Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The amino acid sequences of three fragments obtained on cyanogen bromide cleavage of human transferrin have been determined. Two of the fragments are small (4 and 7 residues) and had not been isolated in previous studies of the CNBr fragments of transferrin. The sequence of the larger fragment (53 residues) was elucidated by examining peptides isolated from digests of the fragment with trypsin, chymotrypsin or thermolysin. This region of transferrin appears to contain the sites of three previously-reported substitutions in the D1 and D-chi genetic variants.
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PMID:The amino-acid sequences of three cystine-free cyanogen-bromide fragments of human serum transferrin. 112 16

The precursor of matrix metalloproteinase 9 (proMMP-9), also known as '92 kDa progelatinase/type IV procollagenase', was purified from the conditioned medium of U937 monocytic leukaemia and HT1080 fibrosarcoma cell lines stimulated with phorbol 12-myristate 13-acetate. ProMMP-9 in these culture media is non-covalently complexed with the 29 kDa tissue inhibitor of metalloproteinases (TIMP), but free proMMP-9 was separated from the TIMP-proMMP-9 complex by chromatography on Green A Dyematrex gel. The final product was homogeneous on SDS/PAGE, with a molecular mass of 88 kDa without reduction and 92 kDa with reduction. Treatment of proMMP-9 with 4-aminophenylmercuric acetate converted the 88 kDa precursor into 80 kDa and 68 kDa forms. Gelatin-containing zymographic analysis showed zones of lysis associated with all three species. However, only the 68 kDa species was shown to be catalytically active by its ability to bind to alpha 2-macroglobulin. In the presence of an equimolar amount of TIMP, only the 80 kDa species was generated by treatment with 4-aminophenylmercuric acetate, but no enzyme activity was detected. This indicates that TIMP binds to the 80 kDa intermediate and inhibits the generation of the active 68 kDa species. Eight endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, cathepsin G, neutrophil elastase and thermolysin) were tested for their ability to activate proMMP-9. Of them, trypsin was the most effective activator of proMMP-9. Only partial activation (10-30%) was observed with plasmin, cathepsin G and chymotrypsin. The active forms generated by trypsin were identified as 80 kDa, 74 kDa and 66 kDa by their abilities to bind to alpha 2-macroglobulin. In the presence of an equimolar amount of TIMP, proMMP-9 was also converted into the same molecular-mass species by trypsin, but they were not proteolytically active. This suggests activated MMP-9 is inhibited by TIMP. Activated MMP-9 digested gelatin, type-V collagen, reduced carboxymethylated transferrin and, to a lesser extent, type-IV collagen and laminin A chain. The specific activity against gelatin was estimated to be 15,000 units/mg (1 unit = 1 microgram of gelatin degraded/min at 37 degrees C) by titration with alpha 2-macroglobulin. Comparative studies on digestion of gelatin and collagen types IV and V by MMP-9 and MMP-2 indicated that both enzymes degrade these substrates into similar fragments. However, the susceptibilities of laminin, fibronectin and reduced carboxymethylated transferrin to these two MMPs were sufficiently different to indicate differences in substrate specificities between these two closely related proteinases.
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PMID:Purification and characterization of matrix metalloproteinase 9 from U937 monocytic leukaemia and HT1080 fibrosarcoma cells. 137 48

The primary structure of p97 (melanotransferrin) has been compared with other members of the transferrin superfamily. A molecular structure of p97 has been modelled based on the crystal structure of diferric rabbit serum transferrin. The most significant amino acid substitutions in p97 are almost exclusively limited to only two regions; the C-lobe iron-binding cleft and the interlobe contact region. The latter includes within the N-terminal lobe a Zn-binding consensus sequence found in metallopeptidases, and in the C-terminal lobe a glutamic acid residue (Glu-394) capable of completing a potential thermolysin-like Zn-binding site. Thus, p97 may have a Zn-binding potential, unique amongst the transferrin superfamily.
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PMID:A molecular model for the tumour-associated antigen, p97, suggests a Zn-binding function. 163 59

Various purified proteins, protein derivatives and two polysaccharides were added individually to a physiological medium in order to effect uptake of 125I-thrombin by the rabbit aorta endothelium. Over a wide range of concentration (0.004-40 mg/ml), the presence of either purified rabbit or bovine albumin during thrombin uptake encouraged an increase (70-110%) in 125I-thrombin binding by the endothelium and subendothelium compared to uptake by aorta segments in the absence of added protein. Pretreatment of aorta segments with albumin before incubation with 125I-thrombin in the absence of albumin did not encourage thrombin uptake to the same extent as having 125I-thrombin and albumin together. Purified human transferrin, rabbit IgG, chicken ovalbumin or denatured bovine casein could replace albumin to produce a similar enhancement of thrombin uptake. Replacing active concentrations of albumin by either reduced-carboxymethylated albumin, defatted albumin, plasmin-treated or thermolysin-treated albumin also caused an increase (50-130%) in thrombin binding, whereas replacement by acid-hydrolysed albumin or with polyglutamic acid was either ineffective or even inhibitory. Lysine-modified or arginine-modified albumins caused a small enhancement (14-32%) and no enhancement of thrombin uptake, respectively. Dextran, at low concentration (0.04-0.4 mg/ml) did not influence thrombin uptake, and at higher concentration (4-40 mg/ml) caused a decrease in uptake by both the endothelium and subendothelial layers. Low concentration of dextran sulphate inhibited thrombin uptake to 20-30% of control values. These data express the importance of accompanying protein in the response of the vascular endothelium during binding of thrombin. The possibility that other protein-cell interactions may be similarly influenced by macromolecular solutes is also discussed.
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PMID:The presence of plasma proteins facilitates the uptake of 125I-thrombin by the rabbit thoracic aorta endothelium in vitro. 242 49

We have investigated the effect in solution of synthetic carrier ampholytes on the saturation of human serum transferrin. By spectrophotometric titrations of human serum transferrin with various Fe3+-carrier ampholyte solutions, we demonstrated that under these conditions carrier ampholytes behave as typical chelators, their binding curves being very similar to that obtained with disodium nitrilotriacetate. On performing titration experiments at three different pH values, carrier ampholytes act like nitrilotriacetate at pH 7.5, but the former are more effective iron donors at pH 8.4 and worse iron donors at pH 5.2. Spectrophotometric titrations of isolated C-terminal and N-terminal fragments obtained from human serum transferrin by thermolysin cleavage show no differences between them, and no differences with respect to the whole protein except that they contain half the number of binding sites. In order to determine a site-specificity of iron in the presence of ampholytes, the classical urea/polyacrylamide-gel-electrophoresis technique was adopted. Under saturating conditions carrier ampholyte solutions act mostly on the C-terminal site, whereas desaturating agents remove iron preferentially from the N-terminal site. Our findings support the hypothesis that Ampholine may chelate Fe3+ as well as many other compounds.
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PMID:Effect of synthetic carrier ampholytes on saturation of human serum transferrin. 273 May 92

A single-sited iron-binding fragment of human transferrin has been obtained by thermolysin cleavage of the protein, selectively loaded with iron in the C-terminal binding site, in a urea-containing buffer. The fragment contains carbohydrate, and hence derives from the C-terminal half of transferrin. Its metal-binding site accepts Fe3+ and Cu2+ with bicarbonate as accompanying anion, but only Fe3+ with oxalate as anion. EPR spectroscopic properties of the fragment are similar to those of the corresponding site in the intact protein. However, iron-binding by the fragment is weaker than by the C-terminal site of the intact protein, particularly at low pH, suggesting that overall as well as local protein conformation influences the metal-binding functions of the site.
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PMID:Preparation and properties of a single-sited fragment from the C-terminal domain of human transferrin. 298 30

The amino acid sequences of seven cyanogen bromide fragments of human serum transferrin have been determined, and the primary structure of transferrin established by determining the order of these and three additional fragments (Sutton, M. R., MacGillivray, R. T. A., and Brew, K. (1975) Eur. J. Biochem. 51, 43-48) in the polypeptide chain. The order of the fragments was deduced from peptides that overlap methionyl residues which were obtained by thermolysin digestion of performic acid-oxidized transferrin or by partial peptic hydrolysis of unmodified transferrin, together with other evidence. The polypeptide chain of transferrin contains 679 amino acid residues, which together with the two N-linked oligosaccharide chains gives a calculated molecular weight of 79,570. Transferrin consists of two homologous domains (residues 1-336, 337-679), each associated with a single Fe-binding site, with both sites of glycosylation in the carboxyl-terminal domain at positions 413 and 611. Consideration of the primary structure in relation to previously published results provides information concerning the evolutionary development of transferrins and related proteins, and the locations of metal-binding residues in the transferrin molecule.
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PMID:The primary structure of human serum transferrin. The structures of seven cyanogen bromide fragments and the assembly of the complete structure. 683 13

The aim of the present study was to determine if human N-terminal half-transferrin (N- fragment), prepared by thermolysin cleavage of diferric transferrin, would bind to the rat hepatocyte transferrin receptor and donate iron to the cell. Competition experiments between 125I-labelled N-fragment and diferric transferrin revealed no receptor binding of the half-transferrin. Still, the N-fragment delivered iron to the cells in amounts approximately 30-fold above what could be accounted for by uptake of the fragment itself. The rate of cellular iron uptake from the fragment was comparable to what is seen with the intact transferrin. The uptake of 125I-labelled N-fragment was not inhibited by excess non-radioactive diferric transferrin. By comparison, the uptake of 59Fe from the N-fragment was inhibited 70% by excess nonradioactive diferric transferrin. This suggests that iron derived from diferric transferrin competes with the iron derived from the N-fragment for a common transport pathway. Although some cellular degradation of the N-fragment occurred, the extent of degradation was too low to explain the amount of iron accumulated by the cells. The results show that the hepatocyte has an effective transferrin-receptor-independent mechanism for accumulation of iron from transferrin.
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PMID:Uptake of iron from N-terminal half-transferrin by isolated rat hepatocytes. Evidence of transferrin-receptor-independent iron uptake. 755 41

The present study was initiated to identify the region(s) of ovotransferrin involved in binding to the bacterial transferrin receptors from Haemophilus paragallinarum and Haemophilus avium. Ovotransferrin was digested with either trypsin or thermolysin to obtain its N-lobe and C-lobe fragments. The individual fragments were then purified by a combination of gel exclusion and ion-exchange chromatography. Solid phase binding experiments with the individual fragments demonstrated that the C-lobe fragments blocked the binding of horse radish peroxidase-conjugated ovotransferrin to the transferrin receptors and that much higher concentrations of the N-lobe fragment were required for any detectable blocking. Affinity isolation of the bacterial transferrin receptor from the two Haemophilus species revealed that both native ovotransferrin and its C-lobe fragment were capable of isolating two iron repressible outer membrane proteins. These 95 and 60 kDa outer membrane proteins correspond to Tbp1 and Tpb2, respectively. In contrast, the N-lobe fragment was capable of isolating Tbp2 of H. paragallinarum but not that of H. avium. The inability of the N-lobe and C-lobe fragments from ovotransferrin and human transferrin to support the growth of iron-limited cultures of H. paragallinarum and Neisseria meningitidis, respectively, suggested that interaction with both lobes is necessary for efficient iron acquisition.
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PMID:Transferrin binding protein two interacts with both the N-lobe and C-lobe of ovotransferrin. 872 96

In acute intraocular inflammation, neutrophils release a variety of agents, that are potentially toxic to the surrounding tissues. The reactive oxygen metabolites, including superoxide are among these injurious agents. In the present study, retinal pigment epithelial (RPE) cells were found to inhibit by 70% to 80% the production of superoxide by neutrophils that were stimulated either by the receptor-coupled activator, N-formyl-methionyl-leucyl-phenylalanine, or by the non-receptor-coupled activator, phorbol myristate acetate. The inhibition is effective with a relatively small number of RPE cells (0.65 x 10(5)) mixed in a large pool of neutrophils (7.6 x 10(5)). The transduction of this effect does not require a neutrophil-RPE cell surface contact since the RPE culture supernatants also effectively inhibit the superoxide production. This protein is secreted specifically by cultured and noncultivated intact RPE cells, but not by fibroblasts, corneal epithelial cells or intact choroidal tissues. The protein is not cytotoxic to the neutrophils, and the inhibitory effect occurs in a dose-dependent manner up to the concentration tested. This factor is unrelated to either transforming growth factor-beta, or transferrin. There was no evidence of RPE scavenging of superoxide generated enzymatically by hypoxanthine-xanthine oxidase system, indicating that this factor does not have superoxide dismutase activity. When RPE cells were preincubated with 10 micrograms ml-1 cycloheximide, 60% of the activity was lost, suggesting that a de novo protein synthesis is required for the activity and that the protein is a significant steady-state product of RPE cells. Incubation of the released RPP with thermolysin (10 micrograms ml-1 15 min, 37 degrees C) also eliminated the activity. The degradation of activity by protease and inhibition of RPP activity by cycloheximide, therefore, confirmed the protein nature of the suppressive factor. This protein from RPE cells appears to act directly on the neutrophils, reducing the release of oxygen metabolites during activation, and thus limiting tissue injury during inflammation.
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PMID:A novel retinal pigment epithelial protein suppresses neutrophil superoxide generation. I. Characterization of the suppressive factor. 906 78


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