Neutral metalloprotease from tendons.
Tendon repair following trauma, rupture, or surgery involves both synthesis and degradation of collagen in order to reweave new collagen bundles in with the old. Using an in situ assay on polyacrylamide gels containing gelatin, we have identified protease activity from tendon tissue and from tendon cells in culture. A population of synovial cells from the epitenon surrounding the tendon as well as the tendon fibroblasts themselves were examined. The cells and the conditioned medium from both cell populations exhibited a major band of gelatin-degrading activity at 70 kdaltons and a minor band of activity at 60 kdaltons. When preparations were reacted with p-aminophenylmercuric acetate (APMA) before electrophoresis, a third band appeared at 63 kdaltons. The main band at 70 kdaltons comigrated with a [35S]methionine-radiolabeled protein band. Inhibitor and pH studies identified the enzymes as neutral metalloproteases requiring disulfide bonds for activity. No proteolytic activity was detected on casein-containing gels, ruling out the presence of stromelysin. Since electrophoresis in the presence of SDS would separate the metalloprotease from the smaller molecular weight inhibitor (TIMP), these in situ assays provide a sensitive screening system for gelatin-degrading enzymes present in tendon without prior removal of TIMP.
Piening
C
C
Department of Orthopaedics, University of Washington, Seattle 98195.
Riederer-Henderson
M A
MA
D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D004591:Electrophoresis, Polyacrylamide Gel; D018093:Gelatinases; D008666:Metalloendopeptidases / Q000378:metabolism*; D003012:Microbial Collagenase / Q000378:metabolism; D010434:Pepsin A / Q000378:metabolism; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives; D013439:Sulfhydryl Reagents; D013582:Synovial Fluid / Q000166:cytology / Q000201:enzymology; D013710:Tendons / Q000166:cytology / Q000201:enzymology*
D013439:Sulfhydryl Reagents; C010024:4-aminophenylmercuriacetate; D010434:Pepsin A; D018093:Gelatinases; D008666:Metalloendopeptidases; D003012:Microbial Collagenase; D010662:Phenylmercuric Acetate