Purification and characterization of a two-chain form of tissue inhibitor of metalloproteinases (TIMP) type 2 and a low molecular weight TIMP-like protein.
Multiple forms of metalloproteinase inhibitors were found in the serum-free conditioned medium of the EJ-1 human bladder carcinoma cell line by reverse zymography assay with gelatinase A as the indicator enzyme. Two novel forms of inhibitor with apparent molecular masses of 18 and 22 kDa on nonreducing SDS-polyacrylamide gel electrophoresis (PAGE), together with tissue inhibitor of metalloproteinases (TIMP) and TIMP-2, were purified from the conditioned medium by a series of chromatographic steps. Structural analysis showed that the 18-kDa inhibitor is a two-chain form of TIMP-2 (tc-TIMP-2) produced by proteolytic processing, and the 22-kDa inhibitor may be a partially glycosylated form of TIMP. The purified tc-TIMP-2 was separated into a 17-kDa peptide and a small peptide of about 2.5 kDa by reducing SDS-PAGE and into four isoforms with pI 7.6, 7.3, 7.2, and 6.8 by isoelectric focusing. tc-TIMP-2 has essentially the same inhibitory activity as TIMP-2 toward gelatinase A, collagenase, stromelysin, and matrilysin. Unlike TIMP-2, however, tc-TIMP-2 does not bind to the latent precursor fo gelatinase A. Similar two-chain forms of TIMP-2 were produced by its partial digestion with trypsin or less effectively with plasmin. These results suggest that proteolytic processing of TIMP-2 plays a role in the regulation of gelatinase A activity in the extracellular matrix.
Miyazaki
K
K
Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Japan.
Funahashi
K
K
Numata
Y
Y
Koshikawa
N
N
Akaogi
K
K
Kikkawa
Y
Y
Yasumitsu
H
H
Umeda
M
M
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D017077:Culture Media, Conditioned; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D006801:Humans; D007525:Isoelectric Focusing; D020778:Matrix Metalloproteinase 2; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D008961:Models, Structural; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology; D010446:Peptide Fragments / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D019716:Tissue Inhibitor of Metalloproteinase-2; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms
D017077:Culture Media, Conditioned; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D019716:Tissue Inhibitor of Metalloproteinase-2; D010450:Endopeptidases; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2