Lack of effect of carbohydrate depletion on some properties of human mast cell chymase

Human chymase from vascular tissues was purified to homogeneity by heparin affinity and gel filtration chromatography. Treatment of human chymase with endoglycosidase F resulted in cleavage of the carbohydrate moiety yielding a deglycosylation product that did not lose its catalytic activity. This e nzymatic deglycosylation product was enough to explore possibilities that N -glycan might modify some properties of human chymase. Substrate specificit y, optimum PH and the elution profile from the heparin affinity gel were no t affected by the deglycosylation. Only a slight but significant difference was observed in the K-m value for conversion of angiotensin I to angiotens in II. Other kinetic constants such as k(cat) were not influenced. The kine tics of conversion of big endothelin-l to endothelin-l(1-31) were not signi ficantly affected. The deglycosylated human chymase was more susceptible to deactivation under alkaline PH and thermal stress. Even at physiological t emperature and pH, the activity of glycosylated human chymase was moro stab le. From these results, it appears that the N-glycan of human chymase contr ibutes to the stability of this enzyme but not to its functional properties . (C) 1999 Elsevier Science B.V. All rights reserved.