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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A plasmalemma-bound NADH oxidation system (Lin 1982 Proc Natl Acad Sci USA 79: 3773-3776) in corn root protoplasts was isolated by a mild treatment of intact protoplasts with trypsin. The majority of NADH stimulated O(2) consumption activity of the protoplasts could be recovered in the supernatant isolated from the intact protoplasts which have been treated with trypsin. The activation energy of NADH oxidation in the supernatant is similar to that of the intact protoplasts (8.7 versus 9.4 kilocalories per mole per degree). Unlike that of the intact protoplasts, an Arrhenius plot of the temperature response (from 5 to 25 degrees C) of the activity in the supernatant shows no transition suggestive of a dissociation of the enzyme from the membrane. Trypsin treatment did not affect K(+) uptake into cell volume of the protoplast. However, the NADH-stimulated K(+) uptake and the increase of cell volume were greatly reduced. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of trichloroacetic acid-precipitated protein from the supernatant showed one extra peptide band with approximately 42 kilodalton molecular weight.
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PMID:Isolation of NADH Oxidation System from the Plasmalemma of Corn Root Protoplasts. 1666 74

The isotherm for isocitrate oxidation by potato (Solanum tuberosum L. var. Russet Burbank) mitochondria in the presence of exogenous NAD is characterized by a hyperbolic phase at isocitrate concentrations below 3 millimolar, and a sigmoid, or positively cooperative phase from approximately 3 to 30 millimolar. The two forms of isocitrate dehydrogenase were separated and characterized following the sonication of mitochondria in 15% glycerol in the absence of buffer, followed by fractionation in a density step gradient to yield inner membrane and matrix components. The membrane-associated isocitrate dehydrogenase was found to have a Hill, or cooperativity, number of 1, while the Hill number of the matrix enzyme was 2.5. Upon digitonin extraction the cooperativity number of the membrane enzyme rose to 3.5. The isocitrate K(m) for the membrane enzyme was calculated to be approximately 5.9 x 10(-4) molar, while the S(0.5) for the matrix was 6.9 x 10(-4) molar. The NAD K(m) for both enzymes was 150 micromolar. Whereas the membrane enzyme proved indifferent to adenine nucleotides, the matrix enzyme was arguably inhibited by AMP and ADP, and inhibited some 25% by 5 millimolar ATP. Both enzymes were negatively responsive to the mole fraction of NADH, the membrane enzyme being 50% inhibited at a mole fraction of 0.26, and the matrix enzyme by a mole fraction of 0.32. The suggestion is offered that the enzymes in question constitute two forms of a single enzyme, one peripherally associated with the inner membrane, and one soluble in the matrix. It is proposed that a degree of regulation may be achieved by the apportionment of the enzyme between the bound and free forms.
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PMID:Isolation and Characterization of Inner Membrane-Associated and Matrix NAD-Specific Isocitrate Dehydrogenase in Potato Mitochondria. 1666 46

Enthalpy changes in the formation of a proton electrochemical potential (Delta mu H+) and its components, DeltapH (proton gradient) and Deltapsi (electrical potential), across two types of E. coli membrane vesicles were investigated. Flow dialysis experiments showed that in 0.1 M KPi, pH 6.6, E. coli GR19N membrane vesicles coupled with d-lactate exhibited 57 mV for DeltapH, 70 mV for Deltapsi, and 127 mV for Delta mu H+. Microcalorimetric measurements revealed that the corresponding enthalpy changes (DeltaH(pH), DeltaH(psi) and DeltaHm) were 3.5, 3.3 and 6.9 kcal/mole, respectively. Moreover, in E. coli ML 308-225 membrane vesicles across which 120mV of Delta mu H+ was generated, values of DeltaH(pH) and DeltaH(psi) were determined as 7.0 and 6.6 kcal/mole, as compared with the previously reported 14.1 kcal/mole for DeltaH(m). Comparisons of these enthalpy data revealed that component enthalpies (DeltaH(pH) and DeltaH(psi)) essentially added up to the total enthalpy (DeltaHm), providing a self-consistent test for the obtained data. In both membranes, the ratio ofDeltaH(psi) to Deltapsi was comparable to that of DeltaH(pH) to DeltapH in the formation of Delta mu H+. These observations indicated that the process of the movement of H+ across the membranes was the major contributor to the observed energetic changes. Moreover, the enthalpy change in the formation of Delta mu H+ was compared with the membranes derived from GR19N and ML 308-225 and coupled with NADH and d-lactate. The results were discussed in terms of trans-membrane phenomena.
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PMID:Enthalpy changes in the formation of the proton electrochemical potential and its components. 1702 Aug 51

Pseudomonas sp. strain C4 metabolizes carbaryl (1-naphthyl-N-methylcarbamate) as the sole source of carbon and energy via 1-naphthol, 1,2-dihydroxynaphthalene, and gentisate. 1-Naphthol-2-hydroxylase (1-NH) was purified 9.1-fold to homogeneity from Pseudomonas sp. strain C4. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme is a homodimer with a native molecular mass of 130 kDa and a subunit molecular mass of 66 kDa. The enzyme was yellow, with absorption maxima at 274, 375, and 445 nm, indicating a flavoprotein. High-performance liquid chromatography analysis of the flavin moiety extracted from 1-NH suggested the presence of flavin adenine dinucleotide (FAD). Based on the spectral properties and the molar extinction coefficient, it was determined that the enzyme contained 1.07 mol of FAD per mol of enzyme. Although the enzyme accepts electrons from NADH, it showed maximum activity with NADPH and had a pH optimum of 8.0. The kinetic constants K(m) and V(max) for 1-naphthol and NADPH were determined to be 9.6 and 34.2 microM and 9.5 and 5.1 micromol min(-1) mg(-1), respectively. At a higher concentration of 1-naphthol, the enzyme showed less activity, indicating substrate inhibition. The K(i) for 1-naphthol was determined to be 79.8 microM. The enzyme showed maximum activity with 1-naphthol compared to 4-chloro-1-naphthol (62%) and 5-amino-1-naphthol (54%). However, it failed to act on 2-naphthol, substituted naphthalenes, and phenol derivatives. The enzyme utilized one mole of oxygen per mole of NADPH. Thin-layer chromatographic analysis showed the conversion of 1-naphthol to 1,2-dihydroxynaphthalene under aerobic conditions, but under anaerobic conditions, the enzyme failed to hydroxylate 1-naphthol. These results suggest that 1-NH belongs to the FAD-containing external flavin mono-oxygenase group of the oxidoreductase class of proteins.
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PMID:Purification and characterization of 1-naphthol-2-hydroxylase from carbaryl-degrading Pseudomonas strain c4. 1723 79

Carbohydrate and fatty acids are major energetic substrates, although amino acid oxidation also permits ATP synthesis. Among several major metabolic differences between lipids and carbohydrate (activation, transport, effect of insulin, etc.), two are of particular importance when considering energy metabolism of critically ill patients: the yield of ATP synthesis and the response to uncoupling. (I) Oxidative phosphorylation yield is higher when NADH is the electron donor (three coupling sites: complex 1, 3 and 4) as compared to FADH2 (two coupling sites: complex 3 and 4). Since the ratio NADH/FADH2 is higher for glycolysis as compared to beta-oxidation, the stoichiometry of ATP synthesis to oxygen consumption is also higher. Lipid oxidation provides more ATP than carbohydrate, but it requires more oxygen per mole of ATP synthesized. (II) The ratio of NADH oxidation versus FADH, oxidation depends on the proton motive force, and lowering proton motive force by uncoupling favours FADH2 oxidation, i.e. lipids versus carbohydrate. In conclusion, lipid oxidation provides a high rate of ATP synthesis even during a mild uncoupling state, but at a high rate of oxygen consumption. If oxygen availability is limited, the major metabolic adaptation to increase the efficiency is represented by a switch from lipid oxidation to glucose oxidation.
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PMID:Choosing the right substrate. 1738 Jul 91

Clostridium acetobutylicum, an obligatory anaerobe, is able to grow microoxically with the accumulation of two functionally unknown O2-induced proteins identified by two-dimensional electrophoresis. One was determined to be a novel type rubrerythrin-like protein, named rubperoxin (Rpr) in this study, that conserves one rubredoxin-type Fe(SCys)(4) site per polypeptide in the N-terminus. Recombinant rubperoxin expressed in E. coli purified in its oxidized form is a dimer with optical absorption maxima at 492, 377, and 277nm. Reduced rubperoxin is rapidly and fully oxidized by a half molar ratio of H2O2 per mole protein, and slowly oxidized by t-butyl hydroperoxide and O2. Cell-free extracts from microoxically grown cells efficiently reduce rubperoxin when NAD(P)H is used as the electron donor (preferentially reduced by NADH). These results strongly suggest that rubperoxin is involved in NAD(P)H-dependent H2O2 detoxification in vivo.
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PMID:An O2-inducible rubrerythrin-like protein, rubperoxin, is functional as a H2O2 reductase in an obligatory anaerobe Clostridium acetobutylicum. 1748 86

Derivatives of Escherichia coli C were engineered to produce primarily succinate or malate in mineral salts media using simple fermentations (anaerobic stirred batch with pH control) without the addition of plasmids or foreign genes. This was done by a combination of gene deletions (genetic engineering) and metabolic evolution with over 2,000 generations of growth-based selection. After deletion of the central anaerobic fermentation genes (ldhA, adhE, ackA), the pathway for malate and succinate production remained as the primary route for the regeneration of NAD+. Under anaerobic conditions, ATP production for growth was obligately coupled to malate dehydrogenase and fumarate reductase by the requirement for NADH oxidation. Selecting strains for improved growth co-selected increased production of these dicarboxylic acids. Additional deletions were introduced as further improvements (focA, pflB, poxB, mgsA). The best succinate biocatalysts, strains KJ060(ldhA, adhE, ackA, focA, pflB) and KJ073(ldhA, adhE, ackA, focA, pflB, mgsA, poxB), produce 622-733 mM of succinate with molar yields of 1.2-1.6 per mole of metabolized glucose. The best malate biocatalyst, strain KJ071(ldhA, adhE, ackA, focA, pflB, mgsA), produced 516 mM malate with molar yields of 1.4 per mole of glucose metabolized.
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PMID:Combining metabolic engineering and metabolic evolution to develop nonrecombinant strains of Escherichia coli C that produce succinate and malate. 1797 30

The NAD-malic enzyme catalyzes the oxidative decarboxylation of l-malate. Structures of the enzyme indicate that arginine 181 (R181) is within hydrogen bonding distance of the 1-carboxylate of malate in the active site of the enzyme and interacts with the carboxamide side chain of the nicotinamide ring of NADH, but not with NAD+. Data suggested R181 might play a central role in binding and catalysis in malic enzyme, and it was thus changed to lysine and glutamine to probe its potential function. A nearly 100-fold increase in the Km for malate and a 30-fold increase in the Ki for oxalate, an analogue of the enolpyruvate intermediate, in the R181Q and R181K mutants are consistent with a role for R181 in binding substrates. The mutant enzymes also exhibit a >10-fold increase in KiNADH, but only a slight or no change in KNAD, consistent with rotation of the nicotinamide ring into the malate binding site upon reduction of NAD+ to NADH. The activity of the R181Q mutant can be rescued by ammonium ion likely by binding in the pocket vacated by the guanidinium group of R181. Results suggest 2 mol of ammonia bind per mole of active sites with a high-affinity KNH4 of 0.7 +/- 0.1 mM and a low-affinity KNH4 of approximately 420 mM. Occupancy of the high-affinity site, likely by NH4+, results in an increase in the affinity of malate, oxalate, and NADH (with no change in NAD affinity), consistent with the above-proposed roles for R181. The second molecule to bind is likely neutral NH3, and its binding increases V/Et approximately 20-fold. Primary deuterium and 13C isotope effects measured in the absence and presence of ammonium ion suggest R181Q predominantly affects the rate of the reaction by changing the rate of the precatalytic conformational change. The isotope effects do not change upon binding the second mole of ammonia in spite of the 20-fold increase in V/Et. Thus, the R181Q mutant enzyme exists as an equilibrium mixture between active and less active forms, and NH3 stabilizes the more active conformation of the enzyme.
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PMID:Multiple roles of arginine 181 in binding and catalysis in the NAD-malic enzyme from Ascaris suum. 1802 82

The optical response from a mechanical stimulus signal oscillating system of NADH, DPIP and O2 with the participant of lactate dehyrogenase (LDH) was studied with UV/Vis Spectrometer in the present paper. The signal transduction efficiency of the system was largely improved by the catalysis of LDH, when DPIP. : NADH was 1 : 4.5 (mole ratio)without lactate, and the average period of system was shortened from 108 to 34 min, and to 29 min with lactate existing. It was presumed that the accelerating effect of LDH was mainly brought by the activation of NADH at its catalysis center, and in addition, brought by the supplement of NADH through the dehydrogenation of lactate. The results indicated that the catalysis of enzyme would be put up also when only one of the two substrates existed.
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PMID:[The spectrum studies of mechano-optical signal transduction with participation of LDH]. 1853 24

Mitochondrial NADH fluorescence has been a useful tool in evaluating mitochondrial energetics both in vitro and in vivo. Mitochondrial NADH fluorescence is enhanced several-fold in the matrix through extended fluorescence lifetimes (EFL). However, the actual binding sites responsible for NADH EFL are unknown. We tested the hypothesis that NADH binding to Complex I is a significant source of mitochondrial NADH fluorescence enhancement. To test this hypothesis, the effect of Complex I binding on NADH fluorescence efficiency was evaluated in purified protein, and in native gels of the entire porcine heart mitochondria proteome. To avoid the oxidation of NADH in these preparations, we conducted the binding experiments under anoxic conditions in a specially designed apparatus. Purified intact Complex I enhanced NADH fluorescence in native gels approximately 10-fold. However, no enhancement was detected in denatured individual Complex I subunit proteins. In the Clear and Ghost native gels of the entire mitochondrial proteome, NADH fluorescence enhancement was localized to regions where NADH oxidation occurred in the presence of oxygen. Inhibitor and mass spectroscopy studies revealed that the fluorescence enhancement was specific to Complex I proteins. No fluorescence enhancement was detected for MDH or other dehydrogenases in this assay system, at physiological mole fractions of the matrix proteins. These data suggest that NADH associated with Complex I significantly contributes to the overall mitochondrial NADH fluorescence signal and provides an explanation for the well established close correlation of mitochondrial NADH fluorescence and the metabolic state.
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PMID:Mitochondrial NADH fluorescence is enhanced by complex I binding. 1870 5


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