Interaction of proteinases and their inhibitors from yeast. Activation of carboxypeptidase Y.

In a crude extract of baker's carboxypeptidase Y is predominantly found in an inactive form. A procedure for the isolation of the inactive form of the enzyme is presented. It is shown that the inactive form is identical to the reconstituted complex of carboxypeptide Y with its inhibitor. This complex is stable above pH 5, i.e., it remains inactive between pH 5 and 9. The conversion to the active enzyme occurs below pH 5, also in the absence of proteolytic enzymes. The inhibitor of carboxypeptidase Y can be removed enzymatically from the complex by treatment with proteinase B (EC 3.4.22.9) at pH 7. At pH 5, the carboxypeptidase Y-inhibitor complex is activated both by proteinase A (EC 3.4.23.6) and B. Yeast proteinases are activated in a crude extract by incubation at pH 5 [3]. Based on the levels of proteinase A and B in an activated extract and on the time required for conversion to active carboxypeptidase Y, proteinase B is at least 10-times more effective than proteinase A. Peptides that arise during the pH 5-incubation procedure did not accelerate the proteolytic activation of carboxypeptidase Y. The inhibitor of carboxypeptidase Y is completely degraded in the proteolytic activation steps, no accumulation of intermediates is observed. Only one form of active carboxypeptidase Y is found to be present in the proteolytically activated extracts, i.e., no polypeptide fragments of carboxypeptidase Y-inhibitor remain bound to the enzyme after it has been activated by proteinase B. In vacuoles prepared from spheroplasts no inactive carboxypeptidase Y can be detected.