Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Endothelin-3 (ET-3), a potent vasoactive peptide, is considered to be produced from big ET-3 by endothelin-converting enzyme (ECE) like the other members of the endothelin family (ET-1 and ET-2). We purified a novel ECE from bovine iris microsomes. The purified enzyme, a 140 kDa protein by SDS-PAGE analysis, converted big ET-3 to ET-3 but not big ET-1, with a Km value of 0.14 microM for big ET-3. The conversion to ET-3 was confirmed with sandwich EIA by monoclonal antibodies, the elution profile of HPLC, and intracellular calcium mobilization in CHO-K1 cells expressing recombinant human ET(B) receptors. The conversion activity was inhibited by an inhibitor of neutral endopeptidase 24.11 (NEP) phosphoramidon. These results show that ECE-3 purified from bovine iris is a novel metalloprotease totally different from ECE-1 or ECE-2, in that the enzyme is highly specific for big ET-3.
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PMID:Purification of a novel endothelin-converting enzyme specific for big endothelin-3. 965 54

A membrane-bound protease induced by sulfur mustard in cultured normal human epidermal keratinocytes (NHEK) was purified and partially characterized. Maximum enzyme stimulation occurred at 16 hr after normal human epidermal keratinocytes were exposed to 300 microM sulfur mustard. Purification to homogeneity of the protease was accomplished by Triton X-100 solubilization, ultracentrifugation, and dialysis, followed by ion-exchange chromatography through DEAE-cellulose and finally hydrophobic column chromatography through phenyl Sepharose. Analysis of the purified enzyme by SDS-PAGE revealed a single polypeptide at the 80 kDa region. Further investigation of biochemical properties showed that a synthetic serine-specific Chromozym TRY peptide and the physiological protein laminin were good substrates for this enzyme. Moreover, this enzyme was inhibited mostly by the serine-protease inhibitors leupeptin and di-isopropyl fluorophosphate and not by the cysteine protease inhibitor E-64 or the metalloprotease inhibitor 1,10-phenanthroline (Component H, CH), indicating the serine protease nature of this enzyme. This enzyme had a pH optimum in the range of 7.0 to 8.0. Amino acid sequencing of the purified enzyme revealed that this enzyme belongs to the endopeptidase family (serine protease), and is homologous with a mammalian-type bacterial serine endopeptidase that can preferentially cleave K-X, including K-P. These results suggest that serine-protease stimulation may be one of the mechanisms of mustard-induced skin blister formation, and that some specific serine-protease inhibitors may be useful for the treatment of this sulfur mustard toxicity.
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PMID:Purification and characterization of protease activated by sulfur mustard in normal human epidermal keratinocytes. 976 22

A non-hemorrhagic proteinase, brevilysin L6 (L6), has been purified to homogeneity from Agkistrodon halys brevicaudus venom by gel filtration and DEAE-Toyopearl 650M chromatography. It is an acidic protein with an isoelectric point of 4.8, and its molecular mass was estimated to be 21.5 kDa by SDS-PAGE. The optimum pH of L6 was about 9. EDTA and o-phenanthroline inhibited the proteolytic activity, suggesting that L6 is a metalloprotease. Cysteine also inhibited the activity of L6, but glutathione did not. The protein was stable in the pH range of 5-8.5 and below 40 degreesC. Calcium ions had no effect on the proteolytic activity of L6 or on its thermal stability. The enzyme preferentially cleaved X-Leu, X-Phe, X-Val, and X-His bonds. L6 showed weak alpha-fibrinogenase activity. The complete amino acid sequence of L6 was also determined by manual Edman degradation. L6 is a non-glycosylated single-chain polypeptide consisting of 203 residues with an N-terminal pyroglutamic acid and a calculated molecular weight of 22,713 Da. Its entire sequence is highly homologous to those of other metalloproteases from various snake venoms. A zinc-binding motif, HEXXHXXGXXH, is located at residues 143-153 in the sequence of L6.
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PMID:Purification and amino acid sequence of brevilysin L6, a non-hemorrhagic metalloprotease from Agkistrodon halys brevicaudus venom. 988 Jul 98

An endopeptidase was purified from Archachatina ventricosa by chromatography on columns of gel filtration, DEAE-Sepharose and phenyl-Sepharose. The preparation was shown to be homogeneous by polyacrylamide gel electrophoresis and capillary electrophoresis. The purified enzyme displayed two protein bands on SDS-polyacrylamide gel electrophoresis with estimated molecular weights of 90,000 and 121,000. The protease exhibited maximum proteolytic activity at 55 degrees C and at pH 8.0, but it retained more than 85% of its activity in the pH range 7.5 to 8.5. It was completely inactivated by the chelating agents EDTA and 1,10-phenanthroline which are metalloprotease inhibitors. Studies on substrate specificity showed that only the amide bonds of peptide substrates having a threonine residue at the P1' position were hydrolyzed by the purified protease. This endopeptidase constitutes a novel tool for the study of proteins in view of its narrow and unique substrate specificity.
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PMID:A novel endopeptidase with a strict specificity for threonine residues at the P1' position. 1008 Sep 15

The prtV gene, encoding a collagenase of Vibrio parahaemolyticus, was expressed in Escherichia coli and purified by affinity chromatography. The transformant E. coli BL21(DE3)(pPRT2) secreted the recombinant PrtV, and the highest enzyme activity was detected in the culture supernatant after 5 h IPTG induction. The molecular mass of purified PrtV was 62 kDa as determined by gel filtration, which was similar to that obtained by SDS-PAGE (64 kDa). This suggested that PrtV was a monomer protein having no subunit structure. The isoelectric point of PrtV was 8.52. In addition, PrtV contained a 27 amino acid signal peptide, and the amino acid composition of the PrtV showed satisfactory agreement with that predicted from the DNA sequence. The optimum temperature and pH of PrtV were 40 degrees C and pH 7.5, respectively. The activity of PrtV was inhibited by chelators such as EDTA, EGTA and 1,10-phenanthroline; however, its activity was restored by the addition of various metal ions (Co2+, Mn2+, Ca2+, Cu2+, Ni2+ and Zn2+), indicating that PrtV is a metalloprotease. PrtV degraded both type I collagen and synthetic substrate FALGPA well, showing that PrtV is indeed a collagenase.
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PMID:Expression and characterization of the prtV gene encoding a collagenase from Vibrio parahaemolyticus in Escherichia coli. 1020 92

Immunization of sheep with a protective antigen complex from the intestinal cells of Haemonchus contortus in Freund's adjuvant stimulated individually variable antibody responses but still conferred significant protection against parasite infection. Correlation between antibody concentration and degree of protection was suggestive of antibody being the effector mechanism. The antigen is known as Haemonchus galactose-containing glycoprotein complex (H-gal-GP) because it binds to lectins with a specificity for N-acetyl-galactosamine. Polypeptide composition analysis by polyacrylamide gel electrophoresis indicated an apparent molecular weight of about 1000 kDa and SDS gels revealed four major polypeptides, containing between 2 and 5 disulphide linked subunits, nearly all being glycosylated. N-terminal amino acid sequence was obtained from 12 subunits, ten showing homologies with cDNAs from Haemonchus encoding either pepsin, metalloprotease or cysteine protease-like enzymes. pH optima, inhibitor and various substrate studies confirmed that the native complex possessed proteolytic activities in agreement with the sequence data. Although the cDNAs predicted water soluble enzymes, little of the complex was solubilized from worm membranes without the use of a detergent, such as Triton X-100. It is hypothesized that H-gal-GP is a gut membrane associated multiprotease complex which is involved in the digestion of the blood meal and which can be neutralized by specific antibodies with drastic consequences for the parasite.
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PMID:Further immunization and biochemical studies with a protective antigen complex from the microvillar membrane of the intestine of Haemonchus contortus. 1032 Jun 16

Endothelin-converting enzyme-1 (ECE-1) is a membrane-bound metalloprotease that catalyses the conversion of inactive big endothelins into active endothelins. Here we have examined whether the three isoforms of human ECE-1 (ECE-1a, ECE-1b and ECE-1c) are modified by the covalent attachment of the fatty acid palmitate and have evaluated a potential functional role of this modification. To do this, wild-type and mutant enzymes were expressed and analysed by metabolic labelling with [3H]palmitate, immunoprecipitation and SDS/PAGE. All three ECE-1 isoforms were found to be palmitoylated via hydroxylamine-sensitive thioester bonds. In addition, the isoforms showed similar levels of acylation. Cys46 in ECE-1a, Cys58 in ECE-1b and Cys42 in ECE-1c were identified as sites of palmitoylation and each of these cysteines accounted for all the palmitoylation that occured in the corresponding isoform. Immunofluorescence analysis demonstrated further that palmitoylated and non-palmitoylated ECE-1 isoforms had the same subcellular localizations. Moreover, complete solubility of the three isoforms in Triton X-100 revealed that palmitoylation does not target ECE-1 to cholesterol and sphingolipid-rich membrane domains or caveolae. The enzymic activities of ECE-1a, ECE-1b and ECE-1c were also not significantly affected by the absence of palmitoylation.
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PMID:Palmitoylation of the three isoforms of human endothelin-converting enzyme-1. 1035 48

The cysteine protease known as "prohormone thiol protease" (PTP) has been identified as a major proenkephalin processing enzyme in secretory vesicles of adrenal medulla (known as chromaffin granules). This study provides the first demonstration that PTP exists as a multicatalytic cysteine protease complex that can be activated by endogenous glutathione present in chromaffin granules. The high molecular mass nature of PTP, of approximately 185 kDa, was demonstrated by elution of a single peak of 35S-enkephalin precursor cleaving activity by Sephacryl S200 gel filtration chromatography and by a single band of 35S-enkephalin precursor cleaving activity detected on radiozymogram gels under native buffer conditions. Importantly, when 0.1% SDS was included in radiozymogram gels, PTP activity was resolved into three bands of proteolytic activity with apparent molecular masses of 88, 81, and 61 kDa. These activities were all cysteine proteases, since they were inhibited by the cysteine protease inhibitor E-64c but not by pepstatin A or EDTA that inhibit aspartyl protease and metalloprotease, respectively. Purification of native PTP by preparative gel electrophoresis indicated that PTP was composed of four polypeptides of 66, 60, 33, and 29 kDa detected on SDS-PAGE gels. These four protein subunits accounted for the three catalytic activities of PTP, as demonstrated on 35S-enkephalin precursor radiozymogram gels. Results also indicated that the electrophoretic mobilities of the four subunits differed under reducing compared to nonreducing conditions. The multicatalytic activities of the PTP complex all require reducing conditions for activity, which can be provided by endogenous reduced glutathione in chromaffin granules. These novel findings provide the first evidence for a role of a multicatalytic cysteine protease complex, PTP, in chromaffin granules that may be involved in the proteolytic processing of proenkephalin and perhaps other precursors into active neuropeptides.
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PMID:Evidence for the proenkephalin processing enzyme prohormone thiol protease (PTP) as a multicatalytic cysteine protease complex: activation by glutathione localized to secretory vesicles. 1036 Sep 39

Strains of Vibrio cholerae O1 (Ogawa, Inaba) and non-O1 serogroups have been found to produce an elastolytic protease that can be detected on 0.3% elastin agar plates or in broth cultures. The elastase enzyme appears to be maximally expressed in late log phase (14-18 h postinoculation) and has optimum activity at a pH range between 7 and 8. Comparative studies indicate that more than 60% of V. cholerae strains analyzed quantitatively produce more elastase in broth (two- to fourfold higher) than other elastase-positive Vibrio species such as Vibrio vulnificus. The V. cholerae elastase enzyme was not inhibited by trypsin, serine-protease, or thiol-protease inhibitors, but was inhibited by phosphoramidon. Ultrafiltration studies indicate the V. cholerae elastase enzyme has a molecular weight >30,000, and a 34K protein with possible elastase activity has been detected by SDS-PAGE for one non-O1 isolate (strain 2396). Cumulative results suggest that the V. cholerae elastase is probably a member of the N-type metalloprotease family and shares similar properties with other elastase enzymes described for pathogenic and nonpathogenic species in this genus.
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PMID:Identification and initial characterization of elastase activity associated with Vibrio cholerae. 1039 30

A protease produced by Staphylococcus aureus, isolated from a chicken suffering from dermatitis, was purified by successive precipitation with ammonium sulfate, ion-exchange chromatography on Q-Sepharose FF, Sp-Sepharose FF and Mono-Q columns. By Mono-Q column chromatography, two proteases (protease 1 and 2) were obtained. The molecular weights of protease 1 and 2 were estimated at 23.1 and 22.7 kDa, respectively, by SDS-polyacrylamide gel electrophoresis. Their isoelectric points were 5.85 and 5.55, respectively, and they possessed antigenic similarity when examined by the immunoblotting. The N-terminal amino acid sequences of both the proteases were identical (RAQYVNQLKNFKIRETQ). The activities of both the proteases were strongly increased by reducing agents such as L-cysteine and sodium thioglycolate. Their activity was inhibited by thiol protease inhibitors, but was not inhibited by metalloprotease or serine protease inhibitors. From the results, it seems likely that these proteases, produced by S. aureus from diseased chickens, might belong to the thiol protease group.
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PMID:Purification and characterization of protease produced by Staphylococcus aureus isolated from a diseased chicken. 1041 73


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