Higher-order complex formation between the 72-kilodalton type IV collagenase and tissue inhibitor of metalloproteinases-2.
The collagenases are a class of matrix degradative enzymes whose actions are important in physiological and pathological processes. The human 72-kDa type IV collagenase (matrix metalloproteinase-2) and its proteinase inhibitor, tissue inhibitor of metalloproteinases-2 (TIMP-2), are produced as a proenzyme-inhibitor complex by numerous cell lines. We analyzed the quaternary structure of and enzyme-inhibitor interactions in the native enzyme-inhibitor complex by studying the pattern of complexes demonstrated by molecular weight determination in nondenaturing polyacrylamide gels and evaluating the products formed by reaction of the native complexes with cross-linking agents. Electrophoresis in native polyacrylamide gels demonstrates that approximately 79% of the latent enzyme is present in a 1:1 bimolecular complex with the inhibitor TIMP-2, with 21% present as a complete tetrameric complex of two molecules of collagenase combined with two molecules of TIMP-2. The enzyme complex activated with organomercurials displays a shift to a higher proportion of the bimolecular complex with only 5% present as higher molecular weight complexes. Cross-linking of the latent and active forms of the complex with bis(sulfosuccinimidyl) suberate (BS3) and bis(sulfosuccinimidyl) tartarate demonstrates both the 1:1 and 2:2 complexes as well as an intermediate form that appears to be a complex composed of two molecules of collagenase and one of TIMP-2. The distribution of cross-linked products is unchanged with the addition of excess TIMP-2 to the reaction mix, implying that the binding sites for TIMP-2 to the initial enzyme-inhibitor complex are all occupied when the stoichiometry is 1 to 1.(ABSTRACT TRUNCATED AT 250 WORDS)
Kleiner
D E
DE
Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland 20892.
Unsworth
E J
EJ
Krutzsch
H C
HC
Stetler-Stevenson
W G
WG
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006801:Humans; D046911:Macromolecular Substances; D020780:Matrix Metalloproteinase 9; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism*; D013388:Succinimides; D019716:Tissue Inhibitor of Metalloproteinase-2
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D009363:Neoplasm Proteins; D013388:Succinimides; C072854:bis(sulfosuccinimidyl)tartrate; D019716:Tissue Inhibitor of Metalloproteinase-2; C035760:bis(sulfosuccinimidyl)suberate; D008666:Metalloendopeptidases; D003012:Microbial Collagenase; D020780:Matrix Metalloproteinase 9