The purification of tissue inhibitor of metalloproteinases-2 from its 72 kDa progelatinase complex. Demonstration of the biochemical similarities of tissue inhibitor of metalloproteinases-2 and tissue inhibitor of metalloproteinases-1.
Human gingival fibroblasts in culture were shown to secrete a 72 kDa progelatinase, of which a proportion in the medium was found to be complexed with tissue inhibitor of metalloproteinases-2 (TIMP-2). A purification procedure was devised to purify free enzyme and inhibitor. We also describe the purification of both 95 kDa progelatinase bound to TIMP-1 and free 95 kDa progelatinase from the medium of U937 cells. A polyclonal antiserum to TIMP-2 was prepared and it was shown that TIMP-1 and TIMP-2 are antigenically distinct. The ability to form stable complexes and the relative inhibitory activities of TIMP-1 and TIMP-2 towards 95 kDa and 72 kDa gelatinases, collagenase, stromelysins 1 and 2 and punctuated metalloproteinase were determined; only minor differences were found. Complex-formation between TIMP-2 and 72 kDa progelatinase was demonstrated not to reduce the metalloproteinase-inhibitory activity of TIMP-2, a finding that led to the characterization of high-molecular-mass TIMP activity. Competition experiments between progelatinases and active gelatinases for TIMPs indicated that the affinity of TIMPs for progelatinases is weaker than that for active gelatinases. In a study of the effects of TIMP-1 and TIMP-2 on progelatinase self-cleavage we found that both TIMP-1 and TIMP-2 inhibit the conversion of 95 kDa and 72 kDa progelatinases and prostromelysin into lower-molecular-mass forms. TIMP capable of complexing with progelatinase was shown to be no more efficient an inhibitor of gelatinase self-cleavage than TIMP not able to complex with progelatinase.
Ward
R V
RV
Department of Cell and Molecular Biology, Strangeways Research Laboratory, Cambridge, U.K.
Hembry
R M
RM
Reynolds
J J
JJ
Murphy
G
G
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002478:Cells, Cultured; D004492:Edetic Acid / Q000494:pharmacology; D004792:Enzyme Precursors / Q000302:isolation & purification / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D018093:Gelatinases; D005881:Gingiva / Q000378:metabolism; D006023:Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D010434:Pepsin A / Q000302:isolation & purification / Q000378:metabolism*; D019716:Tissue Inhibitor of Metalloproteinase-2; D019714:Tissue Inhibitor of Metalloproteinases
D004792:Enzyme Precursors; D006023:Glycoproteins; D009363:Neoplasm Proteins; D019714:Tissue Inhibitor of Metalloproteinases; D019716:Tissue Inhibitor of Metalloproteinase-2; D004492:Edetic Acid; D010434:Pepsin A; D018093:Gelatinases; D008666:Metalloendopeptidases; C069860:progelatinase