The enzymatic evaluation of procollagenase and collagenase inhibitors in crude biological media.
The validity of the enzymatic assay of procollagenase within crude biological media containing also the collagenase inhibitor TIMP (tissue inhibitor of metalloproteinases) as well as other (pro)metalloproteinases and sometimes, metalloproteinase-TIMP complexes, has been reevaluated. To be enzymatically assayed, procollagenase has to be activated. The standard activation procedures by either trypsin or 4-aminophenylmercuric acetate (APMA) both allow an optimal recovery of collagenase from procollagenase when the media do not contain free TIMP. However, they do not destroy TIMP nor do they reactivate the collagenase present in enzyme-inhibitor complexes. Therefore, the collagenase formed by the activation of procollagenase in the presence of free TIMP is immediately inactivated by binding to the inhibitor. As a result, both the bound collagenase and TIMP can no longer be assayed by enzymatic methods. An optimal recovery of collagenase can, however, be obtained if free TIMP is neutralized by the binding of other tissue metalloproteinases (such as those present in culture media of rabbit bone marrow-derived macrophages) prior to the activation and assay of procollagenase. Similarly, it is possible to recover under an active free form a large part of the TIMP present in collagenase- (or other metalloproteinase-)TIMP complexes by heating the complexes at acid pH under conditions which inactivate the collagenase.
Lefebvre
V
V
Laboratoire de Chimie Physiologique, Université de Louvain, Brussels, Belgium.
Vaes
G
G
D000818:Animals; D002358:Cartilage, Articular / Q000032:analysis*; D002478:Cells, Cultured; D017364:Collagenases; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000494:pharmacology; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007700:Kinetics; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D011817:Rabbits; D012867:Skin / Q000032:analysis*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013582:Synovial Fluid / Q000166:cytology*; D019714:Tissue Inhibitor of Metalloproteinases; D014357:Trypsin / Q000494:pharmacology
D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D013439:Sulfhydryl Reagents; D019714:Tissue Inhibitor of Metalloproteinases; C010024:4-aminophenylmercuriacetate; D014357:Trypsin; D017364:Collagenases; D008666:Metalloendopeptidases; C022440:procollagenase; D003012:Microbial Collagenase; D010662:Phenylmercuric Acetate