title: Purification and properties of proteinase B from yeast.
abstract: Proteinase B (EC 3.4.22.9) was purified from commercial baker's yeast and from wild type strains of Saccharomyces cerevisiae and Saccharomyces carlsbergensis. For large scale purification a procedure was developed involving hydrophobic chromatography on octyl-Sepharose 4B and gel filtration on Sephadex G-100. A rapid purification of small amounts of proteinase B was achieved by affinity chromatography on the nitrated proteinase B inhibitor, immobilized on CH-Sepharose according to Bünning and Holzer (Bünning, P. and Holzer, H. (1977). J. Biol. Chem. 252, 5316-5323). The enzyme prepared from all three sources appeared to be homogeneous and exhibited a molecular weight of 33 000 in SDS-polyacrylamide gel electrophoresis. Homogeneity and molecular weight were confirmed for the enzyme from baker's yeast by ultracentrifugation studies. Polyacrylamide gel electrophoresis without SDS and electrofocusing however, indicated microheterogeneity of the proteinase B activity. The aminoterminal residue of the enzyme was found to be glycine. Proteinase B turned out to be a glycoprotein, containing 8-9% neutral sugars and 1.5% amino sugars. The enzyme is blocked by p-hydroxymercuribenzoate and by the serine proteinase inhibitors DFP and PMSF. Among the proteinase inhibitors from microbial origin, chymostatin and antipain were the most powerful inhibitors of proteinase B.
mesh_terms: D000596:Amino Acids / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D010450:Endopeptidases / Q000302:isolation & purification*; D008970:Molecular Weight; D011480:Protease Inhibitors; D012440:Saccharomyces / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012697:Serine Endopeptidases; D014617:Vacuoles / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins
chemical_list: D000596:Amino Acids; D002241:Carbohydrates; D010450:Endopeptidases; D011480:Protease Inhibitors; D012697:Serine Endopeptidases; C023302:yeast proteinase B; D029701:Saccharomyces cerevisiae Proteins