The activating system of chitin synthetase from Saccharomyces cerevisiae. Purification and properties of the activating factor.
The yeast proteinase that causes activation of the chitin synthetase zymogen has been purified by a procedure that includes affinity chromatography on an agarose column to which the proteinaceous inhibitor of the enzyme had been covalently attached. The purified enzyme yielded a single band upon disc gel electrophoresis at pH 4.5 in the presence of urea. At the same pH, but without urea, a faint band was detected in coincidence with enzymatic activity, whereas at pH 9.5, either in the absence or in the presence of sodium dodecyl sulfate, no protein zone could be seen. From sedimentation and gel filtration data, a molecular weight of 44,000 was estimated. The proteinase was active within a wide range of pH values, with an optimum between pH 6.5 AND 7. Titraton of the activity with the protein inhibitor from yeast required 1 mol of inhibitor/mol of enzyme. A similar result was obtained with phenylmethylsulfonyl fluoride, an indication that 1 serine residue is required for enzymatic activity. The enzyme exhibited hydrolytic activity with several proteins and esterolytic activity with many synthetic substrates, including benzoylarginine ethyl ester and acetyltyrosine ethyl ester.A comparison of the properties of the enzyme with those of known yeast proteinases led to the conclusion that the chitin synthestase activating factor is identical with the enzyme previously designated as proteinase B (EC 3.4.22.9). This is the first time that a homogeneous preparation of proteinase B has been obtained and characterized.
D002687:Chitin Synthase / Q000378:metabolism*; D002729:Chloromercuribenzoates / Q000494:pharmacology; D002846:Chromatography, Affinity; D004789:Enzyme Activation; D006602:Hexosyltransferases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000502:physiology*; D011480:Protease Inhibitors; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
D002729:Chloromercuribenzoates; D011480:Protease Inhibitors; D006602:Hexosyltransferases; D002687:Chitin Synthase; D010447:Peptide Hydrolases; D014357:Trypsin