Higher-order complex formation between the 72-kilodalton type IV collagenase and tissue inhibitor of metalloproteinases-2

• Published in: Biochemistry (Easton) Vol. 31; no. 6; pp. 1665 - 1672
• Main Authors: Kleiner, David E, Unsworth, Edward J, Krutzsch, Henry C, Stetler-Stevenson, William G
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 18-02-1992
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Biological and medical sciences; chemical properties; collagenase; complexes; Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzymes and enzyme inhibitors; formation; Fundamental and applied biological sciences. Psychology; Humans; Hydrolases; Macromolecular Substances; man; Matrix Metalloproteinase 9; Metalloendopeptidases - antagonists & inhibitors; Microbial Collagenase - antagonists & inhibitors; Microbial Collagenase - chemistry; Microbial Collagenase - metabolism; Molecular Sequence Data; Molecular Weight; Neoplasm Proteins - chemistry; Neoplasm Proteins - metabolism; Succinimides; tissue inhibition of metalloproteinase 2; Tissue Inhibitor of Metalloproteinase-2; Human; Stoichiometry; Enzyme; Quaternary structure; Enzyme inhibitor; Collagenase; Inhibitor enzyme complex; Metalloproteinase
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00121a013

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.