Studies on New Intracellular Proteases in Various Organs of Rats

Homogenates of the muscle layer of rat small intestine irreversibly inactivated endogenous ornithine aminotransferase at 37°C. Addition to the homogenate of coenzymes and the various keto‐acids which act as substrate inhibited conversion of the holoenzyme to the apoenzyme and its subsequent degradation. Addition of protease inhibitors including soybean trypsin inhibitor, chymostatin and phenylmethylsulfonyl fluoride almost completely prevented inactivation of the enzyme. Immunological activity decreased during inactivation of the enzyme, but its rate of decrease was much slower than that of loss of enzyme activity. Antigen‐antibody precipitates from homogenates containing inactivated enzyme. were separated by electrophoresis on sodium dodecylsulfatepolyacrylamide gels. In this way breakdown products of the enzyme were found, indicating that the enzyme in homogenates was inactivated by limited proteolysis. These results obtained in vitro support our previous suggestion (1975) of a stepwise mechanism for degradation of pyridoxal enzymes. However, in vivo there was no difference between the rates of decrease of enzyme activity and of immunological activity during the decrease of ornithine aminotransferase following inhibition of protein synthesis. Moreover, no degradation products of the enzyme were found under either control, or pyridoxine‐deficient conditions. But the rate of decay of ornithine aminotransferase in the muscle layer of the small intestine was faster in pyridoxine deficiency. The apparent half‐life in vitamin‐B 6 ‐depleted rats was estimated as 5 h, which was shorter than that in control rats (8 h). These results suggest that a step in the conversion of the holoenzyme to the apoenzyte is ratelimiting in degradation of the enzyme in vivo .