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TIMP-1 is a member of the family of tissue inhibitors of metalloproteinases involved in regulating the activity of extracellular matrix degrading metalloproteinases. The TIMP-1 cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR) amplification of the corresponding mRNA from human fibroblasts. Cloning and expression of the TIMP-1 cDNA were performed in Escherichia coli. In the host vector system chosen, rTIMP-1 is stored intracellularly in its denatured, insoluble form in inclusion bodies. We report a new method for the purification and renaturation of rTIMP-1 from E. coli inclusion bodies to an active inhibitor of matrix metalloproteinases (80% yield), presumably containing the correct assignment of the six disulfide bonds. A resin with the covalently bound recombinant catalytic domain of the PMNL-collagenase as the affinity ligand provided an effective means for the separation of correctly folded, active rTIMP-1 from inactive forms with mismatched disulfides. TIMP-1 and TIMP-2, the two most extensively examined members of the family of tissue inhibitors of metalloproteinases, are known to form a complex with the activated forms of most matrix metalloproteinases and the latent forms of the 92-kDa and 72-kDa gelatinases, respectively. In this study, we report on the complex formation of the recombinant catalytic domain of the PMNL-collagenase with TIMP-1, nonglycosylated recombinant TIMP-1, and recombinant TIMP-2. The Ki values for the different inhibitors were determined in a kinetic assay using a fluorogenic substrate peptide. In this assay, rTIMP-2 had a more effective inhibitory capability against the recombinant catalytic domain of the PMNL-collagenase than TIMP-1.(ABSTRACT TRUNCATED AT 250 WORDS)

A tissue inhibitor of metalloproteinases (T1MP)-1 was isolated from human polymorphonuclear leukocytes (PMNL) in a complex with latent 95-kDa gelatinase (matrixmetalloproteinase, . It was separated from the enzyme by gel fitration in the presence of SDS. Using a competitive ELISA procedure, we determined that 10% of the isolated gelatinase was complexed with TIMP-1. The presence of the inhibitor in isolated PMNL could also be demonstrated by indirect immunofluorescence using a specific antibody against TIMP-1. Cellular mRNA was isolated from PMNL, which were highly purified by separation via formylMet-Leu-Pro-stimulated chemotactic migration in a Boyden chamber. Using reversetranscription PCR and Nothern blotting, TIMP-1 mRNA was shown to be present in PMNL, suggesting that these cells are also capable of synthesizing TIMP-1.Keywords. Tissue inhibitor of metalloproteinases (TIMP-1) ; neutrophils ; metalloproteinase.Human polymorphonuclear leukocytes (PMNL) are phagocytic cells with a short life span, estimated to be 24-48 h after release from the bone marrow. They are the primary effector cells in sites of acute inflammatory processes, in which they move along a chemotactic gradient from the bloodstream to the site of inflammation. To penetrate the vessel walls and tissue barrier, they secrete specific proteinases which are stored in their granules. Matrix metalloproteinase (MMP)-9, also called gelatinase B, is assumed to be the required proteinase for leukodiapedesis Bakowski et al., 1992). Gelatinase B is a member of the matrix metalloproteinase family and, amongst other substrates, cleaves type-IV collagen, the main component of basement membranes.The matrix metalloproteinases are secreted as zymogens which undergo extracellular activation. Specific inhibitors exert control over the active enzymes in the extracellular matrix. Two tissue inhibitors of metalloproteinases (TIMPs) are so far known. TIMP-1 is an 28.5-kDa glycoprotein secreted by many cell types, including fibroblasts and macrophages. TIMP-2 is an unglycosylated 21 -kDa protein with significant sequence similarity to TIMP-1. These inhibitors form stoichiometric 1 : 1 complexes with active metalloproteinases (Docherty et al., 1992).In addition to these complexes of the active enzyme with its inhibitor, complexes between the latent 72-kDa gelatinase (MMP-2) and the latent 95-kDa gelatinase (MMP-9) with TIMP-2 and TIMP-1, respectively, have been isolated, which can still be activated by mercurial compounds, as can the noncomplexed latent enzymes. The physiological role and significance of these latent enzyme-inhibitor complexes in vivo is unknown. In human blood cells, TIMP-1 was only detected in platelets and megakaryocytes (Cooper et al., 1985) and macrophages and monocytes (Opdenakker et al., 1991).Here, we report the isolation of TIMP-1 in the free form and in complex with 95-kDa gelatinase from human PMN leukocytes. The detection of TIMP-1 transcripts in highly purified PMNL provided evidence that these cells are capable of regulating metalloprot...

A tissue inhibitor of metalloproteinases (T1MP)-1 was isolated from human polymorphonuclear leukocytes (PMNL) in a complex with latent 95-kDa gelatinase (matrixmetalloproteinase, . It was separated from the enzyme by gel fitration in the presence of SDS. Using a competitive ELISA procedure, we determined that 10% of the isolated gelatinase was complexed with TIMP-1. The presence of the inhibitor in isolated PMNL could also be demonstrated by indirect immunofluorescence using a specific antibody against TIMP-1. Cellular mRNA was isolated from PMNL, which were highly purified by separation via formylMet-Leu-Pro-stimulated chemotactic migration in a Boyden chamber. Using reversetranscription PCR and Nothern blotting, TIMP-1 mRNA was shown to be present in PMNL, suggesting that these cells are also capable of synthesizing TIMP-1.

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