The Isolation And Characterization Of A Yeast Protein Which Specifically Inhibits Yeast Proteinase A

Abstract

A yeast protein was discovered which specifically inhibits proteinase A of Saccharomyces cerevisiae. The inhibitor was purified 8.4-fold using boiling, dialysis and DEAE-cellulose ion-exchange chromatographic steps. The inhibitor, IA, was found to be an unusually thermostable, nondialyzable protein which was not precipitable with trichloroacetic acid, and which adsorbed to DEAE-cellulose at pH 9-10, and CM-Sephadex at pH 6.0. Elution of the inhibitor from DEAE – cellulose was accomplished with 0.01 M Borax buffer, pH 9.1, containing 0.05 M NaCl. Sephadex G-75 gel-filtration and SDS molecular weight disc electrophoresis suggest a molecular weight of about 13,000 for IA. Digestion by trypsin, chymotrypsin, pepsin, subtilisin and yeast carboxypeptidase demonstrated the inhibitor to be of protein nature. IA was found in the greatest concentration in the cytosol of the cell. Possible digestion of IA at pH 2.5 by proteinase A was observed. Digestion of the inhibitor by yeast proteases at pH 6.8 was clearly demonstrated. Lenney's report (6) of proteinase A activation in pH 5.0, yeast autolysates was also confirmed.