title: The inactivation of the cysteinyl exopeptidases cathepsin H and C by affinity-labelling reagents.
abstract: An attempt has been made to extend to the cysteinyl exopeptidases cathepsins H and C affinity-labelling approaches shown to be effective with cysteinyl endopeptidases such as cathepsins B and L and the calcium-activated proteinase. This involved the preparation of amino acid and dipeptide derivatives with unblocked N-termini to satisfy the aminopeptidase and dipeptidyl aminopeptidase characteristics of cathepsins H and C respectively. For covalent reactivity, the possibilities examined included diazomethanes (-CHN2), fluoromethanes (-CH2F) and dimethylsulphonium salt [-CH2S+(CH3)2]. A dipeptidylfluoromethane with a free amino group could not be prepared, perhaps due to inherent instability. Cathepsin H was inactivated by 1 microM-H2N-Phe-CH2F (the 'H2N' indicates a free unblocked amino group) (k2 = 1878 M-1.s-1); this reagent was without effect on cathepsins C and B, even at 100-fold this concentration. Analogous selectivity was shown by H2N-Ser(OBzl)-CHN2 and H2N-Phe-CH2S+(CH3)2, members of other classes of covalently binding reagents. For cathepsin C the dipeptide derivatives H2N-Gly-Phe-CHN2 and H2N-Phe-Ala-CH2S+(CH3)2 caused rapid inactivation near 10(-7) M. Higher concentrations inactivated cathepsins H and B, but the rates were slower by two to three orders of magnitude than for cathepsin C.
authors.affiliation: Friedrich Miescher-Institut, Basel, Switzerland.
authors.forename: H
authors.initials: H
authors.lastname: Angliker
authors.forename: P
authors.initials: P
authors.lastname: Wikstrom
authors.forename: H
authors.initials: H
authors.lastname: Kirschke
authors.forename: E
authors.initials: E
authors.lastname: Shaw
mesh_terms: D000345:Affinity Labels / Q000138:chemical synthesis / Q000494:pharmacology*; D000818:Animals; D002401:Cathepsin B / Q000037:antagonists & inhibitors; D020789:Cathepsin C; D056655:Cathepsin H; D002403:Cathepsins / Q000037:antagonists & inhibitors*; D003546:Cysteine Endopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000037:antagonists & inhibitors*; D004305:Dose-Response Relationship, Drug
chemical_list: D000345:Affinity Labels; D002403:Cathepsins; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D020789:Cathepsin C; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; D056655:Cathepsin H