Activation of latent collagenase purified from human leukocytes.
Latent human leukocyte collagenase was isolated to apparent homogeneity by a simple and rapid method. Isolation was accomplished by gel filtration on Sepharose 6B and ion exchange chromatography on QAE Sephadex A-50 followed by affinity chromatography on Cibacron Blue Sepharose. The purified latent enzyme exhibits an apparent molecular weight of 70 kD as estimated by SDS-polyacrylamide gel electrophoresis. Reduction with dithiothreitol does not change the mobility of the latent human leukocyte collagenase on SDS-polyacrylamide gel electrophoresis, indicating that the enzyme consists of a single polypeptide chain. The enzyme could be activated by trypsin and thiol reagents such as phenylmercuric chloride and N-ethylmaleimide. Upon activation by trypsin a 54 kD polypeptide was formed from the 70 kD latent enzyme. Concomitant with the activation by thiol reagents, no loss of molecular weight was detected. Inactivated trypsin, i.e. phenylmethyl sulfonyl-trypsin or soybean trypsin inhibitor treated trypsin, was not able to activate latent human leukocyte collagenase. The results support the concept that latent human leukocyte collagenase exists as a proenzyme and thiol-dependent activation occurs through conformational perturbation in the proenzyme molecule.
D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006801:Humans; D003012:Microbial Collagenase / Q000302:isolation & purification* / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D012967:Sodium Dodecyl Sulfate; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D013438:Sulfhydryl Compounds; D012967:Sodium Dodecyl Sulfate; D014357:Trypsin; D003012:Microbial Collagenase