Two distinct forms of human mast cell chymase--differences in affinity for heparin and in distribution in skin, heart, and other tissues.
Chymase, a chymotrypsin-like protease secreted by human mast cells, is generally considered to be a single enzyme. However, by heparin-agarose chromatography of high-salt extracts of human skin, we have consistently resolved three peaks of chymotryptic activity, eluting at 0.4 M NaCl (peak A), 1.0-1.2 M NaCl (peak B) and 1.8-2.0 M NaCl (peak C), with peak B containing 75-90% of the recovered activity. Each peak retained its identity upon rechromatography. The three peaks of activity were similar in substrate specificity and inhibitor profile and distinctly different from other chymotryptic enzymes, including cathepsin G and the stratum corneum chymotryptic enzyme. Examination of different tissues revealed that peak C was virtually absent from synovial tissue, was present as a minor component in skin and heart, but constituted the predominant chymotryptic activity in lung. Peaks B and C from skin tissue were further purified by chromatography on Sephacryl S-200. Both had a molecular mass of 28-29 kDa, yielded the N-terminal sequence reported for chymase, and on western blots reacted with a panel of polyclonal, monoclonal and antipeptide antibodies against chymase. Chymase C required higher concentrations of NaCl to overcome the stimulatory effects of heparin than did chymase B, but had a similar pH profile. Thus, human chymase exists in at least two distinct but similar forms, and the differences in heparin binding and tissue distribution could have important consequences for enzyme function.
Immunopharmacology Group, Southampton General Hospital, UK.
A R
AR
McEuen
M D
MD
Gaça
M G
MG
Buckley
S
S
He
M G
MG
Gore
A F
AF
Walls
D002846:Chromatography, Affinity / Q000379:methods; D053818:Chymases; D002918:Chymotrypsin / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry; D008407:Mast Cells / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology*; D017830:Octoxynol / Q000494:pharmacology; D009994:Osmolar Concentration; D011485:Protein Binding / Q000502:physiology; D012685:Sepharose / Q000031:analogs & derivatives / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry*; D012867:Skin / Q000201:enzymology*; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D007527:Isoenzymes; C037405:heparin-sepharose; D017830:Octoxynol; D006493:Heparin; D012685:Sepharose; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D053818:Chymases