Isolation, Characterization and Localization of the Proteinase B Inhibitor I B 3 from Saccharomyces carlsbergensis

A heat‐stable polypeptide has been detected in Saccharomyces carlsbergensis which inhibits specifically proteinase B from yeast. This proteinase B inhibitor I B 3 differs substantially in chemical, physical and antigenic properties from the earlier described proteinase B inhibitors I B 1 and I B 2 from yeast. The inhibitor I B 3 has been purified from S. carlsbergensis and appears to be homogeneous by disc gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. The molecular weight has been estimated at 11500, with no evidence for the existence of subunits. The amino acid analysis shows the absence of tryptophan. No compounds other than amino acids could be detected The isoelectric point is 4.6. The inhibitor is not affected by incubation with proteinase B but is inactivated by proteinase A and carboxypeptidase Y from yeast and by trypsin from bovine pancreas. The proteinase B inhibitor association constant was calculated to be 3.3 × 10 9 M −1 and the enzyme inhibitor complex is stable at 25°C in the pH range 5–10. The inhibitor does not exhibit immunological cross‐reactivity with I B 1 and I B 2. After centrifugal fractionation at 40000 × g of a metabolic lysate from spheroplasts the inhibitor was found to be localized in the supernatant, i.e. the extravacuolar soluble fraction.