Myocardial matrix metalloproteinase(s): localization and activation.
Matrix metalloproteinases (MMPs) and neutrophil elastase (NE) may each contribute to fibrillar collagen degradation in various disease states. Little, however, is known about the activation and localization of MMP in the heart. Accordingly, we extracted MMP and examined mechanisms of proMMP activation in whole tissue extracts of the adult rat myocardium. Incubation of extracts with serine proteases (i.e., trypsin or neutrophil elastase) at 37 degrees C resulted in a time-dependent activation of proMMPs. Based on immunoblot and measurements of MMP activity by zymography, the molecular weight of active MMP was deduced to be 52 kDa. The second-order rate constant for activation of proMMP by serine protease was 5.5 +/- 0.2 x 10(5) M-1min-1 and for oxidized glutathione (GSSG) 1.5 +/- 0.1 M-1min-1. Incubation of the extract with both serine protease and GSSG increased the rate of activation 30-fold. Based on reverse zymographic analysis of collagenase inhibition, tissue inhibitors of metalloproteinases were identified. Indirect immunofluorescence localized proMMPs/MMPs to the endothelium and subendothelial space of the endocardium and throughout the interstitial space found between groups of muscle fibers. These results suggest that the mechanism of activation of MMPs by either a serine protease and by oxidizing, thiol-modifying reagents are mechanistically different and the presence of either a serine protease or GSSG synergistically increase the rate of activation of proMMPs. Our results also suggest that MMPs may be regulated by its own endogenous inhibitors. The contribution of this proteolytic enzyme to tissue remodeling and wound healing responses that occur in various diseases states remains to be established.
Tyagi
S C
SC
Department of Internal Medicine, University of Missouri-Columbia 65212.
Ratajska
A
A
Weber
K T
KT
D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000378:metabolism; D005109:Extracellular Matrix / Q000201:enzymology* / Q000378:metabolism; D005260:Female; D005978:Glutathione / Q000494:pharmacology; D006023:Glycoproteins / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008666:Metalloendopeptidases / Q000187:drug effects / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology*; D051381:Rats; D012697:Serine Endopeptidases / Q000494:pharmacology; D019714:Tissue Inhibitor of Metalloproteinases
D004792:Enzyme Precursors; D006023:Glycoproteins; D019714:Tissue Inhibitor of Metalloproteinases; D012697:Serine Endopeptidases; D008666:Metalloendopeptidases; D005978:Glutathione