Regulation of the activity of human chymase during storage and release from mast cells: the contributions of inorganic cations, pH, heparin and histamine.
Chymase, the major chymotryptic proteinase of human mast cells, can be released in substantial quantities following mast cell activation. As this enzyme is stored in the secretory granules in its fully active form, we have investigated various factors which might regulate its activity in storage and upon release. Chymase was purified from human skin by high salt extraction, cetylpyridinium chloride precipitation, heparin agarose affinity chromatography and gel filtration. Neither the addition of Mg2+ or Ca2+ (0.3-10 mM) nor their sequestration by EDTA had any effect on the rate of cleavage of the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Monovalent cations (Na+,K+) enhanced enzyme activity, but only at non-physiological concentrations (0.5-3.0 M), suggesting an ionic strength effect. At constant I = 0.15, enzyme activity was strongly pH-dependent: at pH 5.5 (the approximate pH of the mast cell granule) the activity was only 10% of that at pH 7.5 (the approximate pH of the extracellular space). Heparin, which is stored with chymase in the mast cell granule, accentuated this difference by enhancing activity at pH 7.5 by 33% and depressing it a pH 5.5 by 40%. Histamine at concentrations up to 50 mM (I = 0.15) had little effect on chymase activity at either pH, although high concentrations did attenuate the actions of heparin. It is concluded that pH and the interaction with heparin are central to the regulation of chymase activity within the granule and following release.
McEuen
A R
AR
Immunopharmacology Group, Southampton General Hospital, UK.
Sharma
B
B
Walls
A F
AF
D000595:Amino Acid Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D053818:Chymases; D004789:Enzyme Activation; D006493:Heparin / Q000494:pharmacology; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008407:Mast Cells / Q000201:enzymology*; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000378:metabolism*
D002413:Cations, Divalent; D006632:Histamine; D006493:Heparin; D012697:Serine Endopeptidases; D053818:Chymases