Purification and properties of three endopeptidases from baker's yeast.
Three endopeptidases, proteinases A, B, and Y, were purified from baker's yeast, Saccharomyces cerevisiae. Two molecular forms of proteinase A (PRA), Mr 45,000 and 54,000, (estimated on SDS-PAGE) were obtained. Both forms were inhibited by pepstatin and other acid proteinase inhibitors. The enzyme digested hemoglobin most rapidly at pH 2.7-3.2 and casein at pH 2.4-2.8 and 5.5-6.0. The optimum pH for hydrolysis of protein substrates could be shifted to about 5 with 4-6 M urea. Urea also stimulated the enzyme activity by 30-50%. As other acid proteinases, the enzyme preferentially cleaved peptide bonds of X-Tyr and X-Phe type. A proteinase B (PRB) preparation of approximately Mr 33,000 possessed milk clotting activity and showed an inhibition pattern typical for seryl-sulfhydryl proteases. The purified enzyme could be stabilized with 40% glycerol and stored at -20 degrees C without significant loss of activity for several months. The third endopeptidase, designated PRY, of Mr 72,000 when estimated by Sephadex G-100 gel filtration, had properties resembling PRA and PRB. Similar to PRB, it could be inhibited by up to 90% with phenylmethylsulfonyl fluoride and para-chloromercuribenzoate and preferentially hydrolyzed the Leu15-Tyr16 peptide bond of the oxidized beta-chain of insulin. On the other hand, contrary to PRB, it had neither milk clotting activity nor esterolytic activity toward N-acetyl-L-tyrosine ethyl ester and N-benzoyl-L-tyrosine ethyl ester and was stable during storage at -20 degrees C without glycerol. The enzyme also showed a lower pH optimum for hydrolysis of casein yellow than PRB.(ABSTRACT TRUNCATED AT 250 WORDS)
Nowak
J
J
Max-Planck-Institut für Experimentelle Medizin, Göttingen, Federal Republic of Germany.
Tsai
H
H
D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008892:Milk / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D006454:Hemoglobins; D011480:Protease Inhibitors; D010450:Endopeptidases