Purification and Characterization of Macromolecular Inhibitors of Proteinase A from Yeast.

Inhibitors for yeast proteinase A have been purified from boiled extracts from baker's yeast. Upon SP-Sephadex C-25 column chromatography, four different peaks of proteinase A inhibitory activity appear. The inhibitors I2,A and I3,A have been further purified by QAE-Sephadex chromatography and preparative disc electrophoresis. Both inhibitors are homogeneous in dodecylsulfate-gel electrophoresis. From the electrophoretic mobilities in dodecylsulfate-polyacrylamide gels, a molecular weight 6100-,+200 was estimated for both inhibitors. In the absence of dodecylsulfate, both inhibitors showed one predominant band and a second faint, diffuse band. As a result of Sephadex G-75 gel filtration, a molecular weight of 23000-,+2000 was obtained for both inhibitors, indicating an aggregation of the monomers to a tetramer. Upon electrofocusing in acrylamide gels the isoelectric points of the inhibitors were found to be 5.7 for I2,A and 6.3 for I3,A. Both inhibitors inhibited proteinase A. neither proteinases B and C were inhibited from yeast or pepsin. Both inhibitors were inactivated after incubation with purified yeast proteinase B, but not with the yeast proteinases A or C. the inhibition of proteinase A at high inhibitor/proteinase ratios was more pronounced at pH 5 and 7 as compared to pH 3. An autocatalytic activation of mixture of inhibited proteinases A, B and C by addition or liberation of traces of active proteinase A or B is discussed. [ABSTRACT FROM AUTHOR]