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

1. The kinetics of the degradation of the kinins bradykinin and Met-Lys-bradykinin, angiotensins I and II and the tachykinin substance P by PMNL-collagenase (MMP 8), PMNL-gelatinase (MMP 9) and by the recombinant catalytic domain of MMP 8 (rcd-PMNL-c) was examined by RP-HPLC. The resulting fragments were identified by automated Edman degradation or by amino acid analysis. 2. The initial degradation rates of substance P at a substrate concentration of 25 microM were 5 min-1 for MMP 9 and 150 min-1 for MMP 8. The kinetic constants KM and kcat were determined by concentration-dependent measurements. For MMP 8/substance P the constants were KM = 78 +/- 14 microM and kcat = 412 +/- 67 min-1. For MMP 9/substance P the constants were KM = 91 +/- 15 microM and kcat = 25 +/- 4 min-1. Both enzymes cleaved substance P between Gln6 and Phe7 and between Gly9 and Leu10. 3. Under the same conditions, MMP 8 degraded angiotensin I at an initial rate of 20 h-1, resulting mainly in the vasoactive fragments angiotensin II and angiotensin(1-7). At a substrate concentration of 25 microM and an enzyme/substrate ratio of 1:100, angiotensin II was degraded very slowly (19% in 24 h) by MMP 8. Under these conditions, MMP 9 degraded angiotensin I to a lesser extent than MMP 8 (25% in 24 h) and was unable to cleave angiotensin II. 4. Under the same conditions, bradykinin and Met-Lys-bradykinin were cleaved by PMNL-collagenase at a rate of 20% in 24 h, producing BK(1-7) and BK(1-8). PMNL-gelatinase was unable to cleave the kinins under these conditions. 5. In all cases, rcd-PMNL-c produced the same fragments as wild type PMNL-collagenase, but at a significantly lower rate.
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PMID:Degradation of kinins, angiotensins and substance P by polymorphonuclear matrix metalloproteinases MMP 8 and MMP 9. 753 73

A galactose-binding protein of M(r) = 30,000 previously described in baby hamster kidney cells (Foddy, L., Stamatoglou, S. C., and Hughes, R. C. (1990) J. Cell Sci. 97, 139-148) has been analyzed by the cloning and sequencing of cDNA clones encoding the complete sequence and an amino-terminal fragment. The intact lectin CBP30 contains 245 amino acid residues, including the initiating methionine residue, and is closely homologous to mammalian S-type lectins of similar size characterized in human, rat, and mouse species. The carboxyl-terminal domain contains the carbohydrate binding activity and the amino-terminal domain, which is extremely sensitive to bacterial collagenase, contains a repetitive sequence rich in glycine, tyrosine, and proline. There are 8 repeats in hamster CBP30, as in the human homologue, compared with about 10 in rat and mouse and > 10 in dog homologues. This repeat sequence is also sensitive to the tissue metalloproteinases, gelatinase B and matrilysin, but, unlike the bacterial collagenase, the mammalian enzymes also cause extensive degradation of the carbohydrate binding carboxyl domain. Physical measurements using CD and tryptophan fluorescence spectroscopy indicate that the two domains of CBP30 are structurally, as well as functionally, distinct and independent. Cross-linking studies indicate that the amino-terminal lectin fragment can efficiently self-assemble into oligomeric species, and less efficient but significant aggregation of the intact lectin is also shown. Domain-specific antibodies to hamster CBP30 have been prepared and used to show that only the full-length, undegraded form of CBP30 is present in whole cell lysates.
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PMID:Structure of baby hamster kidney carbohydrate-binding protein CBP30, an S-type animal lectin. 802 86

The sequence specificities of human 72-kDa fibroblast gelatinase (type IV collagenase), human 92-kDa neutrophil gelatinase (type IV collagenase), and putative metalloproteinase (PUMP or matrilysin) have been examined by measuring the rate of hydrolysis of over 50 synthetic oligopeptides covering the P4 through P4' subsites of the substrate. The peptides investigated in this paper were those employed in our previous study which systematically examined the sequence specificity of human fibroblast and neutrophil collagenases [Netzel-Arnett et al. (1991) J. Biol. Chem. 266, 6747]. The initial rate of hydrolysis of the P1-P1' bond of each peptide has been measured under first-order conditions ([S0] << KM), and kcat/KM values have been calculated from the initial rates. The specificities of these five metalloproteinases are similar, but distinct, with the largest differences occurring at subsites P1, P1', and P3'. The specificities of the two gelatinases are the most similar to each other. They tolerate only small amino acids such as Gly and Ala in subsite P1. In contrast, larger residues such as Met, Pro, Gln, and Glu are also accommodated well by PUMP. All five enzymes prefer hydrophobic, aliphatic residues in subsite P1'. PUMP exhibits a stronger preference for Leu in this subsite than is shown by the other enzymes. The P3' subsite specificities of the gelatinases and collagenases are very similar but different from those of PUMP which particularly prefers Met in this position. The specificity data from this study allow the design of optimized substrates and selective inhibitors for these metalloproteinases.
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PMID:Comparative sequence specificities of human 72- and 92-kDa gelatinases (type IV collagenases) and PUMP (matrilysin). 839 Aug 57

Human matrilysin devoid of its propeptide is expressed in Escherichia coli and purified to homogeneity by heparin chromatography after refolding of the guanidine hydrochloride solubilized protein. Matrilysin autolytically removes its N-terminal tripeptide Met-Tyr-Ser during the refolding process. The enzyme contains 1.91 +/- 0.08 zinc atoms/mol of protein and retains full activity when stored several months at 4 degrees C. It hydrolyzes the fluorescent substrate Dns-PLALWAR at the Ala-Leu bond with a kcat of 3.1 s-1 and K(m) of 1.8 x 10(-5) M at pH 7.5, 37 degrees C, values closely similar to those for the matrilysin produced by activation of the Chinese hamster ovary and E. coli-expressed promatrilysin. The properties of this form of matrilysin demonstrate that the propeptide is not essential for proper folding or stability of the enzyme but likely determines the N-terminal amino acid of the mature enzyme. The pH dependence of kcat/K(m) for Dns-PLALWAR shows that matrilysin has a broad pH optimum (5.0-9.0) and the pKa values obtained are 4.3 and 9.6 at 25 degrees C. The activity is inhibited by several metal binding agents including 1, 10-phenanthroline, OP, but not by the nonchelating isomer, 1,7-phenanthroline. OP inhibits instantaneously by likely forming a transient ternary enzyme.metal.chelator complex. The zinc atom is then removed from the protein in a time-dependent manner. In agreement with the kinetic studies, dialysis in the presence of OP and CaCl2 removes only the catalytic zinc atom. The monozinc enzyme can be reactivated to 90%, 56%, 27%, and 17% of the native activity by addition of zinc, manganese, nickel, and cobalt, respectively. Cadmium, on the other hand, forms an inactive Cd/Zn hybrid. The differences in the chelator accessibility properties of the two zinc sites can thus be exploited to yield metallohybrids of matrilysin.
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PMID:Metal and pH dependence of heptapeptide catalysis by human matrilysin. 896 47

The hatching enzyme (envelysin) of the sea urchin Hemicentrotus pulcherrimus was purified from the medium of hatched blastulae. By cDNA cloning its deduced amino acid sequence and molecular architecture were revealed. The 591-residue precursor with calculated Mr of 66,123 consists of an 18-residue signal sequence, a 151-residue propeptide, and a 422-residue mature enzyme with N-terminal catalytic and C-terminal hemopexin-like domains. As compared with that of Paracentrotus lividus, its amino acid sequence is 69% identical and 10% similar. They share typical structural features with the mammalian MMP gene family members: cysteine switch, zinc-binding signature, methionine-turn, Cys residues near both ends of hemopexin-like domain, etc. However, its propeptide has a 70-residue extra sequence with an Asp- and Glu-rich stretch, supposedly involved in the proenzyme activation by binding Ca2+ ions in seawater. The hinge region is also longer than those of most MMPs, with an extra sequence rich in Thr and Arg residues. Mature 50K enzyme is highly susceptible to autolytic cleavage at Gln(503)-Leu(504), producing the 38K form retaining catalytic activity and substrate specificity against fertilization envelope. The 38K form and 15K fragment were coeluted from a gel-filtration column, suggesting that these two fragments are disulfide-bridged and that the tertiary structure is not much deviated. The 38K form further autolyzed to 32K form by cleaving Tyr(450)-Tyr(451) bond with the loss of protein-substrate specificity, retaining only nonspecific protease activity. Thus, the autolytic release of 2/3 of the C-terminal domain reduced the highly specific enzyme to a common nonspecific protease, implying that the size and structure of almost the entire hemopexin-like domain is essential for the protein substrate specificity. Moreover, autolytic degradation of envelysins from the two species follow quite different pathways despite their high homology in structure. The 38K and 32K forms were inhibited by bovine TIMP-1 with different IC50 values, indicating that its inhibitory activity depends on the extent of the interaction with the C-terminal domain of the enzyme.
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PMID:Sea urchin hatching enzyme (envelysin): cDNA cloning and deprivation of protein substrate specificity by autolytic degradation. 918 24

This paper reports changes to our previously published high-performance liquid chromatographic method for the measurement of 6-methylmercaptopurine (6-MMP) in red blood cell lysates. The extraction procedure and chromatographic conditions have been improved and the range of the calibration curves has been modified. The recoveries of 10 and 100 ng ml(-1) 6-MMP were 99.0+/-6.0% and 96.3+/-4.0% respectively and the limit of quantification was lowered to 5 ng ml(-1). This method, which does not require radioactive S-adenosyl-L-methionine, is more sensitive, specific and reproducible and may prove useful for routine determination of thiopurine methyltransferase activity in red blood cells.
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PMID:Thiopurine methyltransferase activity: new high-performance liquid chromatographic assay conditions. 939 Jul 40

We have searched for the exclusivity of common sequence motifs of the mitochondrial uncoupling proteins (UCP1, UCP2, UCP3, UCP4, BMCP1, and plant UCP [PUMP]) within the gene family of mitochondrial anion carrier proteins. The UCP-specific sequences, "UCP signatures", were found in the first, second, and fourth alpha-helices. First: Ala/Ser-Cys/Thr/n-n/Phe-Ala/Gly-[negatively charged residue]-n/Phe-n/Cys-Thr-Phe/n; second: Gly/Ala-Ile/Leu-Gln/X-[positively charged residue]-NH-n/Cys-Ser/nphi/X-n/Ser-OH/Gly-n-[positively charged residue]-Ile/Met-Gly/Val-n/Thr; fourth: Pro-Asn/ Thr-n-X-[positively charged residue]-Asn/Ser/Ala-n-n-Ile/Leu-n-Asn/Val-Cys/n-n/Thr-[negatively charged residue]-n-n/Thr/Pro-OH/Val (n, nonpolar; phi, aromatic; (positively charged residue/negatively charged residue, charged residue). The second and part of the third signature are also present in the yeast dicarboxylate transporter. The UCP signature excluding BMCP1 was also found in the second matrix segment: [positively charged residue]-(Pro/ del-Leu/del)-[positively charged residue]-phi-X-Gly/Ser-Thr/n-X-NH/[negatively charged residue]-Ala-phi. These UCP signatures are thought to be involved in fatty acid anion binding and translocation.
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PMID:Specific sequence of motifs of mitochondrial uncoupling proteins. 1077 43

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional factor involved both in development and tissue repair, as well as pathological processes such as cancer and metastasis. It has been identified in vivo in many types of tumours together with its tyrosine kinase receptor, Met. We show here that exogenous HGF/SF acts as a strong chemoattractant for human mesothelioma cell lines. The factor also enhanced cell adhesion to and invasion into Matrigel. The mesothelioma cell lines synthesized a panel of matrix metalloproteinases critical for tumour progression such as MMP-1, 2, 3, 9 and membrane-bound MT1-MMP. HGF/SF stimulated the expression of MMP-1, 9 and MT1-MMP and had a slight effect on expression of the MMP inhibitor TIMP-1 but not TIMP-2. However, there was no simple correlation between the levels of MMPs and TIMPs of the cell lines and their different invasion properties or between HGF/SF stimulatory effects on MMP expression and invasion. In addition, effects of protease inhibitors on invasion suggested that serine proteases were also expressed in human mesothelioma cell lines and were involved in HGF/SF-induced invasion. The results show a predominant role for HGF/SF in mesothelioma cell invasion, stimulating simultaneously adhesion, motility, invasion and regulation of MMP and TIMP levels.
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PMID:Hepatocyte growth factor/scatter factor enhances the invasion of mesothelioma cell lines and the expression of matrix metalloproteinases. 1102 27

Matrix metalloproteinase-3 (MMP-3 or stromelysin-1) hydrolyzes the Met(374)-Ser(375) (P3-P2), Glu(416)-Leu(417) and Ser(432)-Leu(433) peptide bonds in human alpha(2)-antiplasmin (alpha(2)-AP), the main physiological plasmin inhibitor. Cleavage is completely abolished in the presence of the MMP inhibitors EDTA or 1,10-phenanthroline. At enzyme/substrate ratio of 1:10 at 37 degrees C, alpha(2)-AP protein cleavage occurs with a half-life of 8 min, and is associated with rapid loss of inhibitory activity towards plasmin with a half-life of 5 min. alpha(2)-AP cleaved by MMP-3 does no longer form a stable complex with plasmin, as shown by SDS-PAGE, and does no longer interact with plasminogen, as shown by crossed immunoelectrophoresis with plasminogen added to the gel. These data are compatible with the removal of a COOH-terminal fragment containing the reactive site peptide bond and the plasmin(ogen)-binding site. In addition, MMP-3 cleaves the Pro(19)-Leu(20) peptide bond in alpha(2)-AP, thereby removing the fibrin-binding site from the inhibitor. A dysfunctional alpha(2)-AP variant (Ala-alpha(2)-AP or alpha(2)-AP Enschede), with an alanine insertion in the reactive site sequence converting it from a plasmin inhibitor into a substrate, was also efficiently cleaved by MMP-3 (half-life of 13 min at 37 degrees C and enzyme/substrate ratio of 1:10). Cleavage and inactivation of alpha(2)-AP by MMP-3 may constitute a mechanism favoring local plasmin-mediated proteolysis.
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PMID:Inactivation of the serpin alpha(2)-antiplasmin by stromelysin-1. 1141 Feb 76

The current article describes a new assay to measure thiopurine methyltransferase (TPMT) activity from red blood cells. This method is based on the measurement of the reaction product 6-methylmercaptopurine (6-MMP) by high-performance liquid chromatography (HPLC). 6-MMP is extracted by ethyl acetate with recoveries of 85%, 80%, 80%, and 92% for 50, 250, 500, and 1,000 ng/100 microL packed red blood cells, respectively. 6-MMP was identified and measured by a Zorbax CN column installed in an HPLC system. The chromatograms were resolved using a mobile phase consisting of 40 mmol/L sodium phosphate buffer (pH 3) and methanol in a gradient from 1% to 20% of methanol. Under these conditions 6-MMP is well resolved from substrates (6-mercaptopurine and S-adenosyl-L-methionine) and endogenous peaks. When the TPMT activity from 20 patients was measured by the HPLC-linked assay and the classic radiochemical method, a linear correlation was obtained between both procedures ( y = 0.99x + 0.33; x-axis, radiochemical assay; y-axis, HPLC-linked assay; r = 0.98). In conclusion, the current report describes a new, reliable, safe, and nonradioactive method to measure TPMT activity that is shorter and simpler than the previously described ones.
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PMID:Determination of thiopurine methyltransferase activity in human erythrocytes by high-performance liquid chromatography: comparison with the radiochemical method. 1159


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