The Purification and Properties of a Yeast Protein Which Specifically Inhibits Yeast Proteinase A

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2014-01-15

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University of Hawaii at Manoa

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In baker's yeast (Saccharomyces cerevisiae) there are three proteolytic enzymes which have been well characterized. These are proteinases A and B and carboxypeptidase Y. These enzymes are localized in the vacuole, which represents the lysosome of the yeast cell. Inhibitors of the proteases are localized in the cytoplasm outside of the vacuoles. The proteases and their inhibitors probably play a role in regulating the turnover of cellular proteins. In this research, the inhibitor of proteinase A (IA) was purified and characterized. It was selectively extracted by boiling the yeast, and was purified by ion exchange and Sephadex chromatography. The purified inhibitor was homogenous by the criterion of gel electrophoresis. It was found to be a protein with a molecular weight of 22,000 which could be dissociated into subunits or polypeptide chains having molecular weights of about 11,000. IA was specific for proteinase A and had no effect on proteinase B or carboxypeptidase Y. Proteinase A was not able to digest and destroy the IA' while Proteinase B destroyed it quite rapidly over a wide pH range. The effectiveness of IA was diminished by lowering the pH below 4.0. At a neutral pH, it was completely stable at 100˚C. It was also found that the Actinomycete inhibitor, pepstatin, inhibits proteinase A selectively without affecting proteinase B or carboxypeptidase Y.

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iv, 37 pages

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