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)

Incubation of rat liver microsomes with norethindrone and a NADPH-generating system leads to the formation of one N-alkylated porphyrin (green pigment, GP1). Administration of this steroid to male rats in vivo results in the formation of three more-polar green pigments (GP2, 3 and 4). A cytosolic protein (green-pigment converting protein) has been purified from rat liver that, when added to liver microsomal mixtures containing norethindrone (0.03 mM) and a NADPH-generating system, results in the formation of all four green pigments (GP1, 2, 3 and 4). Field-desorption mass spectrometry of the purified green pigments gave protonated molecules, [M + H]+, at m/z 905 for GP1, m/z 909 for GP2, m/z 925 for GP3 and 4. The Mr of the purified cytosolic protein on SDS/polyacrylamide-gel electrophoresis or gel filtration was 37000. Polyacrylamide-gel isoelectric focusing gave a pI value of 5.9. Antibodies raised in rabbits against this protein, after preincubation with rat liver cytosol, subsequently prevented the formation of the more-polar norethindrone-induced green pigments (GP2, 3 and 4). The purified protein in the presence of either NADH or NADPH catalysed the reduction of delta 4-ring-reduced norethindrone, 5 alpha-oestran-17 alpha-ethynyl-17 beta-ol-3-one and, with the appropriate cofactor, the oxidation and reduction of steroids lacking the ethynyl function, e.g. androsterone or dihydrotestosterone. Indomethacin inhibited the reduction of dihydrotestosterone by this protein with an I50 (concn. causing 50% inhibition) value of 4.9 microM. From its physical and enzymic properties it is concluded that green-pigment converting protein is the same as 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50).
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PMID:Factors responsible for the formation of different N-alkylated porphyrins in rat liver microsomal systems exposed to norethindrone. The role of 3 alpha-hydroxysteroid dehydrogenase. 346 1

Mannitol-1-phosphate dehydrogenase was purified to homogeneity, and some chemical and physical properties were examined. The isoelectric point is 4.19. Amino acid analysis and polyacrylamide-gel electrophoresis in presence of SDS indicate a subunit Mr of about 22,000, whereas gel filtration and electrophoresis of the native enzyme indicate an Mr of 45,000. Thus the enzyme is a dimer. Amino acid analysis showed cysteine, tyrosine, histidine and tryptophan to be present in low quantities, one, three, four and four residues per subunit respectively. The zinc content is not significant to activity. The enzyme is inactivated (greater than 99%) by reaction of 5,5'-dithiobis-(2-nitrobenzoate) with the single thiol group; the inactivation rate depends hyperbolically on reagent concentration, indicating non-covalent binding of the reagent before covalent modification. The pH-dependence indicated a pKa greater than 10.5 for the thiol group. Coenzymes (NAD+ and NADH) at saturating concentrations protect completely against reaction with 5,5'-dithiobis-(2-nitrobenzoate), and substrates (mannitol 1-phosphate, fructose 6-phosphate) protect strongly but not completely. These results suggest that the thiol group is near the catalytic site, and indicate that substrates as well as coenzymes bind to free enzyme. Dissociation constants were determined from these protective effects: 0.6 +/- 0.1 microM for NADH, 0.2 +/- 0.03 mM for NAD+, 9 +/- 3 microM for mannitol 1-phosphate, 0.06 +/- 0.03 mM for fructose 6-phosphate. The binding order for reaction thus may be random for mannitol 1-phosphate oxidation, though ordered for fructose 6-phosphate reduction. Coenzyme and substrate binding in the E X NADH-mannitol 1-phosphate complex is weaker than in the binary complexes, though in the E X NADH+-fructose 6-phosphate complex binding is stronger.
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PMID:Mannitol-1-phosphate dehydrogenase of Escherichia coli. Chemical properties and binding of substrates. 354 82

Human liver BCKADH complex was purified. On SDS-polyacrylamide gel electrophoresis, the purified enzyme complex gave three major bands having molecular weights of 51,000, 46,000, and 36,000, and one minor band with a molecular weight of 55,000. The minor band corresponded in molecular weight to lipoamide oxidoreductase which was purified separately. The purified BCKADH represented only approximately 20% of the maximum activity when assayed without addition of exogenous lipoamide oxidoreductase, indicating that lipoamide oxidoreductase component was readily dissociable from the complex. The BCKADH effectively oxidized all of KIV, KIC, and KMV, yielding apparent Km values in the range of 14-17 microM for those alpha-keto acids. Vmax values obtained were 0.86, 0.61, and 0.51 mumole NADH produced/min/mg of protein for KIV, KIC, and KMV, respectively, in the presence of excess amount of lipoamide oxidoreductase. This ratio of Vmax values was practically identical to those of specific activities obtained with respective branched-chain alpha-keto acids at each purification step. The enzyme complex also oxidized pyruvate and alpha-ketoglutarate to a lesser extent. Kinetic experiments gave Km values of 0.98 and 2.9 mM for pyruvate and alpha-ketoglutarate, respectively, with Vmax of 0.43 and 0.08 mumole NADH produced/min/mg of protein. NAD and CoASH were absolutely required for the reaction. Km values for NAD and CoASH were estimated to be 47 and 25 microM, respectively.
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PMID:Purification and characterization of human liver branched-chain alpha-keto acid dehydrogenase complex. 359 87

delta 1-Pyrroline-5-carboxylate reductase (L-proline:NAD(P)+ 5-oxidoreductase, EC 1.5.1.2) has been purified from rat lens and biochemically characterized. Purification steps included ammonium sulfate fractionation, affinity chromatography on Amicon Matrex Orange A, and gel filtration with Sephadex G-200. These steps were carried out at ambient temperature (22 degrees C) in 20 mM sodium phosphate/potassium phosphate buffer (pH 7.5) containing 10% glycerol, 7 mM mercaptoethanol and 0.5 mM EDTA. The enzyme, purified to apparent homogeneity, displayed a molecular weight of 240 000 by gel chromatography and 30 000 by SDS-polyacrylamide gel electrophoresis. This suggests that the enzyme is composed of eight subunits. The purified enzyme displays a pH optimum between 6.5 and 7.1 and is inhibited by heavy metal ions and p-chloromercuribenzoate. Kinetic studies indicated Km values of 0.62 mM and 0.051 mM for DL-pyrroline-5-carboxylate as substrate when NADH and NADPH respectively were employed as cofactors. The Km values for the cofactors NADH and NADPH with DL-pyrroline-5-carboxylate as substrate were 0.37 mM and 0.006 mM, respectively. With L-pyrroline-5-carboxylate as substrate, Km values of 0.21 mM and 0.022 mM were obtained for NADH and NADPH, respectively. Enzyme activity is potentially inhibited by NADP+ and ATP, suggesting that delta 1-pyrroline-5-carboxylate reductase may be regulated by the energy level and redox state of the lens.
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PMID:Purification and characterization of rat lens pyrroline-5-carboxylate reductase. 375 84

Microsomal b-type hemoprotein designated, cytochrome b555 of C.roseus seedlings was solubilized using detergents and purified by a combination of ion exchange chromatography and gel filtration to a specific content of 18.5 nmol per mg of protein. The purified cytochrome b555 was homogeneous and estimated to have an apparent molecular weight of 16500 on SDS-PAGE. The absorption spectrum of the reduced form has major peaks at 424, 525 and 555 nm. The alpha-band of the reduced form is asymmetric with a pronounced shoulder at 559 nm. The spectrum of the pyridine ferrohemochrome shows absorption peaks at 557, 524 and 418 nm indicating that the cytochrome has protoheme prosthetic group. The purified cytochrome is autoxidizable and does not combine with carbon monoxide, azide or cyanide. It is reducible by NADH in the presence of NADH-cytochrome b555 reductase partially purified from C.roseus microsomes.
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PMID:Purification and partial characterization of microsomal cytochrome b555 from the higher plant Catharanthus roseus. 375 48

Detergent treatments were examined for their efficacy in purifying trimethylamine-N-oxide (TMAO) demethylase activity from fish muscle microsomes. Tritons X-100 and X-45, deoxycholate, Brijs, Tweens 20, 65, and 80, and SDS were generally ineffective in solubilizing demethylase activity from this membrane fraction, at concentrations up to 10 mg detergent per mg protein. In all of these cases, specific activity became enriched in the particulate fraction obtained post-treatment. Highest fold-purification was achieved by using 10 mg SDS per mg protein in 5 mM histidine, pH 7.0 at 10-14 degrees C. Activity was relatively stable to the presence of SDS at this level, and with this treatment, TMAO demethylase activity became purified in the resultant particulate fraction 28- and 58-fold for activity stimulated by ascorbate-iron-cysteine and FMN-NADH, respectively. The presence of urea or 2-mercaptoethanol, or sonication of the SDS-microsome suspension during purification resulted in significant losses of recovered activity. This partially purified fraction represented about 1% of the original microsomal protein and SDS-PAGE revealed the presence of several protein components. The partially purified demethylase could utilize the same two cofactor systems as the native microsomes. It displayed a curvilinear dependence on iron for activity and a sigmoidal response for cysteine. Utilization of NADH, FMN, and ascorbate differed for the purified fraction as compared to the microsomes. Substrate inhibition by TMAO was observed for the partially purified preparation, whereas saturation kinetics were previously noted for microsomal activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Partial purification of trimethylamine-N-oxide (TMAO) demethylase from crude fish muscle microsomes by detergents. 375 40

Plant mitochondrial NADH dehydrogenases were analysed by two immunological strategies. The first exploited an antiserum raised to a preparation of SDS-solubilized mitochondrial-inner-membrane particles. By using a combination of activity-immunoprecipitation and crossed immunoelectrophoresis, it was shown that Triton X-100-solubilized membranes contain at least three immunologically distinct NADH dehydrogenases. Two of these were subsequently isolated by line immunoelectrophoresis and analysed for polypeptide composition: one contained three polypeptides with molecular masses of 75, 62 and 41 kDa; the other was a single polypeptide with a molecular mass of 53 kDa. The other approach was to probe plant mitochondrial membranes with antibodies raised to a purified preparation of ox heart rotenone-sensitive NADH dehydrogenase and subunits thereof. Cross-reactions were observed with the subunit-specific antisera against the 30 and 49 kDa ox heart proteins. However, the molecular masses of the equivalent polypeptides in plant mitochondria are slightly lower, at 27 and 46 kDa respectively.
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PMID:Immunological analysis of plant mitochondrial NADH dehydrogenases. 379 70

Legionella pneumophila, the etiologic agent of Legionnaires' disease, is phagocytized in an unusual way and multiplies in human mononuclear phagocytes in a novel phagosome. As a first step toward understanding these L. pneumophila-phagocyte interactions, we have studied the envelope of L. pneumophila Philadelphia 1 strain. We isolated cell envelopes by treating whole bacterial cells with lysozyme and EDTA to convert them to spheroplasts, then lysing the spheroplasts osmotically or sonically. We resolved the cell envelopes into two membrane fractions by isopycnic centrifugation. We localized NADH oxidase to the fraction of buoyant density 1.145, which we designated cytoplasmic membrane, and lipopolysaccharide (LPS) to the fraction of density 1.222, which we designated outer membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the L. pneumophila outer membrane contains a single major protein species migrating at 28,000 mol wt; this is the major protein of the bacterium. The cytoplasmic membrane also contains a single major protein species migrating at 65,000 mol wt. Surface iodination of the bacteria and agglutination and immunofluorescence studies with rabbit antibody produced against the purified major outer membrane protein (MOMP) revealed that this protein is exposed at the cell surface. We isolated LPS from L. pneumophila membranes by SDS-EDTA treatment. The pattern obtained by subjecting the LPS to SDS-PAGE and staining the gel with silver nitrate suggests that L. pneumophila LPS might be atypical. We studied patient serologic responses to cell envelope components of L. pneumophila Philadelphia 1, a serogroup 1 organism. Sera from patients with evidence of infection with serogroup 1 L. pneumophila contained large amounts of antibody to this strain. Few of these antibodies recognized the MOMP of L. pneumophila. In contrast, greater than 98% of these antibodies were directed against the LPS. This indicates that LPS is the dominant serogroup antigen and the major antigen responsible for the reactivity of patient sera in the indirect fluorescent antibody assay, currently the principal diagnostic assay for Legionella infection.
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PMID:Isolation and characterization of the cytoplasmic and outer membranes of the Legionnaires' disease bacterium (Legionella pneumophila). 388 79

The particulate activities of Leishmania mexicana mexicana amastigote malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) and phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating) EC 4.1.1.49) have been purified to apparent electrophoretic homogeneity by hydrophobic interaction chromatography using Phenyl-Sepharose CL-4B, affinity chromatography using 5'AMP-Sepharose 4B, and gel filtration using Sephadex G-100. Malate dehydrogenase was purified 150-fold overall with a final specific activity of 1230 units/mg protein and a recovery of 63%. Phosphoenolpyruvate carboxykinase was purified 132-fold with a final specific activity of 30.3 units/mg protein and a recovery of 20%. Molecular weights determined by gel filtration and SDS-gel electrophoresis were 39 800 and 33 300 for malate dehydrogenase and 63 100 and 65 100 for phosphoenolpyruvate carboxykinase, respectively. Kinetic studies with malate dehydrogenase assayed in the direction of oxaloacetic acid reduction showed a Km(NADH) of 41 microM and a Km(oxaloacetic acid) of 39 microM. For malate oxidation there was a Km(malate) of 3.6 mM and a Km(NAD) of 0.79 mM. Oxaloacetic acid exhibited substrate inhibition at concentrations greater than 0.83 mM and malate was found to be a product inhibitor at high concentrations. However, there was no modification of enzyme activity by a number of glycolytic intermediates and cofactors, suggesting that malate dehydrogenase is not a major regulatory enzyme in L. m. mexicana. The results show that these L. m. mexicana amastigote enzymes are in several ways similar to their mammalian counterparts; nevertheless, their apparent importance and unique subcellular organization in the parasite make them potential targets for chemotherapeutic attack.
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PMID:Purification of particulate malate dehydrogenase and phosphoenolpyruvate carboxykinase from Leishmania mexicana mexicana. 397 Sep 41

The mammalian pyruvate dehydrogenase multi-enzyme complex contains a tightly-associated 50 000-Mr polypeptide of unknown function (component X) in addition to its three constituent enzymes, pyruvate dehydrogenase (E1), lipoate acetyltransferase (E2) and lipoamide dehydrogenase (E3) which are jointly responsible for production of CoASAc and NADH. The presence of component X is apparent on sodium dodecyl sulphate/polyacrylamide gel analysis of the complex, performed in Tris-glycine buffers although it co-migrates with the E3 subunit on standard phosphate gels run under denaturing conditions. Refined immunological techniques, employing subunit-specific antisera to individual components of the pyruvate dehydrogenase complex, have demonstrated that protein X is not a proteolytic fragment of E2 (or E3) as suggested previously. In addition, anti-X serum elicits no cross-reaction with either subunit of the intrinsic kinase of the pyruvate dehydrogenase complex. Immune-blotting analysis of SDS extracts of bovine, rat and pig cell lines and derived subcellular fractions have indicated that protein X is a normal cellular component with a specific mitochondrial location. It remains tightly-associated with the 'core' enzyme, E2, on dissociation of the complex at pH 9.5 or by treatment with 0.25 M MgCl2. This polypeptide is not released to any significant extent from E2 by p-hydroxymercuriphenyl sulphonate, a reagent which promotes dissociation of the specific kinase of the complex from the 'core' enzyme. Incubation of the complex with [2-14C]pyruvate in the absence of CoASH promotes the incorporation of radio-label, probably in the form of acetyl groups, into both E2 and component X.
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PMID:Component X. An immunologically distinct polypeptide associated with mammalian pyruvate dehydrogenase multi-enzyme complex. 400 43


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