Preparative purification of the rat mast cell chymase: characterization and interaction with granule components.
The rat mast cell granule chymotrypsinlike enzyme was purified to homogeneity from 1 M NaCl solubilized membrane and granule-rich fractions of concentrated rat peritoneal mast cells by a preparative technique utilizing chromatography on Dowex 1, filtration on Sephadex G-75, and affinity chromatography with D-tryptophan methyl ester. Acid disk gel electrophoresis of the purified chymase disclosed a single stained band with activity being eluted from a replicate sliced gel in the same region. SDS-polyacrylamide gel electrophoresis of purified protein gave a single stained band that did not change in position with reduction and alkylation. Mast cell chymase is thus a cationic protein of 25,000 mol wt composed of a single polypeptide chain. The apparent K(m) of the chymase for BTEE was 1.5 x 10(-3) M and the V(max) was 67.8 mumol/min per mg. The enzyme was inhibited by TPCK and not by TLCK. The chymase complexed with native macromolecular rat mast cell heparin in molar ratios of 12:1 and 16:1, and complete heparin uptake occurred at a 40:1 ratio of chymase to heparin. Chymase activity was partially masked by combination with heparin in the isolated granule or experimental chymase-heparin complex, and soluble purified chymase was inhibited by concentrations of 5-HT comparable to those present in mast cells. It is therefore possible that the active site of chymase in the mast cell granule is largely masked by the combined effects of macromolecular heparin and 5-HT.
D000818:Animals; D001549:Benzamides / Q000378:metabolism; D002845:Chromatography; D012085:Chymosin / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism; D005260:Female; D006493:Heparin / Q000378:metabolism; D006632:Histamine / Q000494:pharmacology; D008297:Male; D008407:Mast Cells / Q000201:enzymology* / Q000648:ultrastructure; D051381:Rats; D012701:Serotonin / Q000494:pharmacology; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D001549:Benzamides; D014107:Tosyllysine Chloromethyl Ketone; D012701:Serotonin; D014108:Tosylphenylalanyl Chloromethyl Ketone; D014443:Tyrosine; D006632:Histamine; D006493:Heparin; D012085:Chymosin