MODES OF PROTEOLYSIS OF CYSTATHIONASE AND ORNITHINE AMINOTRANSFERASE BY SERINE PROTEASE FROM THE RAT SMALL INTESTINE

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The mechanisms of proteolysis of two apo-forms of pyridoxal enzymes, cystathionase [EC 4. 2. 1. 15] and ornithine aminotransferase [EC 2. 6. 1. 13] by serine protease from the rat small intestine were compared. The apo-forms of these two pyridoxal enzymes are susceptible to the serine protease, whereas the holo-forms of the enzymes are not. Pyridoxal phosphate, the coenzyme of these two enzymes, prevented their inactivation by the serine protease. The difference in susceptibility of the apo and holo-forms to the serine protease was due to the difference in their conformations. The time course of inactivation of cystathionase by the protease was apparently biphasic. During the first phase of inactivation, disappearance of the band corresponding to the native protomer, with a molecular weight of 47, 000, was accompanied by accumulation of new material with a molecular weight of 39, 000. In the late stage of proteolysis, extensive degradation of the large molecular weight intermediate was observed. This large intermediate product was isolated and found to compete with intact cystathionase as a substrate for the protease. Proteolysis of cystathionase was accompanied by both dissociation of the enzyme molecule and loss of its antigenicity. Limited proteolysis of ornithine aminotransferase apoenzyme by the serine protease resulted in formation of a large molecular weight product similar to the native apoenzyme, but with nicks in the molecule. Dodecylsulfate-polyacrylamide gel electrophoresis showed that the apoenzyme is degraded to intermediate forms (molecular weight 41, 500 and 15, 000) and later, to stable forms (molecularr weight 25, 500 and 13, 500). and D have no-activity. The intermediate product of ornithine aminotransferase of large molecular weight is formed rapidly in the initial reaction of the serine protease, but the subsequent proteolytic events proceed much more slowly.
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MODES OF PROTEOLYSIS OF CYSTATHIONASE AND ORNITHINE AMINOTRANSFERASE BY SERINE PROTEASE FROM THE RAT SMALL INTESTINE

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