Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) from HT 1080 human fibrosarcoma cells. Purification and activation of the precursor and enzymic properties.
Matrix metalloproteinase 9 (MMP-9) has been purified as an inactive zymogen of M(r) 92,000 (proMMP-9) from the culture medium of HT 1080 human fibrosarcoma cells. The NH2-terminal sequence of proMMP-9 is Ala-Pro-Arg-Gln-Arg-Gln-Ser-Thr-Leu-Val-Leu-Phe-Pro, which is identical to that of the 92-kDa type IV collagenase/gelatinase. The zymogen can be activated by 4-aminophenylmercuric acetate, yielding an intermediate form of M(r) 83,000 and an active species of M(r) 67,000, the second of which has a new NH2 terminus of Met-Arg-Thr-Pro-Arg-(Cys)-Gly-Val-Pro-Asp-Leu-Gly-Arg-Phe-Gln-Thr- Phe-Glu. Immunoblot analyses demonstrate that this activation process is achieved by sequential processing of both NH2- and COOH-terminal peptides. TIMP-1 complexed with proMMP-9 inhibits the conversion of the intermediate form to the active species of M(r) 67,000. The proenzyme is fully activated by cathepsin G, trypsin, alpha-chymotrypsin, and MMP-3 (stromelysin 1) but not by plasmin, leukocyte elastase, plasma kallikrein, thrombin, or MMP-1 (tissue collagenase). During the activation by MMP-3, proMMP-9 is converted to an active species of M(r) 64,000 that lacks both NH2- and COOH-terminal peptides. In addition, HOCl partially activates the zymogen by reacting with an intermediate species of M(r) 83,000. The enzyme degrades type I gelatin rapidly and also cleaves native collagens including alpha 2 chain of type I collagen, collagen types III, IV, and V at undenaturing temperatures. These results indicate that MMP-9 has different activation mechanisms and substrate specificity from those of MMP-2 (72-kDa gelatinase/type IV collagenase).
Okada
Y
Y
Department of Pathology, School of Medicine, Kanazawa University, Japan.
Gonoji
Y
Y
Naka
K
K
Tomita
K
K
Nakanishi
I
I
Iwata
K
K
Yamashita
K
K
Hayakawa
T
T
D000595:Amino Acid Sequence; D015153:Blotting, Western; D056649:Cathepsin G; D002403:Cathepsins / Q000494:pharmacology; D002918:Chymotrypsin / Q000494:pharmacology; D017364:Collagenases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000302:isolation & purification* / Q000378:metabolism; D005354:Fibrosarcoma; D006023:Glycoproteins / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D019278:Matrix Metalloproteinase 3; D020780:Matrix Metalloproteinase 9; D061965:Matrix Metalloproteinase Inhibitors; D008666:Metalloendopeptidases / Q000494:pharmacology; D008969:Molecular Sequence Data; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D012697:Serine Endopeptidases; D013379:Substrate Specificity; D019714:Tissue Inhibitor of Metalloproteinases; D014357:Trypsin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D004792:Enzyme Precursors; D006023:Glycoproteins; D061965:Matrix Metalloproteinase Inhibitors; D019714:Tissue Inhibitor of Metalloproteinases; C010024:4-aminophenylmercuriacetate; D002403:Cathepsins; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C534094:CTSG protein, human; D056649:Cathepsin G; D014357:Trypsin; D017364:Collagenases; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D020780:Matrix Metalloproteinase 9; D010662:Phenylmercuric Acetate