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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The purification and partial characterisation of an NADP(H) dependent artificial mediator accepting pyridine nucleotide oxidoreductase (AMAPOR) from the anaerobic Clostridium thermoaceticum is described. Depending on the redox potential of the artificial mediators the AMAPOR is able to regenerate NADP+ or NADPH rendering the enzyme useful for preparative work applying NADP(H) dependent oxidoreductases. At 37 degrees C crude extracts of C. thermoaceticum have an AMAPOR activity of 5-7 U mg(-1). This is 28 degrees under the optimal growth temperature of this microrganism. Out of apparently more than 10 AMAPOR active proteins in the crude cell extracts visible after electrophoresis and activity staining on the gel, two of these proteins were isolated. They seem to be two different oligomers. According to gel electrophoresis they show apparent molecular masses of about 200 and 400 kDa. These two forms showed after SDS gel electrophoresis two monomers with apparent molecular masses of 42 and 56 kDa which we call alpha and beta. The two oligomers may have the compositions alpha2beta2 and alpha4beta4. They contain Fe/S cluster and FAD. Various amounts of the FAD were lost during the purification procedure. This loss is partially reversible after addition of FAD. The AMAPOR reacts with rather different artificial mediators such as viologens, quinones e.g. 1,4-benzoquinone or anthraquinone-2,6-disulphonate, 2,6-dichloro-indophenol and clostridial rubredoxin. Two different ferredoxins from C. thermoaceticum, oxygen or lipoamide are no substrates indicating the here described AMAPOR is not a diaphorase in the usual sense.
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PMID:On a new artificial mediator accepting NADP(H) oxidoreductase from Clostridium thermoaceticum. 1105 22

Methanosphaera stadtmanae (DSM 3091) is a methanogen that requires H2 and CH3OH for methanogenesis. The organism does not possess an F420-dependent hydrogenase and only low levels of F420. It does however possess NADP+:F420 oxidoreductase activity. The NADP+:F420 oxidoreductase, the enzyme which catalyses the electron transfer between NADP+ and F420 in this organism, was purified and characterized. NAD+, NADH, FMN, and FAD could not be used as electron acceptors. Optimal pH for F420 reduction was 6.0, and 8.5 for NADP+ reduction. During the purification process, it was noted that precipitation with (NH4)2SO4 increased total activity 16-fold but reduced the stability of the enzyme. However, recombination of cell-free extracts with resuspended 65-90% (NH4)2SO4 pellet returned activity to near cell-free extract levels. Neither high salt or protease inhibitors were effective in stabilizing the activity of the partially purified enzyme. The purified enzyme from M. stadtmanae possessed a molecular weight of 148 kDa as determined by gel filtration chromatography and native-PAGE, consisting of alpha, beta, and gamma subunits of 60, 50, and 45 kDa, respectively, using SDS-PAGE. The Km values were 370 microM for NADP+, 142 microM for NADPH, 62.5 microM for F420, and 7.7 microM for F420H2. These values were different from the Km values observed in the cell-free extract.
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PMID:Purification of the NADP+:F420 oxidoreductase of Methanosphaera stadtmanae. 1110 87

The stability of liver and muscle enzymes and proteins in niacin-deficient quail towards trypsin treatment in the presence and absence of coenzymes, NAD or NADP, was characterized. The protection of liver dehydrogenases by coenzymes was low when they are subjected to trypsin digestion for 60 min. In contrast, in the muscle there was substantial protection against trypsin inactivation of glyceraldehyde-3-phosphate dehydrogenase by NAD and of 6-phosphogluconate dehydrogenase by NADP. Among all enzymes tested, glyceraldehyde-3-phosphate dehydrogenase showed the greatest protection against trypsin inactivation by NAD. SDS-polyacrylamide gel electrophoresis demonstrated that muscle proteins from the niacin-deficient group were more substantially protected compared to control and pair-fed groups when liver and muscle extracts were spiked with NAD and subjected to trypsin digestion. Overall results suggest that niacin deficiency exerted specific destabilizing effects on the stability of enzymes and proteins in muscle.
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PMID:Effects of nicotinamide coenzymes on the stability of enzyme activities and proteins in niacin-deficient quail tissues against trypsin treatment. 1116 9

Glucose-6-phosphate dehydrogenase was purified from human placenta using DEAE-Sepharose fast flow, 2',5'-ADP Sepharose 4B column chromatography, and chromatofocusing on PBE 94 with PB 74. The enzyme was purified with 62% yield and had a specific activity of 87 units per milligram protein. The pH optimum was determined to be 7.8, using zero buffer extrapolation method. The purified placental glucose-6-phosphate dehydrogenase gave two activity bands on native PAGE: one band, constituting about 3--5% of total activity, moved slower than the remaining 95%. Among the activity bands only the faster moving band gave a band on protein staining. The slower moving band, which probably corresponded to the higher polymeric form of the G6PD with high specific activity, was not seen on native PAGE due to insufficient protein for Coomassie brilliant blue staining. The observation of one band on SDS--PAGE with an M(r) of 54 kDa and a specific activity lower than expected, suggests the presence of both forms of the G6PD, the high polymeric form at low concentration and the inactive form at high concentration, in our preparation. Measuring the activities of placental glucose-6-phosphate dehydrogenase between 20 and 50 degrees C, the activation energy, activation enthalpy, and Q(10) were calculated to be 8.16 kcal/mol, 7.55 kcal/mol, and 1.57, respectively. It was found that human placental G6PD obeys Michaelis-Menten kinetics. K(m) values were determined using the concentration ranges of 20--300 microM for G6P and 10--200 microM for NADP(+). The K(m) value for G6P was 40 microM; the K(m) value NADP(+) was found to be 20 microM. Double-reciprocal plots of 1/Vm vs 1/G6P (at constant [NADP(+)]) and of 1/Vm vs 1/NADP(+) (at constant [G6P]) intersected at the same point on the 1/V(m) axis to give V(m) = 87 U/mg protein.
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PMID:Purification and some properties of human placental glucose-6-phosphate dehydrogenase. 1123 90

Two types of NADP-dependent isocitrate dehydrogenases (ICDs) have been reported: mitochondrial (ICD1) and cytosolic (ICD2). The C-terminal amino acid sequence of ICD2 has a tripeptide peroxisome targeting signal 1 sequence (PTS1). After differential centrifugation of the postnuclear fraction of rat liver homogenate, approximately 75% of ICD activity was found in the cytosolic fraction. To elucidate the true localization of ICD2 in rat hepatocytes, we analyzed the distribution of ICD activity and immunoreactivity in fractions isolated by Nycodenz gradient centrifugation and immunocytochemical localization of ICD2 antigenic sites in the cells. On Nycodenz gradient centrifugation of the light mitochondrial fraction, ICD2 activity was distributed in the fractions in which activity of catalase, a peroxisomal marker, was also detected, but a low level of activity was also detected in the fractions containing activity for succinate cytochrome C reductase (a mitochondrial marker) and acid phosphatase (a lysosomal marker). We have purified ICD2 from rat liver homogenate and raised a specific antibody to the enzyme. On SDS-PAGE, a single band with a molecular mass of 47 kD was observed, and on immunoblotting analysis of rat liver homogenate a single signal was detected. Double staining of catalase and ICD2 in rat liver revealed co-localization of both enzymes in the same cytoplasmic granules. Immunoelectron microscopy revealed gold particles with antigenic sites of ICD2 present mainly in peroxisomes. The results clearly indicated that ICD2 is a peroxisomal enzyme in rat hepatocytes. ICD2 has been regarded as a cytosolic enzyme, probably because the enzyme easily leaks out of peroxisomes during homogenization. (J Histochem Cytochem 49:1123-1131, 2001)
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PMID:Localization of cytosolic NADP-dependent isocitrate dehydrogenase in the peroxisomes of rat liver cells: biochemical and immunocytochemical studies. 1151 81

The 3-hydroxypropionate cycle has been proposed as a new autotrophic CO(2) fixation pathway for the phototrophic green non-sulfur eubacterium Chloroflexus aurantiacus and for some chemotrophic archaebacteria. The cycle requires the reductive conversion of the characteristic intermediate 3-hydroxypropionate to propionyl-CoA. The specific activity of the 3-hydroxypropionate-, CoA-, K(+)-, and MgATP-dependent oxidation of NADPH in autotrophically grown cells was 0.09 micromol min(-1) mg(-1) protein, which was 2-fold down-regulated in heterotrophically grown cells. Unexpectedly, a single enzyme catalyzes the entire reaction sequence: 3-hydroxypropionate + MgATP + CoA + NADPH + H(+) --> propionyl-CoA + MgAMP + PP(i) + NADP(+) + H(2)O. The enzyme was purified 30-fold to near homogeneity and has a very large native molecular mass between 500 and 800 kDa, with subunits of about 185 kDa as judged by SDS-PAGE, suggesting a homotrimeric or homotetrameric structure. Upon incubation of this new enzyme, termed propionyl-CoA synthase, with the proteinase trypsin, the NADPH oxidation function of the enzyme was lost, whereas the enzyme still activated 3-hydroxypropionate to its CoA-thioester and dehydrated it to acrylyl-CoA. SDS-PAGE revealed that the subunits of propionyl-CoA synthase had been cleaved once and the N-terminal amino acid sequences of the two trypsin digestion products were determined. Two parts of the gene encoding propionyl-CoA synthase (pcs) were identified on two contigs of an incomplete genome data base of C. aurantiacus, and the sequence of the pcs gene was completed. Propionyl-CoA synthase is a natural fusion protein of 201 kDa consisting of a CoA ligase, an enoyl-CoA hydratase, and an enoyl-CoA reductase, the reductase domain containing the trypsin cleavage site. Similar polyfunctional large enzymes are common in secondary metabolism (e.g. polyketide synthases) but rare in primary metabolism (e.g. eukaryotic type I fatty acid synthase). These results lend strong support to the operation of the proposed pathway in autotrophic CO(2) fixation.
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PMID:Propionyl-coenzyme A synthase from Chloroflexus aurantiacus, a key enzyme of the 3-hydroxypropionate cycle for autotrophic CO2 fixation. 1182 99

A gene having high sequence homology (45-49%) with the glycerol-1-phosphate dehydrogenase gene from Methanobacterium thermoautotrophicum was cloned from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820). This gene expressed in Escherichia coli with the pET vector system consists of 1113 nucleotides with an ATG initiation codon and a TAG termination codon. The molecular mass of the purified enzyme was estimated to be 38 kDa by SDS/PAGE and 72.4 kDa by gel column chromatography, indicating presence as a dimer. The optimum reaction temperature of this enzyme was observed to be 94-96 degrees C at near neutral pH. This enzyme was subjected to two-substrate kinetic analysis. The enzyme showed substrate specificity for NAD(P)H-dependent dihydroxyacetone phosphate reduction and NAD(+)-dependent glycerol-1-phosphate (Gro1P) oxidation. NADP(+)-dependent Gro1P oxidation was not observed with this enzyme. For the production of Gro1P in A. pernix cells, NADPH is the preferred coenzyme rather than NADH. Gro1P acted as a noncompetitive inhibitor against dihydroxyacetone phosphate and NAD(P)H. However, NAD(P)(+) acted as a competitive inhibitor against NAD(P)H and as a noncompetitive inhibitor against dihydroxyacetone phosphate. This kinetic data indicates that the catalytic reaction by glycerol- 1-phosphate dehydrogenase from A. pernix follows a ordered bi-bi mechanism.
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PMID:Kinetic study of sn-glycerol-1-phosphate dehydrogenase from the aerobic hyperthermophilic archaeon, Aeropyrum pernix K1. 1184 99

Glucose-6-phosphate dehydrogenase (G6PD) catalyses the first step of the pentose phosphate pathway which generates NADPH for anabolic pathways and protection systems in liver. G6PD was purified from dog liver with a specific activity of 130 U x mg(-1) and a yield of 18%. PAGE showed two bands on protein staining; only the slower moving band had G6PD activity. The observation of one band on SDS/PAGE with M(r) of 52.5 kDa suggested the faster moving band on native protein staining was the monomeric form of the enzyme. Dog liver G6PD had a pH optimum of 7.8. The activation energy, activation enthalpy, and Q10, for the enzymatic reaction were calculated to be 8.96, 8.34 kcal x mol(-1), and 1.62, respectively.The enzyme obeyed "Rapid Equilibrium Random Bi Bi" kinetic model with Km values of 122 +/- 18 microM for glucose-6-phosphate (G6P) and 10 +/- 1 microM for NADP. G6P and 2-deoxyglucose-6-phosphate were used with catalytic efficiencies (kcat/Km) of 1.86 x 10(6) and 5.55 x 10(6) M(-1) x s(-1), respectively. The intrinsic Km value for 2-deoxyglucose-6-phosphate was 24 +/- 4mM. Deamino-NADP (d-NADP) could replace NADP as coenzyme. With G6P as cosubstrate, Km d-ANADP was 23 +/- 3mM; Km for G6P remained the same as with NADP as coenzyme (122 +/- 18 microM). The catalytic efficiencies of NADP and d-ANADP (G6P as substrate) were 2.28 x 10(7) and 6.76 x 10(6) M(-1) x s(-1), respectively. Dog liver G6PD was inhibited competitively by NADPH (K(i)=12.0 +/- 7.0 microM). Low K(i) indicates tight enzyme:NADPH binding and the importance of NADPH in the regulation of the pentose phosphate pathway.
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PMID:Dog liver glucose-6-phosphate dehydrogenase: purification and kinetic properties. 1184 92

Membrane-bound NAD(P)-independent malate dehydrogenase (EC 1.1.99.16) was purified to homogeneity from the membrane of thermotolerant Acetobacter sp. SKU 14, an isolate from Thailand. The enzyme was solubilized from the membrane fraction of glycerol-grown cells with 1% Triton X-100 in the presence of 0.1 M KCl, and purified to homogeneity through steps of column chromatographies on DEAE-Sephadex A-50 and DEAE-Toyopearl in the presence of 0.1% Triton X-100. The purified enzyme showed a single protein band in both native-PAGE and SDS-PAGE. The enzyme was a homodimer with a molecular mass of 60 kDa subunit and had noncovalently bound FAD as the cofactor. The enzyme was stable over pH 5 and had its maximum activity at pH 11.0 when ferricyanide was used as an electron acceptor. The enzyme activity was elevated by the addition of ammonium ions. The substrate specificity was very strict to only L-malate, of which the apparent Km was 10 mM and over 20 compounds involving D-malate were not oxidized by the enzyme.
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PMID:Purification and characterization of membrane-bound malate dehydrogenase from Acetobacter sp. SKU 14. 1199 2

NAD(P)(+)-reducing hydrogenases have been described to be composed of a diaphorase (HoxFU) and a hydrogenase (HoxYH) moiety. This study presents for the first time experimental evidence that in cyanobacteria, a fifth subunit, HoxE, is part of this bidirectional hydrogenase. HoxE exhibits sequence identities to NuoE of respiratory complex I of Escherichia coli. The subunit composition of the cyanobacterial bidirectional hydrogenase has been investigated. The oxygen labile enzyme complex was purified to close homogeneity under anaerobic conditions from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 6301. The 647-fold and 1290-fold enriched purified enzyme has a specific activity of 46 micromol H(2) evolved (min mg protein)(-1) and 15 micromol H(2) evolved (min mg protein)(-1), respectively. H(2)-evolution of the purified enzyme of S. sp. PCC 6803 is highest at 60 degrees C and pH 6.3. Immunoblot experiments, using a polyclonal anti-HoxE antibody, demonstrate that HoxE co-purifies with the hydrogenase activity in S. sp. PCC 6301. SDS-PAGE gels of the purified enzymes revealed six proteins, which were partially sequenced and identified, besides one nonhydrogenase component, as HoxF, HoxU, HoxY, HoxH and, remarkably, HoxE. The molecular weight of the native protein (375 kDa) indicates a dimeric assembly of the enzyme complex, Hox(EFUYH)(2).
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PMID:HoxE--a subunit specific for the pentameric bidirectional hydrogenase complex (HoxEFUYH) of cyanobacteria. 1203 72


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