Isolation, Characterization and Localization of the Proteinase B Inhibitor IB3 from Saccharomyces carlsbergensis

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Abstract
A heat-stable polypeptide was detected in S. carlsbergensis which inhibits specifically proteinase B from yeast. This proteinase B inhibitor IB3 differs substantially in chemical, physical and antigenic properties from the earlier described proteinase B inhibitors IB1 and IB2 from yeast. The inhibitor IB3 was purified from S. carlsbergensis and appears to be homogeneous by disc gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. The MW was estimated at 11,500, 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 [EC 3.4.23.6] and carboxypeptidase Y [EC 3.4.17.4] from yeast and by trypsin from bovine pancreas. The proteinase B inhibitor association constant was calculated to be 3.3 .times. 109/M and the enzyme inhibitor complex is stable at 25.degree. C in the pH range 5-10. The inhibitor does not exhibit immunological cross-reactivity with IB1 and IB2. After centrifugal fractionation at 40,000 .times. g of a metabolic lysate from spheroplasts the inhibitor was localized in the supernatant, i.e., the extravacuolar soluble fraction.

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