Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.23 (MMP)
 4,246 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The latent precursor of matrilysin (EC 3.4.24.23; punctuated metalloproteinase (PUMP) was purified from transfected mouse myeloma cell conditioned medium and was found to contain one zinc atom per molecule which was essential for catalytic activity. Promatrilysin could be activated to the same specific activity by (4-aminophenyl)mercuric acetate, trypsin, and incubation at elevated temperatures (heat activation). Active matrilysin hydrolyzed the fluorescent substrate 2,4-dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond with a maximum value for kcat/Km of 1.3 x 10(4) M-1 s-1 at the pH optimum of 6.5 and pKa values of 4.60 and 8.65. Activity is inhibited by the tissue inhibitor of metalloproteinases-1 in a 1:1 stoichiometric interaction. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis in conjunction with N-terminal sequencing revealed that, as with all other matrix metalloproteinases similarly studied, promatrilysin activation was accompanied by the stepwise proteolytic removal of an M(r) 9000 propeptide from the N-terminus. The intermediates generated were dependent on the mode of activation used but, in all cases studied, activation terminated with an autocatalytic cleavage at E77-Y78 to yield the final M(r) 19,000 active matrilysin. From an analysis of the stability of the various intermediates, we propose that the sequence L13-K33 is particularly important in protecting the E77-Y78 site from autocatalytic cleavage, thereby maintaining the latency of the proenzyme.
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PMID:Biochemical characterization of matrilysin. Activation conforms to the stepwise mechanisms proposed for other matrix metalloproteinases. 139 Jun 35

Metalloproteinase inhibitors were surveyed with the culture media of 19 kinds of human tumor cell lines, using transin (rat stromelysin) as the target enzyme. This survey showed that most of the cell lines more or less secreted inhibitor activity, and that a human hepatoma cell line, HLE, secreted an extremely high inhibitor activity into the culture medium. Two kinds of metalloproteinase inhibitors were purified from the serum-free conditioned medium of HLE cells. The major inhibitor, which showed a single protein band with a molecular weight (Mr) of 21,000 (21k) (nonreduced) or 24k (reduced) on SDS-polyacrylamide gel electrophoresis, was identified as TIMP-2 (tissue inhibitor of metalloproteinases-2) by the analysis of its N-terminal amino acid sequence. The other was immunologically identified as TIMP. Purified TIMP-2 inhibited the activities of transin, matrin (pump-1), Mr 72k gelatinase, and interstitial collagenase with 1:1 stoichiometry. When the latent precursor form (Mr 57k) of transin was incubated with p-aminophenylmercuric acetate as an activating reagent, TIMP-2 inhibited the conversion of the intermediate form (Mr 45k) into the mature enzyme (Mr 42k). This indicated that TIMP-2 regulates not only the activity of the mature enzyme but also the autolytic processing of the proenzyme. TIMP-2 also inhibited in vitro tumor invasion through reconstituted basement membrane (matrigel) in chemotaxis chambers, showing that the metalloproteinase inhibitors as well as the extracellular matrix metalloproteinases are involved in tumor invasion through basement membrane and other extracellular matrices.
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PMID:Efficient purification of TIMP-2 from culture medium conditioned by human hepatoma cell line, and its inhibitory effects on metalloproteinases and in vitro tumor invasion. 166 1

A metalloproteinase with Mr 29,000 was purified to homogeneity as a latent proenzyme from the conditioned medium of a human rectal carcinoma cell line CaR-1. This enzyme hydrolyzed casein more potently than gelatin embedded in polyacrylamide gels in zymography assay. Calcium ion was essential for the activity. It exerted the maximum activity at pH 7-9. Its activity was stimulated by organomercurials, such as p-amino-phenyl mercuric acetate and p-chloromercuric benzoic acid, and was inhibited by 1,10-phenanthroline but was hardly affected by diisopropyl fluorophosphate and pepstatin. When the purified proenzyme was activated by the organomercurials, it effectively hydrolyzed fibronectin, laminin, type IV basement membrane collagen, and several types of gelatins but not interstitial type I and III collagens. The treatment of the purified proenzyme with p-aminophenyl mercuric acetate or trypsin formed an active peptide with Mr 20,000. The structural analysis indicated that it was most likely identical to putative metalloproteinase-1, the complementary DNA of which had been cloned from human tumor mRNAs capable of hybridizing to a rat transin complementary DNA. Based on the fact that this enzyme is secreted extracellularly and degrades the matrix proteins, we propose the name "matrin" for this newly identified enzyme.
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PMID:Purification and characterization of extracellular matrix-degrading metalloproteinase, matrin (pump-1), secreted from human rectal carcinoma cell line. 225 19

Pump-1 cDNA has recently been isolated by screening a human tumor cDNA library with a transin (rat stromelysin) probe under low-stringency hybridization conditions. The cDNA codes for a potential protein with significant sequence similarity to the metalloproteinases collagenase and stromelysin, but which lacks the hemopexin-like domain characteristic of these enzymes. Expression of pump-1 cDNA in cos cells using an expression vector leads to secretion of a protein of Mr 28,000 with latent, organomercurial-activatable proteinase activity. Cos cells transfected with a partial pump-1 cDNA in the vector pPROTA secrete a fusion protein between the IgG-binding domains of staphylococcal protein A and pump-1. The fusion protein binds to IgG-Sepharose, and the bound fusion protein undergoes apparent autocleavage in the presence of 4-aminophenylmercuric acetate with elution of active pump-1 species of Mr 21,000 and 19,000. Active pump-1 degrades casein, gelatins of types I, III, IV, and V, and fibronectin and can activate collagenase. Active pump-1 is inhibited by EDTA, 1,10-phenanthroline, and the tissue inhibitor of metalloproteinases. These results show that, despite the absence of a hemopexin-like domain, pump-1 is a latent secreted metalloproteinase. Postpartum rat uteri contain elevated levels of rat pump-1 mRNA. On the basis of this observation, its size, and its substrate specificity, we suggest that pump-1 might correspond to a previously described uterine metalloproteinase, matrix metalloproteinase 7.
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PMID:Pump-1 cDNA codes for a protein with characteristics similar to those of classical collagenase family members. 255 50

A small metalloproteinase that digests Azocoll was found in the uterus of the rat. Its activity increased to high levels during the postpartum period in parallel with the breakdown of the extracellular matrix exclusive of collagen (Sellers, A., and Woessner, J.F., Jr. (1980) Biochem. J. 189, 521-531). This enzyme has now been purified almost 7,000-fold to homogeneity from 12 g of tissue using molecular sieve chromatography, blue sepharose chromatography, and zinc-chelate chromatography. Gel electrophoresis with sodium dodecyl sulfate and dithiothreitol gives Mr = 28,000 for the latent form of the enzyme and Mr = 19,000 for the active form that arises spontaneously or by treatment with aminophenylmercuric acetate. The enzyme digests components of the extracellular matrix including gelatins of types I, III, IV, and V, fibronectin, and proteoglycan. It digests the alpha 2(I) chain of gelatin in preference to the alpha 1(I) chain and cleaves dinitrophenyl-Pro-Leu-Gly-Ile-Ala-Gly-Pro-D-Arg. It cleaves the B chain of insulin at two points: Ala14-Leu15 and Tyr16-Leu17. It has no action on collagens of types I, III, IV, or V at 26 degrees C and no action on elastin or phenylazo-Pro-Leu-Gly-Pro-D-Arg. The pH optimum is at pH 7 and the pI at 5.9. The enzyme requires zinc and calcium ions for activity; cobalt and strontium can partially replace these metal ions. The enzyme is not inhibited by low levels of phosphoramidon or Zincov. Its properties clearly distinguish it from collagenase, gelatinase (matrix metalloproteinase 2), and stromelysin (matrix metalloproteinase 3); it therefore constitutes a further member of the family of extracellular matrix metalloendopeptidases. The name matrix metalloproteinase 7 is proposed.
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PMID:Purification and properties of a small latent matrix metalloproteinase of the rat uterus. 318 22

Human promatrilysin (matrix metalloproteinase-7) has been produced in Escherichia coli as an N-terminal fusion protein with ubiquitin. The insoluble product was solubilized, refolded, and activated with amino-phenylmercuric acetate. Activation of the fusion protein demonstrated kinetics and intermediates that were very similar to those observed during activation of promatrilysin produced in Chinese Hamster Ovary (CHO) cells. Following activation, matrilysin was purified to > 95% homogeneity using a Sepharose-Pro-Leu-Gly-NHOH affinity column. The matrilysin purified by this procedure is indistinguishable from the enzyme purified from CHO cells with respect to the kinetic parameters for hydrolysis of a peptide substrate and the ability to obtain diffraction quality crystals in the presence of an inhibitor of the enzyme. Additionally, to facilitate detailed kinetic analyses of matrilysin, a new fluorogenic peptide substrate with the optimized sequence Dnp-Arg-Pro-Leu-Ala-Leu-Trp-Arg-Ser (Dnp, dinitrophenyl) has been synthesized. This peptide is the best substrate developed for matrilysin thus far with Km and kcat values of 26 microM and 5.0 s-1, respectively.
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PMID:Purification of human matrilysin produced in Escherichia coli and characterization using a new optimized fluorogenic peptide substrate. 750 60

The initiation of the angiogenic process requires a locally confined and time-limited proteolysis of the basement membrane (BM) components at the site of new vessel sprout. Gelatinase A, a member of the matrix metalloproteinase family, degrades BM type IV collagen and is involved in the BM breakdown by migrating tumor cells and endothelial cells (EC). Gelatinase A is synthesized as latent proenzyme and must be activated in order to express its proteolytic activity. A plasma membrane-dependent mechanism of activation has been described for several tumor and transformed cells lines. In the present study, we show that latent (72 kD) and mature (62-59 kD) forms of gelatinase A are present in EC membrane fraction from Triton X-114 extract while only latent form is found in the cytosolic fraction. The incubation of EC membrane fraction with exogenous latent gelatinase A resulted in a significant activation giving rise to 62-59 kD mature forms. 12-O-tetradecanoylphorbol-13-acetate (TPA), a strong potentiator of angiogenesis in vitro and in vivo, increases the amount of both latent and activated forms of gelatinase A in EC membrane fraction as well as the ability of this latter fraction to activate exogenous latent gelatinase A. We show that the mRNA transcript coding for the membrane-integrated MMP, the MT-MMP, previously described as a potential gelatinase A activator in invasive tumor cells is also expressed in vascular EC and is regulated through a TPA sensitive process. This enzyme may be responsible for membrane-dependent gelatinase A activation in normal vascular EC and may therefore be a determinant in the control of BM proteolysis during angiogenesis.
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PMID:Plasma membrane-dependent activation of gelatinase A in human vascular endothelial cells. 759 26

A small uterine metalloproteinase of the rat has been shown by amino acid and cDNA sequencing to be orthologous to human pump-1. Both proteinases are now designated as matrilysin or matrix metalloproteinase 7. The properties of purified uterine metalloproteinase and recombinant pump-1 were compared. Their specificities on substrates (gelatins, fibronectin, transferrin, elastin, Azocoll, and (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3,[2, 4-dinitrophenyl]-L-2, 3-diaminopropionyl)-Ala-Arg-NH2) are similar and distinct from those of the stromelysins and gelatinases. The two matrilysins have similar sensitivity to hydroxamate and pseudopeptide inhibitors. Rat matrilysin selectively cleaves the alpha 2(I) chain of rat gelatin, producing major cuts at Gly713-decreases-Ile714, Gly775-decreases-Leu776, and Gly809-decreases-Ile810. Rat matrilysin produces maximum activation of latent human interstitial collagenase 1 (pro-matrix metalloproteinase 1) when added in the presence of 4-aminophenylmercuric acetate (APMA) by cleaving the Gln80-decreases-Phe81 bond. Rat and human matrilysin do not directly activate latent rat collagenase 3 (matrix metalloproteinase 13) and do not enhance its activation when added together with APMA. Autoactivation of collagenase 3 in the presence of APMA results in cleavage at Val81-decreases-Tyr82 corresponding to the Gln80-decreases-Phe81 cleavage in collagenase 1. Thus collagenase 3 is capable of maximal autoactivation, whereas collagenase 1 is dependent upon another matrix metalloproteinase in order to be activated to its full potential.
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PMID:Characterization of rat uterine matrilysin and its cDNA. Relationship to human pump-1 and activation of procollagenases. 760 62

The gene expression of five matrix metalloproteinases (MMPs) and two tissue inhibitors of metalloproteinases (TIMPs) was studied in human gliomas in vivo and in vitro to evaluate their roles in glioma invasion. Simultaneous expression of one to four MMP genes and two TIMP genes was found in 17 surgical glioma specimens, and one MMP (gelatinase A) gene and two TIMP genes were simultaneously expressed in tissue of three brains. The concomitant overexpression of gelatinase A, gelatinase B, and occasional matrilysin genes was associated with the malignancy of gliomas and accompanied by overexpression of the TIMP-1 gene. In five human glioma cell lines, gelatinase A, TIMP-1, and TIMP-2 genes were constitutively expressed in alll cell lines: the matrilysin gene in three cell lines; the stromelysin gene in two cell lines; and the interstitial collagenase gene in one cell line. There was a clear difference in the expression of gelatinase B and stromelysin genes between surgical glioma specimens and glioma cell lines: the gelatinase B gene was not expressed constitutively in vitro but was overexpressed in vivo, whereas the stromelysin gene was not expressed in vivo but was expressed in some cell lines. To find the cause of that difference in vivo and in vitro, the transcriptional regulations of MMP and TIMP genes by tumor promoter, growth factors, or cytokines were studied in vitro. Interstitial collagenase, gelatinase B, stromelysin, and TIMP-1 genes were upregulated in many cell lines by phorbol-12-myristate-13-acetate (PMA) and in some cell lines by epidermal growth factor, tumor necrosis factor-alpha, or interleukin-1 beta. Transforming growth factor-beta 1 (TGF beta 1) upregulated gelatinase A and matrilysin genes in some cell lines, and there were no clear responses from any MMP and TIMP genes to interleukin-6. Thus, the transcriptional modulation of MMP genes by these growth factors and cytokines seemed insufficient to explain the difference in gelatinase B and stromelysin gene expressions in vivo and in vitro and was suggestive of the genetic alteration of glioma cells in vitro, the heterogeneous cell population in glioma tissues, or both. Furthermore, the in vitro invasion of glioma cells through Matrigel in response to PMA, TGF beta 1, or TIMP-1 was assessed by chemoinvasion assay. In most cell lines, invasion was significantly stimulated by PMA or TGF beta 1 but suppressed by TIMP-1.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Matrix metalloproteinases and tissue inhibitors of metalloproteinases in human gliomas. 761 76

The precursor of matrix metalloproteinase 9 (pro-MMP-9, progelatinase B) noncovalently binds to tissue inhibitor of metalloproteinases (TIMP)-1 through the C-terminal domain of each molecule. We have isolated the proMMP-9.TIMP-1 complex from the medium of human fibrosarcoma HT-1080 cells and investigated the activation processes of the complex by 4-aminophenylmercuric acetate, trypsin, and matrix metalloproteinase 3 (MMP-3, stromelysin 1). The treatment of the proMMP-9.TIMP-1 complex with 4-aminophenylmercuric acetate or trypsin converts proMMP-9 to lower molecular weight species corresponding to active forms, but no gelatinolytic activity is detected. The lack of enzymic activity results from binding of TIMP-1 to the activated MMP-9. The treatment of the proMMP-9.TIMP-1 complex with a possible physiological proMMP-9 activator, MMP-3, does not reveal any gelatinolytic activity unless the molar ratio of MMP-3 to the complex exceeds 1. This is due to the inhibition of MMP-3 by TIMP-1 forming a ternary proMMP-9.TIMP-1.MMP-3 complex. The formation of the ternary complex weakens the interaction between proMMP-9 and TIMP-1, resulting in partial dissociation of the complex into proMMP-9 and the TIMP-1.MMP-3 complex. When MMP-3 is in excess, the propeptide is completely processed, and the full activity of MMP-9 is detected. Similarly, the proMMP-9.TIMP-1 complex inhibits MMP-1 (interstitial collagenase) and in turn renders the proMMP-9 activable by a catalytic amount of MMP-3. These results suggest that formation of the proMMP-9.TIMP-1 complex regulates extracellular matrix breakdown in tissue by switching the predominant MMP activity from one type to another.
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PMID:Steps involved in activation of the pro-matrix metalloproteinase 9 (progelatinase B)-tissue inhibitor of metalloproteinases-1 complex by 4-aminophenylmercuric acetate and proteinases. 762 79


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