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 f orm, we have investigated various factors which might regulate its act ivity in storage and upon release. Chymase was purified from human ski n by high salt extraction, cetylpyridinium chloride precipitation, hep arin agarose affinity chromatography and gel filtration. Neither the a ddition of Mg2+ or Ca2+ (0.3-10 mM) nor their sequestration by EDTA ha d any effect on the rate of cleavage of the synthetic substrate N-succ inyl-AIa-Ala-Pro-Phe-p-nitroanilide. Monovalent cations (Na+, K+) enha nced enzyme activity, but only at non-physiological concentrations (0. 5-3.0 M), suggesting an ionic strength effect. At constant I = 0.15, e nzyme activity was strongly pH-dependent: at pH 5.5 (the approximate p H 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 s tored with chymase in the mast cell granule, accentuated this differen ce by enhancing activity at pH 7.5 by 33% and depressing it at 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 acti vity within the granule and following release.