Inhibition of erythrocyte plasma membrane NADH dehydrogenase by nucleotides and uncouplers

Erythrocyte ghost NADH dehydrogenase is inhibited in a competitive fashion by ATP and ADP whereas other nucleoside di- and triphosphates, cyclic nucleosides, as well as non-phosphorylating ATP analogs are relatively ineffective. In addition, this enzyme, measured with ferricyanide as electron acceptor, is inhibited by uncouplers of oxidative phosphorylation (proton-conducting reagents), the inhibition being competitive in character (i.e., the uncouplers were without influence upon maximum velocity). The effectiveness of the uncouplers was in the order of their hydrophobic character with the presence of the alkyl side chain rendering nonyl-dinitrophenol much more active than 2,6-dinitrophenol itself. Hydrophobic compounds that are not protonophores (e.g., eosin, proflavin or valinomycin) were not inhibitory. Whereas adenine nucleotides probably inhibit NADH oxidation competitively through structural similarity with the substrate, it appears unlikely that uncouplers compete at the NADH site directly. Rather, the apparently-competitive inhibition in the latter case may reflect competition for proton transfer to an acceptor residing in a hydrophobic region of the enzyme complex.     

Kinetic studies on a 4-methoxybenzoate O-demethylase from Pseudomonas putida.

A direct, sensitive and reliable photometric assay procedure for monitoring the activity of non-specific 4-methoxybenzoate O-demethylases of microorganisms is described. The assay is based on the O-demethylation of 3-nitro-4-methoxybenzoate to the yellow-coloured product 3-nitro-4-hydroxybenzoate. Using this assay and by monitoring the oxidation rate of reduced pyridine nucleotides, the kinetic properties of a purified, reconstituted enzyme system composed of 4-methoxybenzoate monooxygenase (O-demethylating) and a reductase from Pseudomonas putida have been investigated. It has been found that the KM value of the monoxygenase of this enzyme system towards different substrates (i.e. tight couplers, uncouplers and partial uncouplers) rises from the extremely low value of 0.07 muM for the tight couplers to about 55 muM for the uncouplers. The effect of possible inhibitors and metal ions on the reconstituted enzyme system was investigated. The inhibition pattern was almost identical to that found for the purified reductase, only batho-phenanthrolinedisulfonate showing a greater inhibition of the reconstituted enzyme system. The affinity of the reductase towards NADH was found to be approximately 200-fold greater than that towards NADPH. Futhermore, the affinity of this reductase to NADH depended on the nature of the electron acceptor. The affinity to NADH was more than 10 times higher when the monooxygenase-substrate complex was used as the electron acceptor, than when cytochrome c or 2,6-dichloroindophenol was used. These differences are discussed on the basis of enzyme-enzyme interactions between the reductase and the monooxygenase.     

Studies on energy-linked reactions. Energy-linked reduction of oxidized nicotinamide–adenine dinucleotide by succinate in Escherichia coli

1. A method for the preparation of small particles from Escherichia coli is described. 2. These particles catalyse the ATP-driven reduction of NAD⁺ by succinate. 3. ATP utilized/NADH produced ratios below 2.0 were measured and a stoicheiometry of 1 molecule of ATP utilized per molecule of NADH produced is proposed. 4. The reaction is not inhibited by 2,4-dinitrophenol or by oligomycin but is inhibited by other uncouplers such as pentabromophenol and dicoumarol. 5. The specificity of the energy source, the specificity of the electron acceptor, the effects of pH, Mg²⁺, P_i and temperature have been studied.     

Effects of adenosine phosphates and nicotinamide nucleotides on pyruvate carboxylase from baker''s yeast

1. Pyruvate carboxylase from baker's yeast is inhibited by ADP, AMP and adenosine at pH8.0 in the presence of magnesium chloride concentrations equal to or higher than the ATP concentration. The adenine moiety is essential for the inhibitory effect. 2. In the absence of acetyl-CoA (an allosteric activator) ADP, AMP and adenosine are competitive inhibitors with respect to ATP. In the presence of acetyl-CoA, besides the effect with respect to ATP, AMP competes with acetyl-CoA, whereas ADP and adenosine are non-competitive inhibitors with respect to the activator. 3. Pyruvate carboxylase is inhibited by NADH. The inhibition is competitive with respect to acetyl-CoA and specific with respect to NADH, since NAD(+), NADP(+) and NADPH do not affect the enzyme activity. In the absence of acetyl-CoA, NAD(+), NADH, NADP(+) and NADPH do not inhibit pyruvate carboxylase. 4. Pyruvate carboxylase is inhibited by ADP, AMP and NADH at pH6.5, in the presence of 12mm-Mg(2+), 0.75mm-Mn(2+) and 0.5mm-ATP, medium conditions similar to those existing inside the yeast cell. The ADP and NADH effects are consistent with a regulation of enzyme activity by the intracellular [ATP]/[ADP] ratio and secondarily by NADH concentration. These mechanisms would supplement the already known control of yeast pyruvate carboxylase by acetyl-CoA and l-aspartate. Inhibition by AMP is less marked and its physiological role is perhaps limited.     

L-Lactate dehydrogenase from leaves of higher plants. Kinetics and regulation of the enzyme from lettuce (Lactuca sativa L).

1. L-Lactate dehydrogenase from lettuce (Lactuca sativa) leaves was purified to electrophoretic homogeneity by affinity chromatography. 2. In addition to its NAD(H)-dependent activity with L-lactate and pyruvate, the enzyme also catalyses the reduction of hydroxypyruvate and glyoxylate. The latter activities are not due to a contamination of the enzyme preparations with hydroxypyruvate reductase. 3. The enzyme shows allosteric properties that are markedly by the pH. 4. ATP is a potent inhibitor of the enzyme. The kinetic data suggest that the inhibition by ATP is competitive with respect to NADH at pH 7.0 and 6.2. The existence of regulatory binding sites for ATP and NADH is discussed. 5. Bivalent metal cations and fructose 6-phosphate relieve the ATP inhibition of the enzyme. 6. A function of leaf L-lactate dehydrogenase is proposed as a component of the systems regulating the cellular pH and/or controlling the concentration of reducing equivalents in the cytoplasm of leaf cells.     

The inhibition of isocitrate oxidation by palmitoyl-l-carnitine and palmitoyl-C0 A in rat liver mitochondria.

Palmitoyl-L carnitine decreases the oxidation of isocitrate in rat liver mitochondria in state 3 by 25-30%. Palmitoyl-L-carnitine acts as an additional substrate raising the rate of oxidative phosphorylation, NAD reduction and ATP/ADP ratio in mitochondria. Palmitoyl-CoA added to mitochondria oxidizing isocitrate in state 3 causes a strong inhibition of isocitrate oxidation and of oxidative phosphorylation and a considerable elevation of intramitochondrial NADH/NAD and ATP/ADP ratios. The effect of palmitoyl-CoA is dependent on its concentration and is competitive with ADP. Carnitine restores only oxidative phosphorylation, but the oxidation of isocitrate remains inhibited. Evidence is presented that the transport of isocitrate is not affected by palmitoyl-CoA is due to the inhibition of adenine nucleotide translocation. The kinetic studies of NAD-dependent isocitrate dehydrogenase in the soluble fraction of sonicated mitochondria revealed that the enzyme is very sensitive towards the inhibition by NADH and only very slightly affected by ATP (Ki for NADH and ATP are 0.017 and 3.6 mM respectively). On the basis of the kinetic data the relative contribution of NADH and ATP in the inhibition of isocitrate oxidation by fatty acids was calculated. It is concluded that the inhibition of isocitrate oxidation caused by palmitoyl-L-carnitine and palmitoyl-CoA is primarily due to the increased reduction of NAD, whereas the increase of ATP/ADP ratio is much less important.     

Inhibition of hepatic adenylate cyclase by NADH

Adenylate cyclase activity in isolated rat liver plasma membranes was inhibited by NADH in a concentration-dependent manner. Half-maximal inhibition of adenylate cyclase was observed at 120 microM concentration of NADH. The effect of NADH was specific since adenylate cyclase activity was not altered by NAD+, NADP+, NADPH, and nicotinic acid. The ability of NADH to inhibit adenylate cyclase was not altered when the enzyme was stimulated by activating the cyclase was not altered when the enzyme was stimulated by activating the Gs regulatory element with either glucagon or cholera toxin. Similarly, inhibition of Gi function by pertussis toxin treatment of membranes did not attenuate the ability of NADH to inhibit adenylate cyclase activity. Inhibition of adenylate cyclase activity to the same extent in the presence and absence of the Gpp (NH) p suggested that NADH directly affects the catalytic subunit. This notion was confirmed by the finding that NADH also inhibited solubilized adenylate cyclase in the absence of Gpp (NH)p. Kinetic analysis of the NADH-mediated inhibition suggested that NADH competes with ATP to inhibit adenylate cyclase; in the presence of NADH (1 mM) the Km for ATP was increased from 0.24 +/- 0.02 mM to 0.44 +/- 0.08 mM with no change in Vmax. This observation and the inability of high NADH concentrations to completely inhibit the enzyme suggest that NADH interacts at a site(s) on the enzyme to increase the Km for ATP by 2-fold and this inhibitory effect is overcome at high ATP concentrations.     

Studies in the enzymology of glutathione metabolism in human erythrocytes

Spectrophotometric assay methods are described for glutathione synthetase, gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase of erythrocytes. The contents of these enzymes in normal human erythrocytes are reported. Erythrocyte glutathione synthetase is inhibited by ADP; this inhibition is competitive with respect to ATP. gamma-Glutamylcysteine synthetase is subject to feedback inhibition by GSH, and is also inhibited by NADH, and to a lesser extent by NAD(+) and NADPH. This enzyme is irreversibly inactivated by cysteamine.     

Isolation of Capsicum annuum leaf nitrate reductase and characterization of the effect of adenine nucleotides and NADH on its activity

Abstract. In the preliminary purification of Capsicum leaf nitrate reductase (EC 1.6.6.1), treatment of the crude extract on Sephadex G-25 was necessary to prevent a gelling of the extract and sedimentation of the enzyme. Its K_m values for NADH and nitrate were estimated to be 9.3 and 105mmol m⁻³ ADP and ATP gave hyperbolic competitive inhibition, with respect to NADH, while the inhibition by AMP was linear competitive. K_i values calculated were: ADP and ATP approximately lmol m⁻³ and AMP 2.3 mol m⁻³. Inhibition by ADP was not altered by reduced glutathione.
The Capsicum nitrate reduclase was very susceptible to inhibition by NADH (in the absence of nitrate) and an in vivo assay showed that the activity of the enzyme was limited by the supply of nitrate. NADH and adenine nucleotide levels measured in the Capsicum leaf were used to estimate inhibition of nitrate reductase and a prediction was made of the nitrate reductase activity at different times in the photoperiod. This was shown to follow the same trend as the measured in vivo activity of the enzyme. Changes in adenine nucleotide levels had little effect on nitrate reductase activity.     

Inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex by reduced nicotinamide adenine dinucleotide in the presence or absence of calcium ion and effect of adenosine 5'-diphosphate on reduced nicotinamide adenine dinucleotide inhibition

Micromolar Ca2+ markedly reduces NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex [Lawlis, V. B., & Roche, T. E. (1980) Mol. Cell. Biochem. 32, 147-152]. Product inhibition patterns from initial velocity studies conducted at less than 10(-9) M or at 1.5 X 10(-5) M Ca2+ with NAD+, CoA, or alpha-ketoglutarate as the variable substrate showed that NADH was a noncompetitive inhibitor with respect to each of these substrates, except at high NAD+ concentrations, where reciprocal plots were nonlinear and the inhibition pattern for NADH vs. NAD+ changed from a noncompetitive to a competitive pattern. From slope and intercept replots, 2-fold to 12-fold higher inhibition constants were estimated for inhibition by NADH vs. the various substrates in the presence of 1.5 X 10(-5) M Ca2+ than for inhibition at less than 10(-9) M Ca2+. These inhibition patterns and the lack of an effect of Ca2+ on the inhibition of the dihydrolipoyl dehydrogenase component suggested that Ca2+-modulated NADH inhibition occurs at an allosteric site with competitive binding at the site by high levels of NAD+. Decarboxylation of alpha-keto[1-14C]glutarate by the resolved alpha-ketoglutarate dehydrogenase component was investigated in the presence of 5.0 mM glyoxylate which served as an efficient acceptor. NADH (0.2 mM) or 1.0 mM ATP inhibited the partial reaction whereas 15 muM Ca2+, 1.0 mM ADP, or 10 mM NAD+ stimulated the partial reaction and reduced NADH inhibition of this reaction. Thus these effectors alter the activity of the alpha-ketoglutarate dehydrogenase complex by binding at allosteric sites on the alpha-ketoglutarate dehydrogenase component. Inhibition by NADH over a wide range of NADH/NAD+ ratios was measured under conditions in which the level of alpha-ketoglutarate was adjusted to give matching control activities at less than 10(-9) M Ca2+ or 1.5 X 10(-5) M Ca2+ in either the presence or the absence of 1.6 mM ADP. These studies establish that both Ca2+ and ADP decreased NADH inhibition under conditions compensating for the effects of Ca2+ and ADP on S0.5 for alpha-ketoglutarate. ADP was particularly effective in reducing NADH inhibition; further studies are required to determine whether this occurs through binding of NADH and ADP at the same, overlapping, or interacting sites.     

Energy-linked electron transfer reactions in Rhodopseudomonas viridis

The particulate fraction of Rhodopseudomonas viridis when supplied with succinate catalyses the reduction of NAD⁺ by light; this reaction is inhibited by uncouplers of oxidative phosphorylation but not by oligomycin. Formation of NADH takes place in the dark when ATP or PP_i is supplied. Both light and dark reactions are inhibited by valinomycin and nigericin, when added together, but not by either separately. NADH formation in R. viridis appears to take place by an energy-dependent reversal of electron flow and energy may be conserved in the form of a membrane potential. The addition of ATP caused the oxidation of both C553 and C558 in chromatophores; carbonylcyanide p-trifluoromethoxyphenylhydrazone and oligomycin abolished this oxidation.

The NAD⁺ and NADH concentrations at equilibrium in the light-dependent reaction were determined and the oxidation-reduction potential of this couple calculated. From this value it was calculated that under these experimental conditions the energy requirement to form NADH from the succinate/fumarate couple at E_h = o V was 9.4 kcal.

Particles of R. viridis contained an active transhydrogenase, driven by either light or ATP, that was sensitive to uncouplers of oxidative phosphorylation; the light-driven reaction was insensitive to oligomycin and was inhibited by antimycin A and 2-heptyl-4-hydroxyquinone-N-oxide.

R. viridis did not grow aerobically but particles contained NADH oxidase activity that was cyanide sensitive. There was no spectroscopic evidence for cytochromes of the b-type in reduced-minus-oxidised spectra of particles or in pyridine haemochrome spectra of whole cells.     

Steady-state kinetics of formaldehyde dehydrogenase and formate dehydrogenase from a methanol-utilizing yeast, Candida boidinii.

Initial velocity studies and product inhibition studies were conducted for the forward and reverse reactions of formaldehyde dehydrogenase (formaldehyde: NAD oxidoreductase, EC 1.2.1.1) isolated from a methanol-utilizing yeast Candida boidinii. The data were consistent with an ordered Bi-Bi mechanism for this reaction in which NAD+ is bound first to the enzyme and NADH released last. Kinetic studies indicated that the nucleoside phosphates ATP, ADP and AMP are competitive inhibitors with respect to NAD and noncompetitive inhibitors with respect to S-hydroxymethylglutathione. The inhibitions of the enzyme activity by ATP and ADP are greater at pH 6.0 and 6.5 than at neutral or alkaline pH values. The kinetic studies of formate dehydrogenase (formate:NAD oxidoreductase, EC 1.2.1.2) from the methanol grown C. boidinii suggested also an ordered Bi-Bi mechanism with NAD being the first substrate and NADH the last product. Formate dehydrogenase the last enzyme of the dissimilatory pathway of the methanol metabolism is also inhibited by adenosine phosphates. Since the intracellular concentrations of NADH and ATP are in the range of the Ki values for formaldehyde dehydrogenase and formate dehydrogenase the activities of these main enzymes of the dissimilatory pathway of methanol metabolism in this yeast may be regulated by these compounds.     

The microbiol metabolism of C1 compounds. Oxidative phosphorylation in membrane preparations of Pseudomonas AM1

A method is described for preparation of membrane vesicles (diameter 80nm) capable of respiration-linked ATP synthesis. Vesicles prepared from succinate-grown bacteria oxidized NADH, succinate and ascorbate plus NNN'N'-tetramethylphenylenediamine; vesicles prepared from methanol-grown bacteria also oxidized methanol and formaldehyde, but they were otherwise identical. The uncoupling agent carbonyl cyanide chlorophenylhydrazone and the adenosine triphosphatase inhibitor dicyclohexylcarbodi-imide both inhibited ATP synthesis, whereas they had no effect on the rate of respiration. Rotenone inhibited ATP synthesis and respiration with NADH as substrate; antimycin A inhibited with succinate as substrate, and cyanide inhibited with all substrates. P/O ratios were usually 0.7-1.3 with NADH, 0.6-1.0 with succinate and 0.2-0.6 with reduced NNN'N'-tetramethylphenylenediamine or methanol as respiratory substrate. When 2,6-dichlorophenol-indophenol was used as an alternative electron acceptor to O(2) (NADH as donor) the P/2e ratio was 1.65. Although these P/O ratios are minimum values, because they do not take into account unknown amounts of uncoupled O(2) consumption, they are consistent with previous proposals [O'Keeffe & Anthony (1978) Biochem, J.170, 561-567] based on measurements of proton translocation in whole cells. The results also confirm that methanol dehydrogenase and cytochromes c and a/a(3) are arranged so that the first step in methanol oxidation is coupled to synthesis of ATP.     

Inhibition of 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) activity of human lung microsomes by genistein, daidzein, coumestrol and C(18)-, C(19)- and C(21)-hydroxysteroids and ketosteroids

Epidemiologic data suggest a relationship between dietary intake of phytochemicals and a lower incidence of some cancers. Modulation of steroid hormone metabolism has been proposed as a basis for this effect. It has been shown that aromatase, 3beta-hydroxysteroid dehydrogenase and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) are inhibited by the isoflavones, genistein and daidzein, and by coumestrol. In general, the extent of inhibition has been expressed in terms of IC50-values, which do not give information as to the pattern of inhibition, i.e., competitive, non-competitive, or mixed. Less is known of the effects of these compounds on 3alpha-HSD. The human lung is known to have a high level of 17beta-HSD and 3alpha-HSD activity. During the course of studies to characterize both activities in normal and inflamed lung and lung tumors we noted that 3alpha-HSD activity with 5alpha-DHT of microsomes from normal, adult lung was particularly susceptible to inhibition by coumestrol. To clarify the pattern of inhibition, the inhibition constants Ki and K'i were evaluated from plots of 1/v versus [I] and [S]/v versus [I]. Genistein, daidzein and coumestrol gave mixed inhibition patterns versus both 5alpha-DHT and NADH. In contrast, 5alpha-androstane-3,17-dione and 5alpha-pregnane-3,20-dione were competitive with 5alpha-DHT. NAD inhibited competitively with NADH. Our findings demonstrate that phytochemicals have the potential to inhibit 5alpha-DHT metabolism and thereby affect the androgen status of the human lung. The observation of a mixed inhibition pattern suggests these compounds bind to more than one form of the enzyme within the catalytic pathway.     

Inhibition of NADH-dehydrogenase by low concentrations of NAD+]

Low concentrations of NAD+ inhibit the NADH: acceptor reductase reactions catalyzed by soluble NADH dehydrogenase from bovine heart mitochondria. The degree of incomplete inhibition of the enzyme depends on the nature and concentration of artificial electron acceptors and is manifested only at low concentrations of the latter. Marked inhibition was demonstrated for the 2.6-dichlorophenolindophenol-, ferricyanide- and O2-reductase reactions, being weakly pronounced during the measurement of the NADH: cytochrome c reductase activity. The inhibition of the above reactions by oxidized NAD+ isn't competitive towards NADH. A kinetic scheme is proposed, which postulates NADH: acceptor reductase reactions occurrence via two mechanisms, namely, a ping-pong mechanism and oxidation of the product-enzyme complex by the acceptor. It was shown that low concentrations of NAD+ also inhibit the NADH oxidase reaction catalyzed by complex I.     

Oxidative phosphorylation in intact chl-r mutants of Escherichia coli K 12.

The efficiency of oxidative phosphorylation was estimated in intact resting cells of Escherichia coli K 12, strain PA 601 (chl-s) and its chl-r mutants, all of them grown anaerobically in the presence of nitrate. The oxidation of endogenous NADH in intact chl-s cells was accompanied by the formation of ATP whatever the terminal electron acceptor, oxygen or nitrate, so that it was possible to conclude that the energy conservation sites are operating with either of the two acceptors in cells grown anaerobically in the presence of nitrate. For chl-r mutants oxidation of endogenous NADH correlated with ATP-production was found only with oxygen as electron acceptor. It is concluded that the energy-conservation sites are preserved in these mutants, the nitrate respiratory chain of which is altered. This assumption is corroborated by the effects of uncouplers of oxidative phosphorylation on ATP-synthesis.     

Anomalous effect of uncouplers on respiratory chain-linked transhydrogenation in Escherichia coli membranes: evidence for a localized proton pathway?

Energization of the pyridine nucleotide transhydrogenase in everted membrane vesicles from Escherichia coli JM83 was compared with the process in vesicles of the same strain transformed with the plasmid pDC21 overexpressing this enzyme. Proton translocation was assayed by the quenching of the fluorescence of the probe quinacrine. Agents able to discharge transmembrane proton gradients such as nigericin and the uncouplers 3,3',4',5-tetrachlorosalicylanilide and carbonyl cyanide m-chlorophenylhydrazone inhibited ATP-dependent transhydrogenation of NADP by NADH and discharged transmembrane proton gradients generated by transhydrogenation of AcNAD by NADPH, by oxidation of NADH, and by hydrolysis of ATP. This was observed in everted membrane vesicles of both strains JM83 and JM83pDC21. These strains differed significantly in the response of the NADH oxidation-dependent transhydrogenase. This reaction was inhibited by nigericin and uncouplers in membrane vesicles of JM83 but there was little inhibition or the reaction was stimulated in JM83pDC21, in spite of the discharge of the NADH oxidation-generated proton gradient measured by quinacrine fluorescence in the latter strain. It is proposed that the transhydrogenase is energized by direct or local (nonbulk phase) proton translocation in membranes of this strain. Uncouplers might facilitate these routes but would not discharge them. The generality of these observations was shown using other strains. NADH oxidase activity was severalfold lower in membrane vesicles of JM83pDC21 compared with JM83. The levels of ubiquinone and cytochromes, and the activities of NADH dehydrogenases I and II, and of cytochrome oxidase, were similar in the two strains. It is concluded that the NADH oxidase activity of JM83pDC21 is low because of the reduced rate of collision between electron-transferring complexes of the respiratory chain due to the large amount of transhydrogenase protein in the membranes of this strain. The large amount of transhydrogenase favors direct, nonbulk phase proton transfer. Transhydrogenase activity was stimulated by Ca2+, Mg2+, or Mn2+.     

Oxidative phosphorylation in Micrococcus denitrificans. I. Preparation and properties of phosphorylating membrane fragments

A procedure was described to prepare stable membrane fragments from aerobically grown cells of Micrococcus denitrificans. This preparation contained flavins, cytochromes b, c, a and o, and catalyzed the synthesis of ATP coupled to the oxidation of NADH and succinate. The P:O ratios were about 1.0 for NADH and 0.4 for succinate oxidation. The electron-transfer pathways responsible for these oxidations were similar to, though not identical with, those of mammalian mitochondria in their construction and sensitivity to inhibitors. Oxidative phosphorylation by the membrane fragments was uncoupled by the usual uncouplers and energy-transfer inhibitors, though 2,4-dinitrophenol was much less effective and higher concentrations of oligomycin and tributyltin chloride were required for complete inhibition as compared with the mitochondrial system. Oleate also caused uncoupling, which was relieved by serum albumin. Treatment with high concentrations of LiCl yielded an essentially uncoupled preparation, but this treatment as well as many other procedures failed to yield soluble coupling factors. Unlike the mitochondrial ATPase activity, ATP hydrolysis by the membrane fragments was inhibited to about 50% by uncouplers and energy-transfer inhibitors. It seems that the bacterial preparation possessed two types of ATPase, one of which was sensitive to these reagents as well as to LiCl treatment and probably to high concentrations of ADP. The advantage of this preparation for the study of the mechanism of oxidative phosphorylation is discussed.     

Demonstration and possible function of NADH:NAD+ transhydrogenase from ascaris muscle mitochondria.

Mitochondria from the muscle of Ascaris lumbricoides var. suis function anaerobically. NADH is generated in the intermembrane space as a consequence of the "malic" enzyme reaction. It has been suggested that this reducing equivalent in the form of hydride ion, would be translocated across the inner membrane in order to mediate ATP generation via the fumarate reductase reaction. In accord with this suggestion, intact Ascaris mitochondria showed appreciable NADH oxidase activity. Sonication resulted in an approximately 2-fold increase in NADH oxidase activity, whereas "malic" enzyme, fumarase, and NADH:NAD+ transhydrogenase activities increased approximately 7- to 14-fold, respectively. Phosphorylation capabilities and permeability toward pyridine nucleotides also indicated the intactness of the mitochondria. Ascaris mitochondria incubated anaerobically in the presence of fumarate, and [14C]NADH catalyzed a rapid reduction of the fumarate to succinate with the concomitant formation of equivalent quantities of extramitochondrial NAD+. However, very little isotope was recovered from the washed mitochondria, indicating the possibility of hydride ion translocation in the absence of nucleotide translocation. NADH:NAD+ transhydrogenase has been isolated from the muscle mitochondria of the intestinal nematode, Ascaris lumbricoides var. suis. The enzyme seems to have been solubilized from the mitochondrial membrane fraction by treatment with sodium deoxycholate followed by dialysis and subsequent adsorption by and elution from alumina C gamma. No NADPH:NAD+ transhydrogenase activity was detectable, making the Ascaris system unique over others reported. Activity was protected by L-cysteine, reduced glutathione and dithioerythritol, but strongly inhibited by low concentrations of p-chloromercuribenzoate or silver nitrate. The thionicotinamide derivative of NAD+ (thioNAD+) was employed to accept hydride ions from NADH in order to assay spectrophotometrically at 398 nm. Apparent Km values for thioNAD+ and NADH were 1 X 10(-4) M and 8 X 10(-6) M, respectively. That the physiological nucleotide, could act as hydride ion acceptor from NADH was indicated by the findings that NAD+ competitively inhibited the reduction of thioNAD+ when assayed at 398 nm. The additional finding of a noncompetitive inhibition between NAD+ and NADH suggested at least two binding sites on the enzyme, one for NADH and another common site for NAD+ and thioNAD+. More conclusive evidence indicating the participation of NAD+ as acceptor was obtained by incubation of the enzyme with NADH and [14C]NAD+ and demonstrating a rapid formation of [14C]NADH. These findings, in conjunction with those discussed above, suggest a physiological function of this enzyme in hydride ion translocation.     

Inhibition of bovine heart NAD-specific isocitrate dehydrogenase by reduced pyridine nucleotides: modulation of inhibition by ADP, NAD+, Ca2+, citrate, and isocitrate

The activity of NAD-dependent isocitrate dehydrogenase from bovine heart was inhibited by NADH (apparent Ki about 4.3 microM) and NADPH (Ki about 9.8 microM) at subsaturating substrate concentrations at pH 7.4. The inhibition by NADH or NADPH was reversed competitively by magnesium isocitrate in the presence of ADP, but not without ADP. Reversal of inhibition by NADH or NADPH with respect to NAD+ was competitive or of the linear mixed type depending on whether ADP was absent or present. ADP3- (0.2 mM) increased the Ki(app) for NADPH from 9.8 to 27.1 microM; further addition of Ca2+ (0.2 mM) raised the Ki(app) to 127 microM. For the modification of NADPH inhibition by ADP, S0.5 for Ca2+ was approximately 48 microM. This compares to the Km for Ca2+ of 0.3-1 microM for the activation of the enzyme without NADPH [Denton, R. M., Richards, D. A., & Chin, J. G. (1978) Biochem. J. 176, 899-906; Aogaichi, T., Evans, J., Gabriel, J., & Plaut, G. W. E. (1980) Arch. Biochem. Biophys. 204, 350-360]. ADP did not affect the Ki for NADH. Magnesium citrate, which was about 100-fold more effective as a positive modifier of the enzyme with ADP than without ADP [Gabriel, J. L., & Plaut, G. W. E. (1983) Fed. Proc., Fed. Am. Soc. Exp. Biol. 42, 2082], reversed competitively the inhibition by NADPH in the presence of ADP, but not without ADP. Magnesium citrate did not reverse NADH inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)