Partial purification and characterization of latent human leukocyte collagenase.
Latent and active collagenase were extracted from human polymorphonuclear leukocytes. Separation of the two forms of the enzyme was performed by gel filtration on Sepharose 6 B. The latent form of the enzyme was detected from chromatographic fractions after a brief treatment with trypsin or exposure of the fractions to the sulfhydryl reagent phenylmercuric chloride. Latent enzyme eluted before active enzyme from the column, indicating a higher apparent molecular weight. Partially purified latent enzyme exhibited an apparent molecular size of 70-75 kDa as estimated by gel filtration. A value of 50-55 kDa was obtained for active enzyme. Without activation the latent enzyme did not degrade soluble collagen substrate. This was demonstrated by a quantitative viscometric assay and also by sodium dodecyl sulfate polyacrylamide gel electrophoresis, when no typical cleavage products of collagen could be seen. Latent enzyme could not be obtained unless serine protease inhibitors were present during the extraction and purification procedures. The effects of the activators trypsin, phenylmercuric chloride, phenylmethyl sulfonyltrypsin, and N-ethylmaleimide on the latent human polymorphonuclear leukocyte collagenase were studied. Contrary to the suggestion that inactive proteases activate latent human polymorphonuclear leukocyte collagenase, the inactive phenylmethyl sulfonyl-trypsin could not activate latent collagenase.
D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006801:Humans; D003012:Microbial Collagenase / Q000097:blood / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology* / Q000502:physiology; D010663:Phenylmercury Compounds / Q000494:pharmacology; D013450:Sulfones / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D010663:Phenylmercury Compounds; D013450:Sulfones; C049168:phenylmethylsulfonyl-trypsin; D014357:Trypsin; D003012:Microbial Collagenase; C008859:phenylmercuric chloride