THE CONVERSION OF RECOMBINANT HUMAN MAST-CELL PROCHYMASE TO ENZYMATICALLY ACTIVE CHYMASE BY DIPEPTIDYL PEPTIDASE-I IS INHIBITED BY HEPARIN AND HISTAMINE

Citation
Ar. Mceuen et al., THE CONVERSION OF RECOMBINANT HUMAN MAST-CELL PROCHYMASE TO ENZYMATICALLY ACTIVE CHYMASE BY DIPEPTIDYL PEPTIDASE-I IS INHIBITED BY HEPARIN AND HISTAMINE, European journal of biochemistry, 253(1), 1998, pp. 300-308
Citations number
45
Categorie Soggetti
Biology
ISSN journal
00142956
Volume
253
Issue
1
Year of publication
1998
Pages
300 - 308
Database
ISI
SICI code
0014-2956(1998)253:1<300:TCORHM>2.0.ZU;2-H
Abstract
Chymase, a major product of mast cell activation, is secreted as a ful ly active enzyme. We have prepared recombinant human prochymase and ha ve investigated the conditions under which it may be activated by dipe ptidyl peptidase I (DPP I). The gene for human chymase was cloned in a baculovirus vector and expressed in High Five insect cells, and the r ecombinant protein purified by heparin-agarose and Eel-filtration chro matography. The purified prochymase was homogeneous by SDS/PAGE with t he same molecular mass as native human chymase, and its identity confi rmed by N-terminal sequence analysis and Western blotting with chymase -specific antibodies. Treatment with DPP I to remove the N-terminal di peptide prosequence resulted in enzymatically active chymase, with sub strate: and inhibitor profiles very similar to those of the native hum an enzyme. The activation of prochymase by DPP I was strongly inhibite d by heparin (IC50 = 0.5 mu g ml(-1)) and histamine (IC50 = 2 mM), tho ugh these mast cell products had little effect on the action of DPP I towards a low molecular-mass substrate. The pH optimum of DPP I was al so higher and narrower with prochymase. The inhibitory action of hepar in was lost at NaCl or KCl concentrations sufficient to elute prochyma se from a heparin agarose column. Dextran sulphate was as inhibitory a s heparin, whereas chondroitin sulphate C was more than 10-fold less e ffective. Our findings suggest that the activation of prochymase might be restricted to the early stages of vesicle maturation. when the FH is close to neutrality and the histamine and heparin concentrations ar e low.