Purification of an endogenous activator of procollagenase from rabbit synovial fibroblast culture medium.
Procollagenase and a latent activator of procollagenase were found in culture medium from rabbit synovial fibroblasts stimulated either with crystals of monosodium urate monohydrate or with phorbol myristate acetate. Procollagenase (pI 6.8) and activator (pI 5.48) could be separated by isoelectric focusing. Both procollagenase and the latent activator were purified to electrophoretic homogeneity by immunoadsorption chromatography. The activator was found to exist as a doublet of Mr = 53,400 and 51,900, with conversion to a lower Mr form upon treatment with either trypsin or 4-aminophenylmercuric acetate. In the absence of activator, purified procollagenase was not activated either by trypsin or by 4-aminophenylmercuric acetate, although both agents were capable of converting procollagenase to a lower Mr form. Activation of crude procollagenase was inhibited by immunoprecipitation of latent activator prior to addition of trypsin. Procollagenase activation increased with increased concentration of activator and with increased time of incubation with activator. The activator itself had no collagenolytic activity. The activation did not result in an apparent decrease in Mr of the procollagenase molecule as seen on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both high and low Mr procollagenase (trypsin-treated) could be activated by the activator. Purified activator, but not purified procollagenase, contained latent proteolytic activity against azocasein, gelatin, and reduced carboxymethylated bovine serum albumin substrates.
D000818:Animals; D002846:Chromatography, Affinity; D017364:Collagenases; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D005347:Fibroblasts / Q000201:enzymology; D005779:Immunodiffusion; D003012:Microbial Collagenase / Q000276:immunology / Q000378:metabolism*; D008970:Molecular Weight; D011506:Proteins / Q000302:isolation & purification*; D011817:Rabbits; D013582:Synovial Fluid / Q000166:cytology*
D004792:Enzyme Precursors; D011506:Proteins; D017364:Collagenases; C022440:procollagenase; D003012:Microbial Collagenase