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)

A prokaryotic expression plasmid, pKK-DT2, containing the cDNA of rat liver NAD(P)H:quinone-acceptor oxidoreductase (EC 1.6.99.2; DT-diaphorase) was constructed and used to transform Escherichia coli strain JM109. The rat liver quinone reductase was expressed in strain in JM109 and was inducible with isopropyl beta-D-thiogalactopyranoside (IPTG). The expressed rat protein was purified by affinity chromatography and had kinetic and physical properties identical with the protein purified from rat liver in that it could utilize either NADH or NADPH as the electron donor and its activity was inhibited by dicoumarol. In addition, we have generated four mutants, Arg-177----His (R177H), Arg-177----Ala (R177A), Arg-177----Cys (R177C) and Arg-177----Leu (R177L), using this expression system. Several of the mutants behaved anomalously on SDS/PAGE, but all of the mutant proteins had the expected M(r) as determined by electrospray m.s. These results and those obtained from enzyme kinetic analysis, u.v./visible absorption spectral analysis, and flavin and tryptophan fluorescence analysis of the wild-type enzyme and four mutants indicated that mutations at Arg-177 changed the conformation of the enzyme, resulting in a decrease in enzyme activity. Replacing Arg-177 with leucine altered the protein conformation and decreased FAD incorporation.
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PMID:Expression of rat liver NAD(P)H:quinone-acceptor oxidoreductase in Escherichia coli and mutagenesis in vitro at Arg-177. 162 1

Extracts prepared from tissue with granulomatous inflammation experimentally produced in liver of CBA-strain mice showed increased hydrolysis of leukotriene D4 (LTD4), Leu-Leu and Ala-Gly as compared with normal hepatic cells. Two dipeptidases, Leu-Leu dipeptidase and Ala-Gly dipeptidase, were purified from hepatic granulomas, and quantitative conversion of LTD4 into leukotriene E4 (LTE4) by both enzymes was demonstrated. M(r) values of the purified enzymes were 178,000 for Leu-Leu dipeptidase and 183,000 for Ala-Gly dipeptidase. The enzymes showed homogeneity, appearing as a single band on SDS/PAGE, and the M(r) values of the subunits were 56,000 and 57,000 for Leu-Leu and Ala-Gly dipeptidase respectively. The amino acid compositions of the two enzymes differed considerably from each other. The activity of Leu-Leu dipeptidase was inhibited by bestatin and captopril and stabilized with MnCl2. The Km for LTD4 was 25 microM with a V(max.) of 49.0 mumols/min per mg. In contrast, the activity of Ala-Gly dipeptidase was inhibited by cilastatin, cytinylglycine, EDTA and dithiothreitol, and also by captopril. The Km for LTD4 was 5.3 microM with a V(max.) of 50.4 mumols/min per mg. The findings indicate that the conversion of LTD4 into LTE4 by microsomal dipeptidases is elevated during granulomatous tissue reaction. This enzyme activity may become useful for biochemical quantification of the pathological tissue reaction that occurs in organized granulomas.
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PMID:Characterization of two dipeptidases purified from hepatic schistosome egg granulomas in mice. Leukotriene D4 hydrolases of granulomatous tissue. 162 4

beta 1----4 Galactosyltransferase was purified from rat liver microsomes. Catalytic properties of the enzyme resembled those of previously purified soluble and membrane-bound beta 1----4 galactosyltransferases. The enzyme purified in the present study showed a major band around a molecular weight of 53,000 on SDS-PAGE. The NH2-terminal sequence of the enzyme was determined up to the 20th residue. The sequence was identical to the amino acid sequence from Ala-13 to Lys-32 deduced from mouse beta 1----4 galactosyltransferase cDNA. These results suggest that most of the mature enzyme in rat liver microsomes is produced by removal of the NH2-terminal 12 amino acids from a precursor polypeptide.
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PMID:Characterization of beta 1----4 galactosyltransferase purified from rat liver microsomes. 163 51

A new type of mitogenic factor (protein) was purified from the culture supernatant of a strain of Streptococcus pyogenes by SP-Sephadex C-25 column chromatography, preparative isoelectric focusing and reversed-phase high-performance liquid chromatography. The purified factor, showing marked mitogenic activity in rabbit peripheral blood lymphocytes, gave a single-band staining for protein on SDS-PAGE. The molecular weight of the purified mitogenic factor was determined to be 25,370, which was different from those calculated from reported amino acid sequences deduced from 4 different nucleotide sequences of 3 kinds of streptococcal pyrogenic exotoxins (two SPEAs, SPEB and SPEC). The amino acid sequence of the N-terminal region of the purified mitogenic factor was determined to be Gln-Thr-Gln-Val-Ser-Asn-Asp-Val-Val-Leu-Asn-Asp-Gly-Ala-Ser-Lys-Tyr-Leu- Asn-Glu - Ala-, which was also different from the reported N-terminal sequences deduced from the 4 different nucleotide sequences. These data indicate that this mitogenic factor is distinct from the already described streptococcal pyrogenic exotoxins.
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PMID:A new type of mitogenic factor produced by Streptococcus pyogenes. 164

A fibrinogenolytic proteinase has been isolated from Aspergillus fumigatus culture filtrate by ammonium sulfate precipitation followed by successive chromatographies on Sephadex G-75 and immobilized phenylalanine. The purified proteinase exhibited a molecular weight of about 33 kDa. When analysed by SDS-polyacrylamide gels containing co-polymerized fibrinogen, the proteinase appeared as a broad band at the top of the gels, which could correspond to polymerization of the enzyme, as suggested by SDS-PAGE analysis of the unboiled eluate. The isoelectric point was 8.75 and the enzyme was not glycosylated. Proteinase activity was optimum at pH 9 and between 37 and 42 degrees C, although a decrease in activity was observed above 37 degrees C. PMSF and chymostatin markedly inhibited the proteinase activity, and good kinetic constants were obtained for the synthetic substrate, N-Suc-Ala-Ala-Pro-Phe-pNA. These results provide direct evidence that this enzyme belongs to the chymotrypsin-like serine proteinase group.
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PMID:Purification and characterization of a fibrinogenolytic serine proteinase from Aspergillus fumigatus culture filtrate. 164 3

A metal-dependent peptidase was isolated from the homogenate of human uterus by standard chromatographic techniques and purified to apparent homogeneity. The peptidase hydrolysed the synthetic vertebrate collagenase substrate 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg (Dnp-peptide), the synthetic bacterial collagenase substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (PZ-peptide) and gelatinolytic peptides of gelatin, but was inactive against collagen type I, gelatin and casein. The cleavage site for the Dnp-peptide was the Gly-Ile bond. The enzyme was not only inhibited by metal chelators, such as EDTA, 1,10-phenantroline and dithiothreitol but also by thiol reagents, such as mersalylic acid and N-ethylmaleimid. However, E-64, an inhibitor for thiolproteinases, and leupeptin, an inhibitor for thiol- and serine proteases, did not exhibit any inhibitory activity. Pepstatin, an inhibitor for aspartate proteinases, and inhibitors for serine proteinases like phenylmethanesulfonyl fluoride and Trasylol were ineffective as well. The purified peptidase displayed a single band in the SDS-PAGE with an apparent molecular mass of 65 kDa. Employing isoelectric focusing an IP of 5.0 could be determined. The enzyme's properties are discussed in relation to the proteinase EC 3.4.24.11 and to proteinases of the collagenase family as well as the possibility to discriminate these three metalloproteinase classes by employing the Dnp-peptide.
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PMID:Isolation and properties of a metal-dependent endopeptidase from human uterus hydrolysing synthetic collagenase substrates. 165 Feb 34

An enzyme which catalyzes the transamination of L-alanine with 2-oxoglutarate has been purified 157-fold to electrophoretic homogeneity from the unicellular green alga Chlamydomonas reinhardtii 6145c. The enzyme showed maximal activity at pH 7.3 and 50 degrees C, has an apparent molecular mass of 105 kDa as estimated by gel filtration, and consists of two identical subunits of 45 kDa each as deduced from PAGE/SDS studies. A stoichiometry of two moles pyridoxal 5-phosphate/mole enzyme was calculated. The enzyme has an isoelectric point of 8.3 and its absorption spectrum exhibits a maximum at 412 nm which is shifted to 330 nm upon addition of L-alanine. Pyridoxal 5-phosphate protected activity against heat inactivation and, to a minor extent, L-alanine and 2-oxoglutarate, but not L-glutamate. Spectral data and activity inhibition and protection studies strongly support the involvement of pyridoxal 5-phosphate in enzyme catalysis through a Schiff's base formation. The purified enzyme was able to transaminate only L-alanine and L-glutamate with glyoxylate out of ten amino acids tested. L-Alanine aminotransferase exhibited hyperbolic kinetic for 2-oxoglutarate, pyruvate, and L-glutamate, and nonhyperbolic behaviour for L-alanine. Apparent Km values were 0.054 mM for 2-oxoglutarate, 0.52 for L-glutamate, 0.24 mM for pyruvate, and 2.7 mM for L-alanine. Transamination of L-alanine in C. reinhardtii is a bisubstrate reaction with a bi-bi ping-pong mechanism, and is not inhibited by substrates.
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PMID:Purification and properties of L-alanine aminotransferase from Chlamydomonas reinhardtii. 166 17

Myelin basic proteins (MBPs) from 6-day-old, 10-day-old, 20-day-old and adult normal mouse brain were compared with those from 20-day-old jimpy (dysmyelinating mutant) mouse brain to determine the effect of reduced levels of proteolipid protein (PLP) on MBPs. Alkaline-urea-gel electrophoresis showed that 6-day-old and 10-day-old normal and jimpy MBPs lacked charge microheterogeneity, since C8 (the least cationic of the components; not be confused with complement component C8) was the only charge isomer present. In contrast, MBPs from 20-day-old and adult normal mouse brain displayed extensive charge microheterogeneity, having at least eight components. A 32 kDa MBP was the major isoform observed on immunoblots of acid-soluble protein from 6-day-old and 10-day-old normal and 20-day-old jimpy mouse brain. There were eight bands present in 20-day-old and adult normal mouse brain. Purified human MBP charge heteromers C1, C2, C3 and C4 reacted strongly with rat 14 kDa MBP antiserum, whereas the reaction with human C8 was weak. This suggested that MBPs from early-myelinating and jimpy mice did not react to MBP antisera because C8 was the major charge isomer in these animals. Purification of MBPs from normal and jimpy brain by alkaline-gel electrophoresis showed that both normal and jimpy MBPs have size heterogeneity when subjected to SDS/PAGE. However, the size isoforms in normal mouse brain (32, 21, 18.5, 17 and 14 kDa) differed from those in jimpy brain (32, 21, 20, 17, 15 and 14 kDa) in both size and relative amounts. Amino acid analyses of MBPs from jimpy brain showed an increase in glutamic acid, alanine and ornithine, and a decrease in histidine, arginine and proline. The changes in glutamic acid, ornithine and arginine are characteristic of the differences observed in human C8 when compared with C1.
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PMID:Myelin basic protein is affected by reduced synthesis of myelin proteolipid protein in the jimpy mouse. 169 71

Antibodies have been raised to synthetic peptides, corresponding to a region in the loop spanning helices 4 and 5 of D 1 protein (Ala 250-Phe 265) and to a region anticipated to be near the C terminus of mature D 1 (His 332-Ala 345). Polyclonal antibodies to the sequence His 332-Ala 345 reacted with a 32 kDa polypeptide in thylakoid preparations identified as D 1 from its resistance (pea) or susceptibility (wheat) to lysine-C degradation. A monoclonal antibody to His 332-Ala 345 reacted preferentially with a faster migrating polypeptide in SDS electrophoresis, a putative conformer of D 1. Polyclonal antibodies to the sequence Ala 250-Phe 265 also reacted with the faster running polypeptide but not with the population of molecules running at 32 kDa. The putative conformer of D 1 from wheat appears to be more resistant than the main D 1 population to lysine-C degradation. Peptide analyses by Takahashi et al. [(1988) FEBS Lett, 240, 6-8] suggest Asn 335-Ala 344 lies at the processed C terminus. The present report provides immunological confirmation that this sequence is retained in mature D 1.
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PMID:Characterization of conformers of D 1 of photosystem II using site-directed antibodies. 169 61

Bovine brain inositol monophosphatase is rapidly cleaved by endoprotease lys-C at a single site in the absence of SDS. Further sites are revealed only after prolonged incubation with high concentrations of protease. The initial cleavage occurs near one end of the enzyme, generating an N-terminally-derived 36-residue peptide, which is blocked, and a large 28 kDa fragment bearing a free N-terminus. The start sequence of this fragment was found to be Xaa-Ser-Pro-Ala-Asp-Leu-Val, consistent with the cDNA sequence, and Lys-36-Ser-37 was identified as the cleavage site. The activity of the cleaved enzyme was markedly decreased to 3% of that of the native enzyme, although its dimeric structure was preserved. The 36-residue peptide was not covalently associated with the large fragment after proteolytic cleavage, although the possibility of non-covalent association could not be excluded. Finally, the epitope for the inhibitory monoclonal antibody G-2A4 [Gee, Howell, Ryan & Ragan (1989) Biochem J. 264. 793-798] was found to lie proximal to the endoprotease lys-C cleavage site. In vitro mutagenesis further mapped the epitope for monoclonal antibody G-2A4 to residues around Cys-8 of the enzyme. These results suggest that the N-terminal region of the enzyme is important for activity.
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PMID:Limited proteolysis and 'in vitro' mutagenesis of bovine brain inositol monophosphatase identifies an N-terminal region important for activity. 170 24


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