Isolation and properties of a metal-dependent endopeptidase from human uterus hydrolysing synthetic collagenase substrates.
A metal-dependent peptidase was isolated from the homogenate of human uterus by standard chromatographic techniques and purified to apparent homogeneity. The peptidase hydrolysed the synthetic vertebrate collagenase substrate 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg (Dnp-peptide), the synthetic bacterial collagenase substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (PZ-peptide) and gelatinolytic peptides of gelatin, but was inactive against collagen type I, gelatin and casein. The cleavage site for the Dnp-peptide was the Gly-Ile bond. The enzyme was not only inhibited by metal chelators, such as EDTA, 1,10-phenantroline and dithiothreitol but also by thiol reagents, such as mersalylic acid and N-ethylmaleimid. However, E-64, an inhibitor for thiolproteinases, and leupeptin, an inhibitor for thiol- and serine proteases, did not exhibit any inhibitory activity. Pepstatin, an inhibitor for aspartate proteinases, and inhibitors for serine proteinases like phenylmethanesulfonyl fluoride and Trasylol were ineffective as well. The purified peptidase displayed a single band in the SDS-PAGE with an apparent molecular mass of 65 kDa. Employing isoelectric focusing an IP of 5.0 could be determined. The enzyme's properties are discussed in relation to the proteinase EC 3.4.24.11 and to proteinases of the collagenase family as well as the possibility to discriminate these three metalloproteinase classes by employing the Dnp-peptide.
Kolkenbrock
H
H
Institut für Biochemie, Freie Universität Berlin.
Wieder
T
T
Ulbrich
N
N
D002852:Chromatography, Ion Exchange; D004140:Dinitrophenols / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007525:Isoelectric Focusing; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000302:isolation & purification; D003012:Microbial Collagenase / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism*; D010618:Phenanthrolines / Q000494:pharmacology; D013379:Substrate Specificity; D014599:Uterus / Q000201:enzymology*
D004140:Dinitrophenols; D009842:Oligopeptides; D010618:Phenanthrolines; C043091:PZ-peptide; C024118:DNP-peptide; D004492:Edetic Acid; D008666:Metalloendopeptidases; D003012:Microbial Collagenase; D004229:Dithiothreitol; C025205:1,10-phenanthroline