Partial purification and characterization of gelatinase and metal dependent peptidase from rabbit uterus and their synergistic action on gelatin in vitro.
Two metal dependent proteases were investigated in rabbit uterus using a synthetic substrate, 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gin-D-Arg (Dnp-peptide). One was extracted by homogenization in 50 mM Tris-HCl/0.25% Triton X-100/100 mM CaCl2, pH 7.4, from rabbit uterus, and the other from the insoluble fraction by heating at 60 degrees C for 4 min in 50 mM Tris-HCl/100 mM CaCl2, pH 7.4. Both enzymes were partially purified by gel filtration, ion-exchange chromatography and chromatofocusing, and further characterized. The soluble enzyme was a metal dependent peptidase, and hydrolysed 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg as well as Dnp-peptide. Its molecular weight was about 7.0 X 10(4), and the cleavage sites for Dnp-peptide were Gln-Gly and possibly Gly-Ile in the ratio of 3:1. On the other hand, the enzyme extracted from the insoluble fraction was present as a latent form, and was found to be activated by 4-aminophenylmercuric acetate but not by trypsin. The activated enzyme hydrolysed gelatin, in addition to Dnp-peptide, indicating that the enzyme is a gelatinase. The molecular weight was about 7.4 X 10(4) for the active form, and the cleavage site for Dnp-peptide was only the Gly-Ile bond. The rabbit uterine metal dependent peptidase obtained here had negligible activity on gelatin, but once it had been cleaved by the above gelatinase, the presence of metal dependent peptidase accelerated the action of gelatinase. Thus, the actions of both enzymes on gelatin were found to be synergistic.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D005260:Female; D005780:Gelatin; D018093:Gelatinases; D007700:Kinetics; D008666:Metalloendopeptidases; D008970:Molecular Weight; D010434:Pepsin A / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D013379:Substrate Specificity; D014599:Uterus / Q000201:enzymology*
D005780:Gelatin; D010450:Endopeptidases; D010434:Pepsin A; D018093:Gelatinases; D008666:Metalloendopeptidases