Phage display of tissue inhibitor of metalloproteinases-2 (TIMP-2): identification of selective inhibitors of collagenase-1 (metalloproteinase 1 (MMP-1))

Tissue inhibitor of metalloproteinases-2 (TIMP-2) is a broad spectrum inhibitor of the matrix metalloproteinases (MMPs), which function in extracellular matrix catabolism. Here, phage display was used to identify variants of human TIMP-2 that are selective inhibitors of human MMP-1, a collagenase whose unregulated action is linked to cancer, arthritis, and fibrosis. Using hard randomization of residues 2, 4, 5, and 6 (L1) and soft randomization of residues 34-40 (L2) and 67-70 (L3), a library was generated containing 2 × 10(10) variants of TIMP-2. Five clones were isolated after five rounds of selection with MMP-1, using MMP-3 as a competitor. The enriched phages selectively bound MMP-1 relative to MMP-3 and contained mutations only in L1. The most selective variant (TM8) was used to generate a second library in which residues Cys(1)-Gln(9) were soft-randomized. Four additional clones, selected from this library, showed a similar affinity for MMP-1 as wild-type TIMP-2 but reduced affinity for MMP-3. Variants of the N-terminal domain of TIMP-2 (N-TIMP-2) with the sequences of the most selective clones were expressed and characterized for inhibitory activity against eight MMPs. All were effective inhibitors of MMP-1 with nanomolar K(i) values, but TM8, containing Ser(2) to Asp and Ser(4) to Ala substitutions, was the most selective having a nanomolar K(i) value for MMP-1 but no detectable inhibitory activity toward MMP-3 and MMP-14 up to 10 μM. This study suggests that phage display and selection with other MMPs may be an effective method for discovering tissue inhibitor of metalloproteinase variants that discriminate between specified MMPs as targets.     

Designing TIMP (tissue inhibitor of metalloproteinases) variants that are selective metalloproteinase inhibitors

The tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of the matrix metalloproteinases (MMPs), enzymes that play central roles in the degradation of extracellular matrix components. The balance between MMPs and TIMPs is important in the maintenance of tissues, and its disruption affects tissue homoeostasis. Four related TIMPs (TIMP-1 to TIMP-4) can each form a complex with MMPs in a 1:1 stoichiometry with high affinity, but their inhibitory activities towards different MMPs are not particularly selective. The three-dimensional structures of TIMP-MMP complexes reveal that TIMPs have an extended ridge structure that slots into the active site of MMPs. Mutation of three separate residues in the ridge, at positions 2, 4 and 68 in the amino acid sequence of the N-terminal inhibitory domain of TIMP-1 (N-TIMP-1), separately and in combination has produced N-TIMP-1 variants with higher binding affinity and specificity for individual MMPs. TIMP-3 is unique in that it inhibits not only MMPs, but also several ADAM (a disintegrin and metalloproteinase) and ADAMTS (ADAM with thrombospondin motifs) metalloproteinases. Inhibition of the latter groups of metalloproteinases, as exemplified with ADAMTS-4 (aggrecanase 1), requires additional structural elements in TIMP-3 that have not yet been identified. Knowledge of the structural basis of the inhibitory action of TIMPs will facilitate the design of selective TIMP variants for investigating the biological roles of specific MMPs and for developing therapeutic interventions for MMP-associated diseases.     

Engineered sarafotoxins as tissue inhibitor of metalloproteinases-like matrix metalloproteinase inhibitors

The sarafotoxins and endothelins are approximately 25-residue peptides that spontaneously fold into a defined tertiary structure with specific pairing of four cysteines into two disulfide bonds. Their structures show an interesting topological similarity to the core of the metalloproteinase interaction sites of the tissue inhibitors of metalloproteinases. Previous work indicates that sarafotoxins and endothelins can be engineered to eliminate or greatly reduce their vasopressive action and that their structural framework can withstand multiple sequence changes. When sarafotoxin 6b, which possesses modest matrix metalloproteinase inhibitory activity, was C-terminally truncated to remove its toxic vasopressive activity, the metalloproteinase inhibitory activity was essentially abolished. However, further changes, based on the sequences of peptides selected from libraries of sarafotoxin variants or suggested by analogy with tissue inhibitors of metalloproteinases, progressively enhanced the matrix metalloproteinase inhibitory activity. Peptide variants with multiple substitutions folded correctly and formed native disulfide bonds. Improvements in matrix metalloproteinase affinity have generated a peptide with micromolar K(i) values for matrix metalloproteinase-1 and -9 that are selective inhibitors of different metalloproteinases. Characterization of its solution structure indicates a close similarity to sarafotoxin but with a more extended C-terminal helix. The effects of N-acetylation and other changes, as well as docking studies, support the hypothesis that the engineered sarafotoxins bind to matrix metalloproteinases in a manner analogous to the tissue inhibitors of metalloproteinases.     

The N-terminal domain of tissue inhibitor of metalloproteinases retains metalloproteinase inhibitory activity

Recombinant tissue inhibitor of metalloproteinases (TIMP-1) and a truncated version containing only the three N-terminal loops, delta 127-184TIMP, have been expressed in myeloma cells and purified by affinity chromatography and gel filtration. delta 127-184TIMP was found to exist as two main glycosylation variants of molecular mass 24 kD and 19.5 kDa and an unglycosylated form of 13 kDa. All forms of the truncated inhibitor were able to inhibit and form complexes with active forms of the matrix metalloproteinases, indicating that the major structural features for specific interaction with these enzymes resides in these three loops. Stable binding of delta 127-184TIMP to pro 95-kDa gelatinase was not demonstrable under the conditions for binding of full-length TIMP-1.     

The effect of matrix metalloproteinase complex formation on the conformational mobility of tissue inhibitor of metalloproteinases-2 (TIMP-2)

The backbone mobility of the N-terminal domain of tissue inhibitor of metalloproteinases-2 (N-TIMP-2) was determined both for the free protein and when bound to the catalytic domain of matrix metalloproteinase-3 (N-MMP-3). Regions of the protein with internal motion were identified by comparison of the T(1) and T(2) relaxation times and (1)H-(15)N nuclear Overhauser effect values for the backbone amide (15)N signals for each residue in the sequence. This analysis revealed rapid internal motion on the picosecond to nanosecond time scale for several regions of free N-TIMP-2, including the extended beta-hairpin between beta-strands A and B, which forms part of the MMP binding site. Evidence of relatively slow motion indicative of exchange between two or more local conformations on a microsecond to millisecond time scale was also found in the free protein, including two other regions of the MMP binding site (the CD and EF loops). On formation of a tight N-TIMP-2. N-MMP-3 complex, the rapid internal motion of the AB beta-hairpin was largely abolished, a change consistent with tight binding of this region to the MMP-3 catalytic domain. The extended AB beta-hairpin is not a feature of all members of the TIMP family; therefore, the binding of this highly mobile region to a site distant from the catalytic cleft of the MMPs suggests a key role in TIMP-2 binding specificity.     

Clinical significance of serum levels of matrix metalloproteinase 2 (MMP-2) and its tissue inhibitor (TIMP-2) in gastric cancer

Matrix metalloproteinase 2 (MMP-2) is able to degrade type IV collagen, and thus plays a key role in the migration of tumor cells. MMP-2 activity is inhibited by its tissue inhibitor (TIMP-2). The imbalance between MMPs and TIMPs may facilitate progression of cancer cells. The aim of this study was to compare the clinical importance of MMP-2 and TIMP-2 to that of classical tumor markers, namely carcinoembryonic antigen (CEA) and carbohydrate antigen (CA 19-9) in the diagnosis of gastric cancer (GC) by calculating the diagnostic criteria and estimating the levels of MMP-2, TIMP-2, CEA and CA 19-9 in GC patients in relation to clinicopathological features of cancer. We found that serum levels of MMP-2 and TIMP-2 were significantly lower, whereas serum tumor markers were higher, in GC patients than in healthy subjects. Moreover, concentrations of TIMP-2 and CEA correlated with gastric wall infiltration, while CA 19-9 levels correlated with gastric wall infiltration and the presence of nodal metastasis. None of the proteins tested was found to be an independent prognostic factor for GC patients' survival. The percentage of true positive results of TIMP-2 (61%) was higher than those of MMP-2 (54%) and the classical tumor markers CEA (21%) and CA 19-9 (31%). The highest diagnostic sensitivity was observed for the combined use of TIMP-2 with MMP-2 (77%). The results suggest the greater importance of serum MMP-2 and TIMP-2 than of the classical tumor markers CEA and CA 19-9 in the diagnosis of GC. But this issue requires further investigation.     

Hyaluronan synthase 2 (HAS2) promotes breast cancer cell invasion by suppression of tissue metalloproteinase inhibitor 1 (TIMP-1)

Invasion and metastasis are the primary causes of breast cancer mortality, and increased knowledge about the molecular mechanisms involved in these processes is highly desirable. High levels of hyaluronan in breast tumors have been correlated with poor patient survival. The involvement of hyaluronan in the early invasive phase of a clone of breast cancer cell line MDA-MB-231 that forms bone metastases was studied using an in vivo-like basement membrane model. The metastatic to bone tumor cells exhibited a 7-fold higher hyaluronan-synthesizing capacity compared with MDA-MB-231 cells predominately due to an increased expression of hyaluronan synthase 2 (HAS2). We found that knockdown of HAS2 completely suppressed the invasive capability of these cells by the induction of tissue metalloproteinase inhibitor 1 (TIMP-1) and dephosphorylation of focal adhesion kinase. HAS2 knockdown-mediated inhibition of basement membrane remodeling was rescued by HAS2 overexpression, transfection with TIMP-1 siRNA, or addition of TIMP-1-blocking antibodies. Moreover, knockdown of HAS2 suppressed the EGF-mediated induction of the focal adhesion kinase/PI3K/Akt signaling pathway. Thus, this study provides new insights into a possible mechanism whereby HAS2 enhances breast cancer invasion.     

Crystal structure of the catalytic domain of matrix metalloproteinase-1 in complex with the inhibitory domain of tissue inhibitor of metalloproteinase-1

The mammalian collagenases are a subgroup of the matrix metalloproteinases (MMPs) that are uniquely able to cleave triple helical fibrillar collagens. Collagen breakdown is an essential part of extracellular matrix turnover in key physiological processes including morphogenesis and wound healing; however, unregulated collagenolysis is linked to important diseases such as arthritis and cancer. The tissue inhibitors of metalloproteinases (TIMPs) function in controlling the activity of MMPs, including collagenases. We report here the structure of a complex of the catalytic domain of fibroblast collagenase (MMP-1) with the N-terminal inhibitory domain of human TIMP-1 (N-TIMP-1) at 2.54 A resolution. Comparison with the previously reported structure of the TIMP-1/stromelysin-1 (MMP-3) complex shows that the mechanisms of inhibition of both MMPs are generally similar, yet there are significant differences in the protein-protein interfaces in the two complexes. Specifically, the loop between beta-strands A and B of TIMP-1 makes contact with MMP-3 but not with MMP-1, and there are marked differences in the roles of individual residues in the C-D connector of TIMP-1 in binding to the two MMPs. Structural rearrangements in the bound MMPs are also strikingly different. This is the first crystallographic structure that contains the truncated N-terminal domain of a TIMP, which shows only minor differences from the corresponding region of the full-length protein. Differences in the interactions in the two TIMP-1 complexes provide a structural explanation for the results of previous mutational studies and a basis for designing new N-TIMP-1 variants with restricted specificity.     

The complex between a tissue inhibitor of metalloproteinases (TIMP-2) and 72-kDa progelatinase is a metalloproteinase inhibitor.

Human rheumatoid synovial cells in culture secrete both 72-kDa progelatinase and a complex consisting of 72-kDa progelatinase and a 24-kDa inhibitor of metalloproteinases, TIMP-2. In addition, the culture medium contains TIMP-1, the classical inhibitor of metalloproteinases, with a molecular mass of 30 kDa. TIMP-1 does not form a complex with free 72-kDa progelatinase. Free progelatinase and progelatinase complexed with TIMP-2 can be activated with the organomercury compound p-aminophenylmercury acetate. The activated complex shows less than 10% the enzyme activity of activated free gelatinase. The progelatinase-TIMP-2 complex could be shown to be an inhibitor for other metalloproteinases, such as gelatinase and collagenase secreted by human rheumatoid synovia fibroblasts, as well as for the corresponding enzymes from human neutrophils.     

Exogenously added GPI-anchored tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) displays enhanced and novel biological activities

The family of tissue inhibitors of metalloproteinases (TIMPs) exhibits diverse physiological/biological functions including the inhibition of active matrix metalloproteinases, regulation of proMMP activation, cell growth, and the modulation of angiogenesis. TIMP-1 is a secreted protein that can be detected on the cell surface through its interaction with surface proteins. The diverse biological functions of TIMP-1 are thought to lie, in part, in the kinetics of TIMP-1/MMP/surface protein interactions. Proteins anchored by glycoinositol phospholipids (GPIs), when purified and added to cells in vitro, are incorporated into their surface membranes. A GPI anchor was fused to TIMP-1 to generate a reagent that could be added directly to cell membranes and thus focus defined concentrations of TIMP-1 protein on any cell surface independent of protein-protein interaction. Unlike native TIMP-1, exogenously added GPI-anchored TIMP-1 protein effectively blocked release of MMP-2 and MMP-9 from osteosarcoma cells. TIMP-1-GPI was a more effective modulator of migration and proliferation than TIMP-1. While control hTIMP-1 protein did not significantly affect migration of primary microvascular endothelial cells at the concentrations tested, the GPI-anchored TIMP-1 protein showed a pronounced suppression of endothelial cell migration in response to bFGF. In addition, TIMP-1-GPI was more effective at inducing microvascular endothelial proliferation. In contrast, fibroblast proliferation was suppressed by the agent. Reagents based on this method should assist in the dissection of the protease cascades and activities involved in TIMP biology. Membrane-fixed TIMP-1 may represent a more effective version of the protein for use in therapeutic expression.     

Tissue inhibitor of metalloproteinase (TIMP-2). A new member of the metalloproteinase inhibitor family

Human melanoma cells secret a 21-kDa protein, termed CSC-21K, which binds with 1:1 molar stoichiometry to the matrix metalloproteinase type IV collagenase proenzyme (70-kDa gelatinase) secreted by the same cells. This binding protein has been purified and its complete primary structure determined by sequencing overlapping peptides which span the entire protein. The amino acid sequence demonstrates that this protein shares significant homology with human TIMP (tissue inhibitor of metalloproteinase), including conservation of the positions of the 12 cysteine residues and 3 of 4 tryptophan residues. The identification of CSC-21K now indicates that a family of TIMP-related proteins exists. Individual members of this family may possess selective affinities for different members of the matrix metalloproteinase family. CSC-21K produced by tumor cells is isolated as a 1:1 molar complex with type IV procollagenase, as demonstrated by amino acid composition analysis. Addition of purified CSC-21K to the activated metalloproteinase results in inhibition of the collagenolytic activity in a stoichiometric fashion. Based on its sequence homology to TIMP and ability to inhibit type IV collagenolysis we propose the name TIMP-2 for this inhibitor.     

Membrane type-1 matrix metalloproteinase and TIMP-2 in tumor angiogenesis.

The matrix metalloproteinases (MMPs) constitute a multigene family of over 23 secreted and cell-surface associated enzymes that cleave or degrade various pericellular substrates. In addition to virtually all extracellular matrix (ECM) compounds, their targets include other proteinases, chemotactic molecules, latent growth factors, growth factor-binding proteins and cell surface molecules. The MMP activity is controlled by the physiological tissue inhibitors of MMPs (TIMPs). There is much evidence that MMPs and their inhibitors play a key role during extracellular remodeling in physiological situations and in cancer progression. They have other functions that promoting tumor invasion. Indeed, they regulate early stages of tumor progression such as tumor growth and angiogenesis. Membrane type MMPs (MT-MMPs) constitute a new subset of cell surface-associated MMPs. The present review will focus on MT1-MMP which plays a major role at least, in the ECM remodeling, directly by degrading several of its components, and indirectly by activating pro-MMP2. As our knowledge on the field of MT1-MMP biology has grown, the unforeseen complexities of this enzyme and its interaction with its inhibitor TIMP-2 have emerged, often revealing unexpected mechanisms of action.     

Metalloproteinases and tissue inhibitor of metalloproteinase 1 (TIMP-1) in endometrial flushings from pre- and post-menopausal women and from women with endometrial adenocarcinoma.

The presence of metalloproteinase activity in endometrial flushings obtained from premenopausal women, during the proliferative and secretory phases of the menstrual cycle, control post-menopausal women and women with post-menopausal bleeding (PMB) with or without adenocarcinoma was analysed by zymography. In addition, quantitative measurements of matrix metalloproteinase 2 (MMP-2), MMP-3, MMP-9 and tissue inhibitor of metalloproteinase 1 (TIMP-1) in the flushings were obtained by ELISA. The zymography results showed eight bands of activity, with molecular weights ranging from 51 to 208 kDa in the flushings from pre-menopausal women and post-menopausal women, particularly those with adenocarcinoma. Both zymography and ELISA showed that MMP-2 and MMP-9 were the major metalloproteinases found in the flushings and only low concentrations of MMP-3 were found. Concentrations of MMP-2 in pre-menopausal women were higher in flushings obtained during the secretory phase of the menstrual cycle than those obtained in the proliferative phase (P < 0.05), suggesting that it may play a role in embryo implantation. Concentrations of MMP-2 (P < 0.001), MMP-9 (P < 0.05) and TIMP-1 (P < 0.001) in the flushings from post-menopausal control women were lower than those from pre-menopausal women. Concentrations of MMP-2 (P < 0.05) and TIMP-1 (P < 0.05) were higher in flushings from women with PMB without carcinoma compared with post-menopausal controls and concentrations of MMP-9 (P < 0.01) and TIMP-1 (P < 0.05) in flushings from women with adenocarcinoma were higher than in post-menopausal controls. Among subjects with PMB, concentrations of MMP-9 in women with adenocarcinoma were higher than those without carcinoma (P < 0.05). Our results show that concentrations of MMP-2, MMP-9 and TIMP-1, but not MMP-3, are associated with endometrial activity and, therefore, may have a role in the breakdown of endometrial tissue. In addition, the increased concentrations of MMP-9 in flushings of women with adenocarcinoma indicate that this particular proteinase is associated with the presence of endometrial neoplastic cells.     

Both tissue inhibitors of metalloproteinases-1 (TIMP-1) and TIMP-2 activate Ras but through different pathways

Tissue inhibitors of metalloproteinases-1 (TIMP-1) and TIMP-2 have growth-stimulating activity for a wide range of cell types. Ras, which comprises a family of three members, i.e, Ha-Ras, Ki-Ras, and H-Ras, is known to participate in growth control in all its facets, including cell proliferation, transformation, differentiation, and apoptosis. In this study, we tested the hypothesis that Ras might be involved in the cell growth-promoting activity of TIMPs. Using MG-63 human osteosarcoma cells, we demonstrated that both TIMP-1 and TIMP-2 caused an increase in the Ras-GTP level in a dose-dependent manner. Our previous results indicated that TIMP-1 activity is mediated through the tyrosine kinase (TYK)/mitogen-activated protein kinase (MAPK) pathway. Here, we demonstrated that Ras activation by TIMP-1 was inhibited by a specific TYK inhibitor, herbimycin A, suggesting that the TYK/MAPK signaling pathway was involved in Ras activation by TIMP-1. However, the activation of Ras by TIMP-2 was inhibited by an inhibitor specific for cyclic AMP-dependent protein kinase (PKA), H89, suggesting the involvement of the PKA-mediated pathway. Furthermore, TIMP-2 promoted the formation of a complex between Ras-GTP and phosphoinositide 3-kinase.     

肺结核并发纤维化患者BALF和血清MMP及TIMP-1检测的临床意义

目的:探讨肺结核并发纤维化患者支气管肺泡灌洗液(BALF)及血清中基质金属蛋白9(MMP-9)及基质金属蛋白酶组织抑制剂l(TIMP-1)含量与肺纤维化及肺功能的关系.方法:酶联免疫(ELISA)法检测我院2009年3月至2009年9月68例肺结核并发纤维化患者(纤维化组)及30例无肺部疾病患者(对照组)的支气管肺泡灌洗液及血清中MMP-9及TIMP-1的含量,同时进行肺功能检查.结果:纤维化组患者的BALF及血清MMP-9的含量与对照组怠者相比无统计学意义,但纤维化组患者的BALF及血清TIMP-1的含量显著高于对照组(P＜0.05),且纤雏化组患者的BALF及血清MMP-9/TIMP-1比值低于对照组(P＜0.05).患者的动脉血氧分压、肺活量等肺功能指标与TIMP-1呈正相关,而与MMP-9/TIMP-1比值呈负相关(P＜0.05).结论:肺结核患者肺纤维化的发生可能与TIMP-1水平升高及MMP-9/TIMP-1比值降低相关,降低TIMP-9的水平可能是防止肺结核惠者并发肺纤维化的一种新途径.     

Isolation and characterization of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) from human rheumatoid synovial fluid.

The tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 were purified to apparent homogeneity from human rheumatoid synovial fluid (HRSF). The inhibitors were isolated by dissociation of non-covalent gelatinase/TIMP complexes. TIMP-1 migrated as a single polypeptide with Mr 28,500 on SDS-PAGE, while the Mr of TIMP-2 was 21,000. The inhibitory activity was stable under heat and acid pH. N-terminal sequence data were obtained for the first 15 residues of both inhibitors and showed identity to the human fibroblast inhibitors TIMP-1 and TIMP-2. This is the first demonstration that TIMP-1 and TIMP-2 can be directly purified from human rheumatoid synovial fluid. The complex formation between the metalloproteinase inhibitors and leucocyte metalloproteinases was shown by immunoblotting.     

The expression of tissue inhibitor of metalloproteinase 2 (TIMP-2) is required for normal development of zebrafish embryos

MMP activities are controlled by a combination of proteolytic pro-enzyme activation steps and inhibition by endogenous inhibitors like alpha2-macroglobulin and the tissue inhibitors of metalloproteinases (TIMPs). TIMPs are the key inhibitors in tissue. The expression of both MMPs and TIMPs is controlled during tissue remodeling to maintain a balance in the turnover of extracellular matrix. Disruption of this balance may result in a broad spectrum of diseases. Additionally, TIMP-2 has been reported to have growth factor activities. To further study the function of TIMP-2 in development, we utilized zebrafish as an experimental model system. We have successfully isolated a TIMP-2 homologue from zebrafish (zTIMP-2). This zebrafish TIMP-2 showed high similarity to human TIMP-2 with all critical features conserved. Whole-mount in situ analysis showed that zTIMP-2 was expressed as early as the one-cell stage indicating a maternal origin. This expression continued through later stages of development. RT-PCR analysis confirmed the early expression pattern from the 16-cell stage through blastula, gastrula and 24-h stages. In addition, at the protein level, immunoreactive zTIMP-2 was detected using antibody against recombinant human TIMP-2. RFP-reporter analysis indicated that TIMP-2 can be secreted into the extracellular space where ECM is forming. Functional studies showed that the balance of TIMP-2 expression is important to normal development as reflected by the fact that both blockage of TIMP-2 translation using antisense morpholino oligonculeotides or increased translation of TIMP-2 using a mRNA microinjection approach resulted in abnormal zebrafish development. This is in contrast to murine knockout studies that indicate that TIMP-2 does not have a major role in mouse embryogenesis.     

Functional roles of the tissue inhibitor of metalloproteinase 3 (TIMP-3) during ascorbate-induced differentiation of osteoblastic MC3T3-E1 cells

The tissue inhibitor of the metalloproteinase-3 (TIMP-3) gene was isolated as a gene involved in the process of ascorbate-induced differentiation of mouse MC3T3-E1 cells by the differential display method. The functional roles of TIMP-3 were characterized by establishing stable cell lines, which constitutively expressed the TIMP-3 gene. The TIMP-3 transfectants produced type I collagen at the same level as that of normal cells in response to ascorbic acid 2-phosphate (AscP). However, the expression of the other osteoblastic marker proteins such as alkaline phosphatase (ALPase), osteopontin (OP), osteocalcin (OC), osteonectin (ON) and matrix metalloproteinases (MMPs) remained at a low level even in the presence of AscP. Furthermore, no mineralization of the extracellular matrix (ECM) occurred with the transfectants. Remodeling ECM through TIMPs and MMPs is concluded to be required for osteoblastic differentiation.     

Identification,purification and partial characterization of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in bovine pulmonary artery smooth muscle

Bovine pulmonary artery smooth muscle tissue possesses the tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) as revealed by immunoblot studies of the cytosolic fraction with polyclonal TIMP-1 antibody. In this report, we described the purification and partial characterization of the inhibitor from the cytosolic fraction of the smooth muscle. This inhibitor was purified by a series of anion-exchange, gel filtration and affinity chromatographic procedure. The purified inhibitor showed an apparent molecular mass of 30 kDa in SDS-PAGE. Amino terminal sequence analysis for the first 22 amino acids of the purified inhibitor was also found to be identical to bovine TIMP-1. This glycosylated inhibitor was found to be active against matrix metallorpoteinase-9 (MMP-9, gelatinase B), the ambient matrix metalloproteinase in the pulmonary smooth muscle. The purified TIMP-1 was also found to be sensitive to pure rabbit and human fibroblast collagenase and type IV collagenase. In contrast, it had minimum inhibitory activity against bacterial collagenase. It was also found to be inactive against the serine proteases trypsin and plasmin. The inhibitor was heat and acid resistant and it had the sensitivity to trypsin degradation and reduction-alkylation.