1 was predominant in livers animals fed on a high carbohydrate diet, as seen in Fig. 14. Ratio Type L to Type Al Enyine in Liver under Various Physiological Pathological Conditions From results shown in Fig. 14, it seemed likely that L M enzymes are regulated by different mechanisms. Table V shows ratio M to L in liver extracts under various physiological pathological conditions. As described in methods, antibody against muscle kinase ( M) neutralized not only enzyme activity muscle enzyme, but also that M enzyme from or sources. However even when 60 times in excess, this antibody had no effect on I. activity. Because this, rapid assay FIG. 14. Neutralization liver kinase after electrophoresis with antibodies against M L1 kinase. Starch zone electrophoresis was carried out as Fig. 4. Upper figure : Crude extract from livers rats fed on laboratory chow. Enzyme extracted after 16 hours assayed+ antibody to muscle kinase. Total activity : Type M activity Lower figure : Crude extract from livers rats fed on high carbohydrate diet. Enzyme extracted after 16 hours assayed } antibody to L1 enzyme. Ttotal activity Type L activity FR;. 15. Changes kinase L M activities after partial hepatectomy. After hepatectomy two thirds liver, rats were fed on laboratory chow ad libitum. Type M L activities were estimated with without antibody against muscle kinase as described in text at times indicated.

15 Two Types Pyruvate Kinase 85 M L enzymes in tissue homogenates was possible. The level L enzymes in creased very markedly to normal values after administration insulin to diabetic animals or a high carbohydrate diet, whereas level M enzyme showed only slight changes. Similar results have been reported for enzymes catalyzing phosphorylation glucose in liver by Sols et al. (16) Walker (17 ). Glucokinase [EC ], corresponding to L, is induced by insulin, whereas hexokinase [EC ], corresponding to M, does not fluctuate under hormonal or dietary conditions. Pyruvate kinase M was found to increase in liver under several conditions. In ascites hepatoma cells (AH 130), which are believed to originate from liver parenchymal cells, all kinase was M its specific activity was markedly elevated. In regenerating liver, most kinase was M. In experiment shown in Table V, kinase in regenerating liver was assayed 10 days after removal two thirds liver mass. The time course change in amounts M L kinase for 2 weeks after hepatectomy can be seen in Fig. 15. Type M began to increase only one day after hepatectomy remained at a high level for 7 days. In foetal liver also, M was found to be predominant. Distributions Type M Type L Pyru- Kinase in Various Organs Rat-Using antibodies against M L1 enzymes, amounts two s enzyme were assayed (Table VI). Liver was only organ which contained both s kinase, erythrocytes were only tissue or than liver which contained L enzyme. It was unexpected by found that kidney enzyme was not neutralized with antibody against L1, in spite fact that crude kidney extract showed two peaks on starch zone electrophoresis, one which had same mobility as L1 enzyme (Fig. 4). With organs cited in Table VI, electrophoresis patterns agreed well with results neutralization experiments with antibodies. Kinetic Properties Type M Type Li Pyruvate Kinase-As described above, M TABLE Change in Ratio Type L to Type M in Liver under Various Physiological Pathological Conditions V Total kinase activity was assayed in absence antibody against muscle kinase. Type L activity was measured in presence 5 times excess amount antibody. Type M activity was expressed as difference between total L activity. Diabetic animals were fed on 10% casein diet, fifteen units insulin per 100 g. body weight were injected subcutaneously 48, hours before sacrifice. The cancer cells were harvested one week after inoculation into peritoneal cavity. For regenerating liver, animals were sacrificed ten days after removal two thirds liver. Activity is expressed as units enzyme per mg. protein crude extract. The values shown are means error. The enzyme was assayed by hydrazone method.

16 86 T. TANAKA, Y. HARANO, F. SUE H. MORIMURA TABLE Distributions Pyruvate Kinase Type L Type M in Various Organs Rats Fed on Laboratory Chow VI The enzyme was assayed by hydrazone method as described in text, in presence antibody against muscle enzyme, or against L, enzyme or without any antibody. Specific activity is expressed as total activity per mg. protein crude extract. Neutralized activity is expressed as a percentage total activity. L kinases behaved quite differently in electrophoresis, in immunological reactions, in ir response to hormones composition diet. In addition to se differences, it was found that two s had characteristic kinetic properties. Fig. 16 shows plots percentage maximum velocities as a function concentration phosphoenolpyruvic acid in purified muscle enzyme Li. enzyme. The activity L1 enzyme was very low with concentrations phosphoenolpyruvic acid below about 0.3 ~ 10-3M, increased abruptly above this. Lineweaver-Burk plots two s kinase to phosphoenolpyruvic acid are shown in Fig. 17. The Michaelis constant M enzyme for phosphoenolpyruvic acid was 0.75 ~ 10-4 did not change with concentration ADP. In contrast Lineweaver-Burk plots L1 enzyme for phosphoenolpyruvic acid was a second order curve rar than a straight line as expected from sigmoid curve in Fig. 15,, furrmore, apparent Michaelis constant was 0.83 ~ 10-8, which was much higher than that M enzyme. The kinetic curve observed for L1 enzyme seemed to be due to activation by substrate itself, as suggested by Dixon Webb (18). As can be seen from Fig. 18, two s enzyme showed different kinetic characteristics towards ADP. The Michaelis constants M L1 were 0.28 ~ ~ 10-s, which were not very different from each or, whereas L1 was inhibited by ADP at concentrations more than 0.5 x 10-3M. It was found that this apparent inhibition was not caused by ATP which could have contaminated ADP preparation or have been formed during enzyme reaction, since same inhibition was observed even when enzyme reaction was coupled with muscle hexokinase glucose, to remove ATP from reaction mixture. Inhibition Type M Type L1 Enzymes by ATP p-chloromercuribenzoic Acid-It has been reported by Meyerh Oecper (19), that rabbit muscle kinase is inhibited by ATP. Experiments on inhibition purified kinase M L1 by ATP are shown in Fig. 19. Of particular interest was marked difference in sensitivity M L1 enzymes to ATP. The apparent half inhibitory concentration ATP for L1, was 0.15 ~ 10-3M,

17 Two Types Pyruvate Kinase 87 which was about one twentieth that for M (3.5 ~ 10-3M). Both s kinase were also inhibited by p-chloromercuribenzoic acid (Fig. 20). The sensitivities M L1 enzymes to this sulfhydryl inhibitor were also quite different. Type L1 enzyme was about 30 times more sensitive than M. This result can be explained to some extent by fact that L1 is much more labile than FIG. 18. Lineweaver-Burk plots M L1 kinase for ADP. The experimental conditions were as in Fig. 17. FIG. 16. Type M L1 kinase activities as a function phosphoenolpyruvic acid concentration. The results in Fig. 17 are plotted as a function concentration phosphoenolpyruvic acid. FIG. 19. Inhibition M L1 kinase by ATP. The enzyme was assayed by coupling with lactic dehydrogenase. The apparent Ki was taken as concentration at which enzyme was 50 per cent inhibited. FIG. 17. Lineweaver-Burk plots M L1 kinase for phosphoenolpyruvic acid. Crystalline preparations both s enzyme were used. Enzyme activity was assayed with a recording spectrophotometer by following decrease absorption at 340 mp in coupled reaction lactic dehydrogenase system with kinase system. The composition assay mixture is described in text. The reaction was started by addition ADP solution after termination small initial decrease absorption due to contaminating phosphoenolpyruvic acid solution. FIG. 20. Inhibition M L1 kinase by p-chloromercuribenzoic acid. The enzyme was preincubated with p-chloromercuribenzoic acid for 3 minutes before reaction was started. The enzyme was assayed by hydrazone method as described in text. The apparent Ki was taken as concentration, at which enzyme was 50 per cent inhibited.

18 88 T. TANAKA, Y. HARANO, F. SUE H. MORIMURA M is protected from inactivation by Clel's reagent (Fig. 6). DISCUSSION Three kinds mechanism regulating level kinase in rat liver are described in present paper. As shown in Fig. 1, a seasonal change in level kinase in rat liver was observed. The possibility that temperature is essential factor in this seasonal change was excluded, since animals were bred at a constant temperature all year round. The nature diet also did not change with season. It also seems unlikely that light or pineal gl plays a role in this seasonal rhythm, as in diurnal rhythm (12, 13). It should be noted that a seasonal change was also observed in maximum level kinase induced by a high carbohydrate diet. Studies are now in progress in our laboratory on seasonal changes L M kinases results will be reported in a few years. Studies on relationship between this hormonal secretion autonomic central nervous system would be one approach to this problem. A second regulatory mechanism is closely related to cell maturity or cell differentiation. Type M kinase in liver, which showed only a slight response to change in diet hormones, was observed to increase under several conditions. Type M enzyme only was present in ascites hepatoma cells (AH 130), which originate from liver parenchymal cells. Type M kinase (Fig. 15), as well as hexokinase (20), increased in regenerating liver, was high in foetal liver. Recently, in this laboratory, M enzyme was also found to increase in intact liver tumor-bearing rats. Under se conditions, M increased in liver, L usually decreased or remained unchanged. The third regulation is hormonal dietary regulatory mechanism, three mechanisms this has been studied most extensively with or key enzymes in glucose metabolism. Type L kinase plays a major role in third regulatory mechanism. It is L, that increases in livers rats fed on a high carbohydrate diet or in diabetic rats after insulin injection (Table V). Recently, it was found that kinase was induced in diabetic rats which were fed on a 83% fructose diet ( 4 ). It is concluded that this induction was. independent action insulin for two reasons. First, induction by fructose was observed in diabetic animals second, insulin secretion was not stimulated as much by fructose as by glucose (21). This induction could be a " substrate induction " (22). Recently, T a k e d a et al. (23) reported that kinase was induced by administration glycerol, y excluded possibility that kinase was induced by insulin. Insulin-independent induction kinase occurs in liver. However it is not possible to exclude possibility induction by insulin, until true rates biosynsis degradation liver kinase have been measured using antibodies. Pyruvate kinase in crude liver extracts. was separated electrophoretically into two major peaks two minor peaks. A peak, which showed same mobility as that muscle kinase was neutralized with antibody against muscle enzyme, was. named M. Liver M enzyme was immunologically indistinguishable from muscle kinase. However it is still uncertain,. wher se two kinases are completely identical, especially in ir kinetic properties. It is possible that M enzyme is not located in parenchymal cells, since liver consists a heterogeneous population cells. Using fluorescent antibody method, it was observed, that M enzyme was located in liver parenchymal cells.* Two minor peaks in liver extracts were named L2 L3 simply because y were neutralized with antibodies against L1, but not with those against M. The enzyme chemical, kinetic properties L2 L3 enzymes have not yet been investigated. K o l e r et al. (24) reported that kinases present in erythrocytes * Harano, Y., Tanaka, T., unpublished data..

19 Two Types Pyruvate Kinase 89 leucocytes were different from each or, since two were eluted from a DEAE cellulose column at different salt concentrations had different kinetic properties. As shown in Table VI, kinase in erythrocytes was L that in leucocytes was M. In contrast to L in liver, L in erythrocytes did not show any response to change in diet or to hormones. The physiological significance presence L enzyme in erythrocytes is unknown. There are at least two s kinase in kidney, which is a gluconeogenetic organ. In previous paper (5), it was reported that L enzyme was present in kidney, since its mobility one peaks was identical to that liver L enzyme. Later it was found that this peak was neutralized with antibody against muscle kinase, but not with that against L1. Thus name this material should be changed to LM. Pyruvate kinase in kidney was not induced by a high carbohydrate diet. The kinetic properties L1 M kinases were markedly different from each or (Figs. 16, 17 19). Rat muscle kinase is quite similar to rabbit muscle kinase in its properties as described in review by Boyer (6). The apparent half maximum concentration phosphoenolpyruvic acid for L1 enzyme was 0.83 ~ 10-3 M, which was about 10 times that for muscle enzyme, it is suggested from second order curve obtained in a Lineweaver-Burk plot, that enzyme is activated by substrate. Type L1 enzyme was very sensitive to ATP inhibition, concentration ATP causing 50% inhibition was 0.15 ~ 10-3 M. It was reported by Hern brook et al. (25), that actual concentration phosphoenolpyruvic acid in liver rats after 24 hours starvation was Đmoles per g. liver. On feeding a high carbohydrate diet, this concentration would increase. The exact concentration ATP in liver is still unknown, since it is supposed to change with diet, but it TABLE Summary Difference between Pyruvate Kinase Type M Type L VII

20 90 T. TANAKA, Y. HARANO, F. SUE H. MORIMURA was reported to be about one Đmole per ml. soluble fraction in liver (26, 27). From kinetic properties, it is concluded that concentrations substrate inhibitor in liver were far from optimum for maximal activity L1 enzyme. Only when concentration phosphoenolpyruvic acid in liver was elevated that ATP was lowered, enzyme could actually function. A summary differences between two s kinase is shown in Table VII. Type L enzyme is similar in many respects to glucokinase whereas M is similar to hexokinase (16, 17). From parallelism between two groups kinases, it seems likely that re may be two kinds pathway glycolysis in liver. One se is catalyzed by hexokinase M kinase might be called basal pathway. The or is catalyzed by glucokinase L kinase is a regulatory pathway. The rate basal pathway would not be influenced by dietary hormonal conditions, it would meet minimum dems cell. The rate regulatory pathway would fluctuate in response to dietary hormonal conditions, it would meet special dems cell. The authors wish to express ir deep thanks to Pr. M. Suda for his valuable advice encouragement, without which this work could not have beem accomplished. The authors are grateful to Dr. S. Isojima Tokushima University for his skillful assistance in immunological experiments, to Dr. K. Kakiuchi for carrying out ultracentrifugal analyses. The authors also express ir thanks to Takeda Pharmaceutical Co., Ltd., for kindly supplying experimental animals. REFERENCES (1) Utter, M.F., Keech, D.B., Sutton, M.G., " Advances in Enzyme Regulation," ed. by G. Weber, Pergamon Press Inc., New York, Vol.11, 49 (1964) p. (2) Krebs, K.A., Eggleston, L.A., Biochem. J., 94, 3c (1965) (3) Weber, G., Singhal, R.L., Stamm, N.B., Strivastava, S.K., Federation Proc., 24, 745 (1965) (4) Tanaka, T., Harano, Y., Morimura, H., Sue, F.,' Proceedings Symposium on Enzyme Chemistry'(in Japanese), Vol.17, p.341 (1965) Vol.18, p.337 (1966) (5) Tanaka, T., Harano, Y., Morimura, H., Mori, R., Biochem. Biophys. Research Communs., 21, 55 (1965) (6) Boyer, P.D.," The Enzymes," 2nd Ed., ed. by P.D., Boyer, H. Lardy K. Myrback, Academic Press Inc., New York, Vol.VI, p.95 (1962) (7) Kimberg, D.V., Yielding, K.L., J. Biol. Chem., 237, 3233 (1962) (8) Layne, E.," Methods in Enzymology," ed. by S.p.Colowick N.O. Kaplan, Academic Press Inc., New York, Vol.III, p.447 (1957) (9) Leskowitz, S., Waksman, B.H., J. Immunol., 84, 58 (1960) (10) Ouchterlony, O., Acta Pathol. Microbiol. Sc., 26, 507 (1949) (11) Campbell, D.H., Garvey, J.S., Cremer, N.E., Sussdr, D.H.," Methods in Immunology," ed. by D.H., Campbell, W.A. Benjamin Inc., New York, p.149 (1963) (12) Holmquest, D.L., Retience, K., Lipscomb, H.S., Science, 152, 662 (1965) (13) Axelord, J., Wurtman, R.J., Snyder, S.H., J. Biol. Chem., 240, 949 (1965) (14) Tietz, A., Ochoa, S.," Methods in Enzymology," ed. by S.p.Colowick N.O. Kaplan, Academic Press Inc., New York, Vol.V, p.365 (1962) (15) " Instruction Manual," Beckmann Instruments Inc., Spinco Division, California, Part IX, p.3 (1961) (16) Sols, A., Salas, M., Vinuela, E.," Advances in Enzyme Regulation," ed. by G. Weber, Pergamon Press Inc., New York, Vol.11. p.177 (1963) (17) Walker, D.G., Biochem. J., 88, 179 (1963) (18) Dixon, M., Webb, E.G.," Enzymes," Academic Press Inc., New York, p.89 (1958) (19) Meyerh, O., Oecper, P., J. Biol. Chem., 179, 1371 (1949) (20) Suda, M., Tanaka, T., Sue, F., Harano, Y., Morimura, H.," Gann Monograph "(in Japanese), ed. by T. Yoshida, Japan Cancer Assoc., Tokyo, Vol.1, p.127 (1966) (21) Grodsky, G.M., Batts, A.A., Bennet, L.L., Vcella, C., McWilliams, N.B., Smith, D.F., Am. J. Physiol., 201, 1073 (1961) (22) Knox, W.E.," Advances in Enzyme Regulation," ed. by G. Weber, Pergamon Press Inc., New York, Vol.11, p.311 (1966) (23) Inoue, H., Honjo, K., Tanioka, H., Daikubara, K., Takeda, Y., J. Japan. Biochem. Soc. (in Japanese), 38, 673 (1966) (24) Koler, R.D., Bigley, R.H., Jones, R.T., Rigas, D.A., Vanbellinghen, P., Thompson, P.,