Purification and characterization of a collagenase extracted from rabbit tumours.
A collagenase was purified from homogenates of V2 ascites-cell carcinoma growing in rabbit muscle. (NH4)2SO4 precipitation, ion-exchange and gel-filtration chromatography, and affinity chromatography (by using the CB7 CNBr) cleavage fragment of alpha 1(I) collagen linked to agarose) gave a 268000-fold purification and a sevenfold increase in total enzyme units recovered. The specific activity, defined as mumol of collagen in solution cleaved/h per mg of enzyme at 35 degrees C, WAS 1.74.2. The collagenase had a broad pH optimum from pH7.0 to 9.5, and a mol.wt. of between 33000 and 35000. It was inhibited by dithiothreitol, L-cysteine, D-penicillamine, EDTA and 1,10-phenanthroline, and by both rabbit and human serum. 3. Removal of cations by a chelating resin (Chelex 100) produced as inactive enzyme that could be reactiviated by the addition of Ca2+ ions at concentrations as low as 1muM. Other bivalent cations were not effective. 4. The purified collagenase cleaved peptides alpha2 and alpha1-CB7 (denatured polypeptides of collagen) at 37 degrees C at one site only. [alpha1 (I)]2alpha2 and [alpha1(III)]3 collagens in solution were cleaved at the same site approximately five times more rapidly than [alpha1 (II)]3. 5. An inhibitor of the enzyme in the tumour extracts, which was dissociable from the enzyme at the (NH4) 2SO4 precipitation step of purification, had a mol. wt. of between 40000 and 50000 but was distinct from the alpha1 trypsin inhibitor. 6. Studies with zonal density-gradient centrifugation suggested that the enzyme was bound to fibrillar substrate (collagen) extracellularly, but that it was not associated with enzymes originating in cell mitochondria, microsomal preparations or lysosomes.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002499:Centrifugation, Density Gradient; D002614:Chelating Agents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D008970:Molecular Weight; D009374:Neoplasms, Experimental / Q000201:enzymology*; D011817:Rabbits; D013583:Synovial Membrane / Q000201:enzymology
D002614:Chelating Agents; D003012:Microbial Collagenase; D002118:Calcium