cathepsin-g and 4-aminophenylmercuriacetate

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).

Topics: Amino Acid Sequence; Blotting, Western; Cathepsin G; Cathepsins; Chymotrypsin; Collagenases; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzyme Precursors; Fibrosarcoma; Glycoproteins; Humans; Kinetics; Matrix Metalloproteinase 3; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Metalloendopeptidases; Molecular Sequence Data; Phenylmercuric Acetate; Serine Endopeptidases; Substrate Specificity; Tissue Inhibitor of Metalloproteinases; Trypsin; Tumor Cells, Cultured

Neutrophils are known to contain several metalloproteinases that can damage collagen, a major structural component of the extracellular matrix. Here a neutrophil serine proteinase secreted from activated neutrophils was shown to cleave denatured collagen (gelatin). This serine proteinase was not inhibited by synthetic inhibitors of elastase (elastatinal or Me-O-suc-Ala-Pro-Val-CH2Cl). However, a synthetic inhibitor of cathepsin G (Z-Gly-Leu-Phe-CH2Cl) was able to inhibit the serine proteinase having gelatinolytic activity, indicating that cathepsin G, a major serine proteinase, from neutrophils is responsible for cleaving gelatin. Purified cathepsin G was also shown to degrade gelatin. In further experiments, oxidized glutathione was able to enhance the gelatinolytic activity of cathepsin G. These results show that cathepsin G is capable of cleaving denatured collagen, and its activity is enhanced or stabilized in the presence of glutathione. The data support the concept that cathepsin G released from neutrophils could play a major role in degrading collagen during inflammation and may in part account for the degradation of extracellular matrix during inflammation.

Topics: Animals; Cathepsin G; Cathepsins; Cells, Cultured; Chick Embryo; Collagen; Edetic Acid; Gelatin; Humans; Neutrophils; Phenylmercuric Acetate; Phenylmethylsulfonyl Fluoride; Serine Endopeptidases