Tryptase in rat mast cells: properties and inhibition by various inhibitors in comparison with chymase.
Previous studies with trans-4-(guanidinomethyl)cyclohexanecarboxylic acid 4-tert-butylphenyl ester (GMCHA-OPhBut), a trypsin inhibitor, strongly suggested the involvement of a trypsin-like protease in histamine release from mast cells induced by various secretagogues (Takei, M., Matumoto, T., Endo, K. & Muramatu, M. (1988) Agents and Actions, in press; Takei, M., Matumoto, T., Ito, T., Endo, K. & Muramatu, M.; Takei, M., Matumoto, T., Endo, K. & Muramatu, M. and Takei, M., Matumoto, T., Urashima, H., Endo, K. & Muramatu, M., unpublished results). Two serine proteases, chymase (Benditt, E.F. & Arase, M. (1959) J. Exp. Med. 110, 451-460) and tryptase Kido, H., Fukusen, N. & Katunuma, N. (1985) Arch. Biochem. Biophys. 239, 436-443) were demonstrated in rat peritoneal mast cells. Both enzymes were purified and the effects of inhibitors for trypsin and chymotrypsin on these proteases were examined. The trypsin-like protease was found in saline extract and purified by successive chromatographies on Sephadex G-100 and DEAE-cellulose columns. The molecular mass of this protease was apparently 120,000 Da. This protease showed maximal activity at pH 7.1 and was named pH 7 tryptase. Chymase was obtained from 1.5M NaCl extract. pH 7 Tryptase markedly hydrolysed Boc-Phe-Ser-Arg-NH-Mec and Boc-Val-Pro-Arg-NH-Mec among the various substrates containing arginyl and lysyl bonds but did not cleave Tos-Arg-OMe. Tos-Lys-CH2Cl and diisopropylfluorophosphate strongly inhibited this protease. Various inhibitors for trypsin inhibited pH 7 tryptase, and those for chymotrypsin inhibited chymase. Among the esters of GMCHA examined, GMCHA-OPhBut most strongly and competitively inhibited pH 7 tryptase but it had no effect on chymase.
Muramatu
M
M
Faculty of Pharmacy, Tokushima Bunri University.
Itoh
T
T
Takei
M
M
Endo
K
K
D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D053818:Chymases; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D008407:Mast Cells / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D011480:Protease Inhibitors; D015842:Serine Proteinase Inhibitors; D010447:Peptide Hydrolases; C023073:tosylarginine methyl ester hydrolase; D012697:Serine Endopeptidases; D053818:Chymases