OF TRANSAMINASE IN RAT TISUES

Transcription

1 OF TRANSAMINASE IN RAT TISUES KOZO YAMADA, SHUNJI SAWAKI, AKIRA FUKUMURA AND MASARU HAYASHID epartment of Internal Mcdicine, Faculty of Medicine, Nagoya University, agoya Showa-ku, N (Received July 30, 1962) The heterogeneity of transaminase activity in animal tissues has been observed in several laboratories (1-6). It has recently been shown that these enzymes appear to be present separately in subcellular fractions. The results suggested that the mitochondrial transaminase differs from the supernatant transaminase in the substrate affinity, ph-dependence, electrophoresic mobility and their mechanism. It was observed in our present work that the enzymes (acetone-stable trans aminases) from different tissues of the rat appeared to act differently in several properties, EXPERIMENTAL Materials ante Methods Rats were decapitated and the tissues were washed thoroughly with cold water and the homogenates were prepared with 10ml of barbital buffer, ph 8.6, ionic strength 0.1, or ice-cold water, and submitted to freezing and thawing, followed by treatment with Triton X100 (final concentration, 0.5g/100ml) for 1-2 hours at 4. Centrifugation of these extracts for 60 minutes at 15,000 ~g produced a super - natant fluid which was decanted. 5-6 ml of the supernatant was separated electrophoretically on a zone, starch being used as a supporting medium. After electrophoresis at 4 for 20 hours at 200V and 10mA, each of the section of the starch block was cut into 1.0-cm segments, from which the protein was eluted with 3ml water or physiological saline and its concentration determined (7). An aliquot from each was then assayed for transaminases, glutamic-oxaloacetic transaminase (GOT), and glutamic-pyruvic transaminase (GPT), according to a modi fication of the method of Cabaud et al. (8). On the other hand, the transaminase activities were observed by the three methods given in the previous report (9). Mitochondria were prepared by Schneider's method (10), washed twice and the enzyme was extracted with Triton X100. Further, the same procedure was adopted. In addition, certain properties, e. g., the inhibition by heating, trypsin, lipase and isonicotinic acid hydrazide, of the enzyme purified electrophoretically from the extracts of acetone-dried powders of various rat tissues were also examined. 2 86

2 Vol. 8 YAMADA, SAWAKI, FUKUMUPA AND HAYASHI 287 RESULT Four electrophoretically-distinguishable peaks of transaminase activity (GOT1, GOT2, GPT1 and GP'T2) were observed in the homogenates of rat liver and three peaks (GOT1, GOT2 and GPT2) in those of other organs (Fig. 1-3). There was a large similarity between the results obtained by the colorimetric method of Cabaud et al. (8) and those by the paper-chrornatograpllic or mano FIG. 1 Distribution of Glutanic-Oxalacetic and Glutainic-Pyruric Transaminase after Zone Electrophoresis of Crude Liver Extract Reaction system: 0.5ml of the reagent (for GOT, 2.66g of DL-aspartic acid, 0.60g of ƒ -ketoglutaric acid and 2.00g of secondary potassium phosphate were dissolved in distilled water and the ph of the solution was adjusted to 8.0 with 1 N potassium hydroxide. The solution was brought to a final volume of 100ml. For GPT, DL-aspartic acid was replaced by 1.78g of DL-alanine) and the enzyme was in cubated for 20 minutes at 37. After the reaction, a drop of 100per cent trichloro acetic acid and for GOT aniline-citrate solution were added to the solution. Then it was incubated for 20 minutes at 37 for GOT and 0.5ml of 0.1per cent dinitrophenylhydrazine was added. After 5 minutes at room temperature, 2.0ml of toluene was added and the whole was shaken vigorously. Then the mixture was centrifuged for 5 minutes. 1.0ml of the toluene layer and 3.0ml of potassium hydroxide-alcohol solution were mixed. The optical density at 490 mƒê was determined. Determination of glutamic acid by paper chromatography; After the reaction, 5.0ml of absolute alcohol was added and filtered. The filtrate was concentrated under reduced pressure. An aliquot was spotted on a filter paper, de veloped with water-saturated phenol and the ninhydrin reagent was sprayed on it. indicate the intensity of ninhydrin reaction of the spot of Iutarnic acid.

3 288 ISOZYMES OF TRANSAMINASE 1962 Fro. 2 Distribution of Transaminase Activity in the Homogenates of Rat Heart, Kidney, Pancreas and Skeletal Muscle Reaction system is the same as that given in Fig, 1, These tissue homogenates were separated electrophoretically on the same starch block. FIG. 3 Distaibntion of Transaminase Activity in Acetone Ponder Extracts of Rat Brain, Heart, Liver, Pancreas, Intes tine, Kidney and skeletal Muscle Reaction system is the same as that given in Fig. 1. metric method. It is suggested that the two types of the enzyme may be simply converted into the other by various treatments, The solubilized extracts obtained by treating with Triton X100 showed the increase in GPT1 activity, but scarcely any in GOT1 activity. Furthermore, each tissue had the three acetone-stable transaminases (GOT1. GOT2 and GPT2). These peaks were obtained with 50mg of the tissue protein. GOT1 and GPT1 are supposed to be derived from mitochondria, as described by Fleisher and Coworkers (1, 5). The two peaks (GOT2, GPT2) were not far from the point of application,

4 Vol. 8 YAMADA, SAWAKI, FUKUMURA AND HAYASHI 289 whereas the other peaks (GOT1, GPT1) were found far from the point. The ratio of the total GOT2 activity to the total GOT1 activity was about and that of the total GPT2 activity to the total GPT1 activity in the fractions of rat liver was also about The values obtained (Tables I-IV) revealed significant differences among the peaks (GOT1, GOT2 and GPT2) by transaminase inhibitors. TABLE I Inhibitory Effect of Temperature on the Transaminase Actict ities of Various Rat Tissues Fractionated by Zone Electrophoresis after Acetone Treatment The figures represent percentage inhibition as a mean of 3 cases. The reaction system is the same as that given in Fig. 1. The enzymes (about the same activity) after preincubation at 50 for 10 minutes were incubated for 20 minutes at 37, and the enzyme activity was compared with that of the control. TABLE II Inhibitory Effect of Trypsin on the Transaminase Activities of Various Rat Tissues Fractionated by Zone Electrophoresis after Acetone Treatment The figures show the percentage inhibition as a mean of 3 cases. The reaction system is the same as that given in Table 1, except for the addition of trypsin prior to the preincubation at 37 for 30 minutes. TABLE III Inhibitory Effect of Lipase on the Transaminase Activities o f Various Rut Tissues Fractionated by Zone Electrophoresis after Acetone Treatmetlt The figures show percentage inhibition as a mean of 3 cases. Reaction system is the same as that given in Table I, except for the addition of lipase prior to the preincubation at 37 for 30 minutes.

5 2 90 ISOZYMES OF TRANSAMINASE 1902 TABLE IV Inhibitory Effect of Isonicotinic Acid Hydrazide on the Transaminase Activities of Various Rat 'issues Fractionated by Zone Electrophoresis after Acetone Treatment The figures show percentage inhibition, a mean of 3 cases. Reaction system is the same as that given in Table I, except for the addition of isonicotinic acid hydrazide (final concentration, M/1000) prior to the preincubation at 37 for 10 minutes. GPT1 was easily destroyed by heating and acetone treatment. On the other hand, GOT1 was more labile than GOT2 at 50 heating, but GOT2 was relatively stable to heating, each tissue having a characteristic difference of transaminase activity. The amount of crystalline trypsin (1000 units supplied from Motida Corpora tion) inhibited 32% of the GOT1 from the heart, 24% of that from the intestine, 30% of that from the skeletal muscle and only 6% of that from the liver. On the contrary, GPT2 was completely inhibited by trypsin. The amount of pancreatic lipase (supplied from Nutritional Biochemical Corpo ration, using tributyrin as a substrate, 50% of the substrate was decomposed by the enzyme after incubation for one hour at 20 ) inhibited 56% of the GOT1 from the heart, 72% of that from the intestine, 41% of that from the skeletal muscle and only 5% of that from the liver. GPT2 was also more strongly inhibited by the lipase than GOT2. Isonicotinic acid hydrazide relatively strongly inhibited several tissue GPT2's and GOT1's, but little inhibited GOT2 except for the transaminases of some tissues. DISCUSSION Each of the homogenates or the acetone powders of rat tissues examined electrophoretically exhibited heterogeneity in transaminase activities. Further - more, each tissue had a constant number of activity peaks and a similar distribution of transaminase activity peaks. Recently Fleisher et al. (1, 5), Borst et al. (3) and Boyd (4) reported that water (or buffer) extracts from different animal tissues contain two GOT's differing in electrophoretic mobility, K for aspartate, ph activity curves, and behaviors on diethyl aminoethyl- and carboxymethyl-cellulose columns. In agreement with our findings, electrophoretic mobilities of Fleisher's transaminase were the same and GPT1, GPT2 and GOT1 were relatively labile to heating. On the contrary, GOT2 being present particularly extramito - chondrially in various tissues was stable to heating. In addition, it is important to stress that heating, trypsin, lipase and isonicotinic acid hydrazide inhibition in

6 Vol various tissue transaminases appear to vary quantitatively. Thus it is evident that the liver transaminase is distinct from the hart enzyme. These results indicate a possibility of determining the organ from which the serum enzyme is derived by using inhibitions by various substances. These observations suggest that the molecular heterogeneity of the transaminases may differ in amino acid sequence from each other. The immunological approach to detect the similarities of the enzymes has also attracted much interest, and the immunological heterogeneity of the trans -aminases will also be used to test the differences. It is our hope that the studies with crystalline transaminases may enable us to ascertain what part of the enzyme molecule is different from each other. SUMMARY 1. Heterogeneity of transaminase activities was proved by electrophoretical separation of the homogenates of the heart, kidney, liver, skeletal muscle, brain, pancreas and intestine of the rat. Four peaks of transaminase activities were found in the liver, three peaks in the hart, kidney, skeletal muscle, brain, pancreas and intestine. Each tissue exhibited a similar distribution of trasaminase activities. 2. The activity peak electrophoretically separated had the same mobility as the peak in the liver having the greatest transaminase activity. The acetone-dried powder extracts showed higher transaminase (GOT,, GOT2 end GPT2) activities than homogenate extracts. 3. The enzymes isolated from rat tissue acetone powders were shown to differ in sensitivity to inhibition by heating, trypsin, lipase and isonicocinic acid hydra -zide. REFERENCES 1. Fleisher, G. A., Potter, C. S., and Wakim, K. G., Proc. Soc. Exptl. Biol, Med. 103, 229 (1960). 2. Fleisher, GA, Fed. ES cc. 19, 6 (1960). 3. Borst, P., and Teeters, E. M., Biochem. Biopnys. Acta. 54, 188 (1961). 4. Royd, J, W., Biochem. J. 81, 434 (1961). 5. Fleisher. G. A., and Wakim, K. G., Proc. Soc. Exptl. Riol. Med. 106, 283 (1961). 6. Rendall, D. S., and de Duve, C., Biochem..J. 74, 444 (1960). 7. Folin, O., and Ciocalteu, V., J. Biol. Chem. 73, 629 (1927). 8. Cabaud, P., Leeper, R., and Wroblewski, F., Am. J. Ctiu. Pathol. 26, 1101 (1956). 9. Yainada, K., Hayashi, M., Sawaki, S., Yazaki, C., and Fukumura, A., J. Vitaminol. 7, 265 (1961). 10. Schneider, W. C. J. Bicl. Chem. 176, 259 (0948).