Pyridine Nucleotide Transhydrogenase Research Articles

Glucagon treatment stimulates the metabolism of hepatic submitochondrial particles.

Hepatic submitochondrial particles, prepared at neutral pH from rats pretreated with glucagon, exhibited stimulated rates of State 3 and uncoupled respiration when succinate or NADH were the substrates, but not when ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine were employed. Measurements of 8-anilino-1-naphthalenesulfonic acid fluorescence in the particles indicated that glucagon treatment resulted in a stimulation of energization supported by succinate respiration or ATP hydrolysis. Similarly, the energy-linked pyridine nucleotide transhydrogenase and reverse electron flow reactions driven by succinate oxidation or ATP were also stimulated. The results indicate that mitochondrial substrate transport is not the prime locus of glucagon action. It is suggested that the increased level of energization in particles prepared from glucagon-treated rats is a reflection of a stimulation of the respiratory chain, possibly between cytochromes b and c, and the ATP-forming reactions.

Dependence of [formula omitted] pyridine nucleotide transhydrogenase on phospholipids and its sensitivity to N-ethylmaleimide

A partially purified preparation of pyridine nucleotide transhydrogenase (E.C. 1.6.1.1.) (energy-independent) has been obtained from membranes of Escherichia coli by means of deoxycholate extraction and DEAE-cellulose chromatography in the presence of Triton X-100. The enzyme was lipid-depleted by treating with cholate and ammonium sulfate. The preparation was reactivated by various phospholipids, in particular, bacterial cardiolipin and phosphatidyl glycerol. Phosphatidyl ethanolamine, the major phospholipid in the outer membrane of E. coli , was relatively ineffective in stimulating activity. The membrane-bound pyridine nucleotide transhydrogenase is slowly inhibited by N-ethylmaleimide. Protection against inhibition was achieved with NAD + and NADP +, but NADPH served to accelerate the rate of inhibition.

Regulatory properties of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa. Active enzyme ultracentrifugation studies.

Active enzyme ultracentrifugation studies of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa (EC 1.6.1.1.) show that the enzymatic reaction is catalyzed by a molecular species characterized by an S20,W value of about 34 S, whatever the reduced substrate may be (tri- or diphosphopyridine nucleotide). The filamentous aggregated form of the enzyme (S20,W = 121 S and higher), identified by previous investigations (Cohen, P. T., and Kaplan, N. O. (1970), J. Biol. Chem. 245, 2825-2836; Louie, D. D., Kaplan, N. O., and Mc Lean, J. D. (1972), J. Mol. Biol. 70, 651-664), appears, therefore, to be an inactive species. The physiological implications of the enzyme are discussed. Several lines of evidence lead to the conclusion that the transhydrogenase might act as an essential link between carbohydrate catabolism and the respiratory chain.

Regulatory properties of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa. Kinetic studies and fluorescence titration.

Mechanisms involved in the action of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa (EC 1.6.1.1) have been investigated by means of kinetic studies and fluorescence titration. Our results, as well as those from previous investigations, suggest that the allosteric MWC model (Monod, J., Wyman, J., and Changeux, J. P. (1965), J. Mol. Biol. 12, 88-118) may be used as a first step for the explanation of the properties of the transhydrogenase. The basic reaction of the enzyme is the oxidation of reduced triphosphopyridine nucleotide (TPNH) by diphosphopyridine nucleotide (DPN+). In terms of the model, the functional R state is favored by TPNH, whereas the product triphosphopyridine nucleotide (TPN+) behaves as an allosteric inhibitor, and is therefore assumed to favor the nonfunctional T state. To a slight extent, the T state is also favored by inorganic phosphate. On the other hand, adenosine 2'-monophosphate and several other 2'-phosphate nucleotides function as activators, and hence are presumed to shift the allosteric equilibrium toward the R state. The studies in this paper suggest a specific regulatory site for the transhydrogenase.

Thyroid hormone action on mitochondria

Measurements of fluorescence at >420 nm and extracted NADPH in mitochondria obtained from the livers of hypothyroid rats show that the addition of Pi, ADP and glutamate rapidly reduces over 90% of the total reducible intrinsic pyridine nucleotides in State 3, compared with 20% in normals. The total fluorescence intensity change and reducible NADP+ is about twice normal in hypothyroid mitochondria. Adding 6-30 microM L-thyroxine to hypothyroid mitochondria in vitro decreases and delays the substrate-induced reduction of pyridine nucleotides, and excludes both NADP+ from such reduction and NADPH from oxidation by added ADP + Pi, without changing the high NADP(H) content. The correcting actions of the hormone are rapidly reversed by albumin, probably by binding free hormone. Changes in respiration do not appear to account for these observations. There is indirect evidence for decreased phosphorylation of added ADP in hypothyroid mitochondria, and a correction by added hormone. The hormonal actions on NADP(H) redox reactions are not reproduced by 1 to 6 microM dinitrophenol in vitro. L-Thyroxine appears to specifically block the participation of NADP(H) in redox reactions in mitochondria from hypothyroid rats, perhaps by effecting a sequestration of the nucleotide, by inhibiting the pyridine nucleotide transhydrogenase, or by activating an energy-linked process that competes with transhydrogenation.

Pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa: Purification by affinity chromatography and physicochemical properties

Pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa was purified 150-fold by affinity chromatography on immobilized 2′-AMP. The binding of the enzyme is pH dependent. Elution was achieved with 2′-AMP, NADP +, or NADPH but not with 5′-AMP, NAD +, or NADH. The enzyme preparations appeared to be homogeneous in gel chromatography and ultracentrifugation, but only if these procedures were carried out in the presence of 2′-AMP or NADP +. With 2′-AMP a sedimentation coefficient of 34 S, a molecular weight of 1.6–1.7 million, and a Stokes' radius of 11.7 nm were determined. In the presence of NADP + the sedimentation coefficient was 42 S and the molecular weight was 6.4 million. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed one kind of subunit with a molecular weight of 54,000. This was consistent with results from amino acid analyses and paper chromatography of peptides. Eight molar urea inactivated the enzyme but did not dissociate it into subunits. Full activity was restored after dialysis against urea-free buffer by mercaptoethanol and flavin-adenine dinucleotide.

Pyridine nucleotide transhydrogenases of parasitic helminths

Mitochondria from the parasitic helminth, Hymenolepis diminuta, catalyzed both NADPH:NAD + and NADH:NADP + transhydrogenase reactions which were demonstrable employing the appropriate acetylpyridine nucleotide derivative as the hydride ion acceptor. Thionicotinamide NAD + would not serve as the oxidant in the former reaction. Under the assay conditions employed, neither reaction was energy linked, and the NADPH:NAD + system was approximately five times more active than the NADH:NADP + system. The NADH:NADP + reaction was inhibited by phosphate and imidazole buffers, EDTA, and adenyl nucleotides, while the NADPH:NAD + reaction was inhibited only slightly by imidazole and unaffected by EDTA and adenyl nucleotides. Enzyme coupling techniques revealed that both transhydrogenase systems functioned when the appropriate physiological pyridine nucleotide was the hydride ion acceptor. An NADH:NAD + transhydrogenase system, which was unaffected by EDTA, or adenyl nucleotides, also was demonstrable in the mitochondria of H. diminuta. Saturation kinetics indicated that the NADH:NAD + reaction was the product of an independent enzyme system. Mitochondria derived from another parasitic helminth, Ascaris lumbricoides, catalyzed only a single transhydrogenase reaction, i.e., the NADH:NAD + activity. Transhydrogenase systems from both parasites were essentially membrane bound and localized on the inner mitochondrial membrane. Physiologically, the NADPH:NAD + transhydrogenase of H. diminuta may serve to couple the intramitochondrial metabolism of malate (via an NADP linked “malic” enzyme) to the anaerobic NADH-dependent ATP-generating fumarate reductase system. In A. lumbricoides, where the intramitochondrial metabolism of malate depends on an NAD-linked “malic” enzyme which is localized primarily in the intermembrane space, the NADH:NAD + transhydrogenase activity may serve physiologically in the translocation of hydride ions across the inner membrane to the anaerobic energy-generating fumarate reductase system.

Yeast glutathione reductase: Steady-state kinetic studies of its transhydrogenase activity

Yeast glutathione reductase catalyzes a pyridine nucleotide transhydrogenase reaction using either NADPH or NADH as the electron donor and thionicotinamideadenine dinucleotide phosphate as the electron acceptor. Competitive substrate inhibition of the transhydrogenase reaction by NADPH ( K i = 11 μM) is observed when NADPH is the electron donor. Competitive substrate inhibition by thionicotinamide-adenine dinucleotide phosphate ( K i = 58 μM) is observed with NADH as the electron donor. The turnover numbers of the two transhydrogenase reactions are similar and are equal to about 1% of the turnover number for the NADPH-dependent reduction of oxidized glutathione catalyzed by the enzyme. The transhydrogenase kinetics are analyzed in terms of a pingpong mechanism. It is concluded that the substrate inhibition results from formation of abortive complexes of NADPH with the reduced form of the enzyme and of thionicotinamide-adenine dinucleotide phosphate with the oxidized form of the enzyme. With NADPH as the electron donor, the apparent Michaelis constant for thionicotinamide-adenine dinucleotide phosphate is sensitive to the ionic composition of the assay medium. The data are interpreted to support the existence of a general pyridine nucleotide-binding site at the active site of the enzyme and separate from the binding site for oxidized glutathione.

Bacterial Respiration‐Linked Proton Translocation and Its Relationship to Respiratory‐Chain Composition

1. The relationship between chain composition and the efficiency of respiration-linked proton translocation was studied in nine bacterial species of widely differing taxonomic and ecological status. 2. All the bacteria investigated contained respiratory chain dehydrogenases, ubiquinone and/or menaquinone, cytochrome b and cytochrome oxidase aa3 and/or o. In addition, some of these organisms also contained pyridine nucleotide transhydrogenase and/or cytochrome c. 3. leads to H+/O ratios of whole cell suspensions oxidising endogenous substrates were in the approximate range 4-8 mol H+ translocated per g-atom oxygen consumed. It was concluded from the observed leads to H+/O ratios of cells loaded with specific substrates that proton-translocating loops 1 and 2 were present in all of the organisms investigated, but that loops 0 and 3 were dependent upon the presence of pyridine nucleotide transhydrogenase and cytochrome c respectively. 4. The wide range in energy conservation efficiency which was observed in these organisms is discussed in relation to their respiratory chain composition and natural habitat.

The reconstitution of the mitochondrial energy-linked transhydrogenase

Complex I (NADH-ubiquinone reductase) catalyzes pyridine nucleotide transhydrogenase at rates several fold higher than those found in submitochondrial particles from bovine heart. An ATP-dependent reduction of NADP + by NADH was demonstrated after combination of Complex I with phospholipids, hydrophobic proteins derived from bovine heart mitochondria, and mitochondrial ATPase (F 1) 1 1 Abbreviations: F 1, coupling factor 1 (ATPase); FCCP p -trifluoromethoycarbonylcyanide phenylhydrazone. . The reaction was inhibited by oligomycin, uncoupling agents and low concentrations of Triton X-100.

Studies on pyridine nucleotide transhydrogenase in fish muscle. II. Purification and some physico-chemical properties.

Pyridine nucleotide transhydrogenase was extracted from mitochondria of yellowtail muscle prefrozen below -20°C. The extract was applied to DEAE-cellulose column chro-matography, resulting in the appearance of two isozymes. The first peak named isozyme-I exhibited both T. D and D. D activities, whereas the second peak named isozyme-II exhibited only D. D activity. Both isozymes were highly purified by repetition of Sephadex G-200 gel filtration and DEAE-cellulose column chromatography. The specific activities of isozyme-I preparation thus obtained were about 500 (T. D) and 140 (D. D) times as high as those of the extract. The specific activity of the isozyme-II preparation was about 600 times in respect of D. D activity. Molecular weights of isozyme-I and -II were estimated at about 37, 000 and 48, 000 respectively by SDS-disc electrophoresis. Absorption spectra of both isozymes showed a maximum at 280nm. Neither of them, however, exhibited any flavin specific maximum around 442nm. This seems to indicate that neither of the yellowtail enzymes are flavopro-teins, unlike the enzymes of bacterial origin.

Studies on pyridine nucleotide transhydrogenase in fish muscle. III. Enzymatic properties and kinetics.

Enzymatic properties of the two isozymes (I and II) of yellowtail pyridine nucleotide transhydrogenase were examined. Isozyme-I showed a pH optimum at 7.8 in respect of T.D activity, and two optima at 6.5 and 7.8 in respect of D. D activity. Isozyme-II exhibiting only D. D activity showed a pH optimum at 6.5. Neither isozyme was activated by 2'-AMP, which activates Pseudomonas transhydrogenase. Some nucleotides and lecithin which affect in various ways the enzymes from other sources did not show any effect on the activity of isozyme-II. In addition, calcium chloride enhanced effectively the activity of isozyme-II. On the contrary, the activity was to some extent inhibited by manganese and cupric chloride. Kinetic constants, KTNAD, KNADH2, and V1, were 13 μM, 12 μM, and 9.1 μmole-min-1. mg protein-1, respectively. Based on the results of kinetic studies, the reaction mechanism of isozyme-II was classified from among various mechanisms proposed by CLELAND as a Theorell-Chance mechanism.

Studies on pyridine nucleotide transhydrogenase in fish muscle. I. Distribution of the enzyme activity in fish.

The distribution of pyridine nucleotide transhydrogenase was examined in various tissues, chiefly in skeletal muscle, of 21 species of marine and freshwater fishes. The activity of this enzyme was detected irrespective of fish species or of source tissues. The activity in the muscle of several species of fishes such as yellowtail and lizard fish was particularly high. The D. D activity of this enzyme generally was several times stronger than the T. D activity in the muscle. It was noticed that the piscine enzyme, unlike that of mammalian origin, is easily solubilized from muscle prefrozen for several days below -20°C. As for the intracellular distri-bution of the enzyme activity, it was found that it is localized in the mitochondrial fraction as it is in mammals.

Energy conservation in Bacillus megaterium.

1. The respiratory chain energy conservation systems of Bacillus megaterium strains D440 and M have been investigated following growth in batch and continuous culture. Respiratory membranes from these strains contained cytochromes b, aa3, o and b, c, a, o, respecitvely; both readily oxidised NADH but neither showed any pyridine nucleotide transhydrogenase activity. 2. Whole cells of both strains exhibited endogenous leads to H+/O ratios of approximately 4; when loaded with specific substrates the resultant leads to H+/O ratios indicated that proton translocating loops 1 and 2 were present in strain D440 and that loops 2 and 3 were present in strain M. 3. In situ respiratory activities were measured as a function of dilution rate during growth in continuous culture. True molar growth yields with respect to oxygen (Y02) of approximately 50 g cells-mole oxygen-1 were obtained for most of the nutrient limitations employed. Average values for YATP of 12.7 and 10.8 g cells-mole ATP equivalent-1 were subsequently calculated for strains D440 and M respectively. 4. Energy requirements for maintenance purposes were low in energy-limited cultures but were substantially increased when growth was limited by nitrogen source (NH+/4). Under the latter conditions there is probably a partial uncoupling of energy-conserving and energy-utilising processes leading to energy wastage.

Pyridine nucleotide transhydrogenase activity of soluble cardiac NADH dehydrogenase and particulate NADH-ubiquinone reductase

Pyridine nucleotide transhydrogenase activities of a highly purified soluble NADH dehydrogenase and particulate NADH-ubiquinone reductase (Complex I) differ in their pH optima (5.0 and 6.0, respectively) and in their sensitivity to inhibition by Mg 2+ and ATP. The oxidation of NADPH with ferricyanide as acceptor is very similar in both preparations with a pH optimum around 5.0. It is concluded that Complex I possesses two types of transhydrogenase activity, whereas only one has been found in the soluble dehydrogenase.

On a slow inhibitory effect of free fatty acids on the respiratory chain of non-phosphorylating submitochondrial particles from beef heart

Free fatty acids (FFA) exert a variety of inhibitory effects on the energy metabolism of mitochondria. They uncouple the oxidative phosphorylation [l-4], inhibit the ATP-Pi exchange [2,3], stimulate the Mg-dependent ATPase [ 1,2] and act as endogenous ionophores for potassium ions [ 51. Moreover, the CoA-esters of fatty acids are strong inhibitors of the adenine nucleotide translocase [6] and the pyridine nucleotide transhydrogenase system [7]. FFA exhibit an instantaneous multi-site inhibition of the electron transport system of non-phosphorylating submitochondrial particles (ETP) which is relieved or prevented by serum albumin [8,9]. In this report a new inhibitory effect of FFA is described which appears slowly.

Biochemical aspects of the endometrium

The distinctive biochemical properties of the endometrium are superimposed on a large number of properties and enzymatic capabilities that the endometrium shares with other tissues in the body. Endometrial cells have all of the enzymes to catalyze the glycolytic pathway, the pentose phosphate pathway, the β oxidation of fatty acids, oxidative phosphorylation, and the synthesis of glycogen, neutral fats, phospholipids, nucleic acids and proteins. Many of these enzymes undergo characteristic changes in activity correlated with the menstrual cycle and with pregnancy. The endometrium, like several other tissues, has specific, high affinity, low-capacity protein receptors which take up and bind estrogens noncovalently, together with other receptors that take up and bind progesterone. Prostaglandins, which have marked effects on uterine motility, also stimulate the synthesis of RNA when instilled into the lumen of the uterus. The synthesis of prostaglandins by human endometrium was demonstrated using endometrial explants grown in organ culture. Estradiol increased and progesterone decreased the synthesis of prostaglandin F 2α. The effects of estrogen appear to be mediated in large part by increased synthesis of RNA and protein, and of specific enzymes such as glucose-6-phosphate dehydrogenase and ornithine decarboxylase. The increase in protein and enzyme synthesis is evoked not only by the injection of estradiol, but by the instillation of RNA extracted from the uterus of an estrogen-treated animal. The RNA is purified by chromatography on a Sepharose polyuridylic acid column which binds only the RNA with a polyadenylic acid tail. Endometrium responds to estradiol in at least three independent fashions —by protein receptors which bind estradiol and transport it to the nucleus where it affects the genome producing RNA with specific information for the synthesis of specific enzymes, by an estrogen-dependent pyridine nucleotide transhydrogenase that mediates certain prompt metabolic effects, and by an effect on the synthesis or release of histamine which plays a role in the rapid appearance of hyperemia and water imbibition by endometrium.

Energy Conservation in Bacillus megaterium

1. The respiratory chain energy conservation systems of Bacillus megaterium strains D440 and M have been investigated following growth in batch and continuous culture. Respiratory membranes from these strains contained cytochromes b, aa3, o and b, c, a, o, respectively; both readily oxidised NADH but neither showed any pyridine nucleotide transhydrogenase activity. 2. Whole cells of both strains exhibited endogenous →H-/O ratios of approximately 4; when loaded with specific substrates the resultant →H+/O ratios indicated that proton translocating loops 1 and 2 were present in strain D440 and that loops 2 and 3 were present in strain M. 3. In situ respiratory activities were measured as a function of dilution rate during growth in continuous culture. True molar growth yields with respect to oxygen (YO2) of approximately 50 g cells·mole oxygen-1 were obtained for most of the nutrient limitations employed. Average values for YATP of 12.7 and 10.8 g cells·mole ATP equivalents-1 were subsequently calculated for strains D440 and M respectively. 4. Energy requirements for maintenance purposes were low in energy-limited cultures but were substantially increased when growth was limited by nitrogen source (NH4+). Under the latter conditions there is probably a partial uncoupling of energy-conserving and energy-utilising processes leading to energy wastage.

An NAD(P) reductase derived from Chlorobium thiosulfa-tophilum: Purification and some properties

A highly purified preparation of an NAD(P) reductase was obtained from Chlorobium thiosulfatophilum and some of its properties were studied. The enzyme possesses FAD as the prosthetic group, and reduces benzyl viologen, 2,6-dichloro-phenolindophenol and cytochromes c, including cytochrome c-555 ( C. thiosulfato-philum), with NADPH or NADH as the electron donor. It reduces NADP + or NAD + photosynthetically with spinach chloroplasts in the presence of added spinach ferredoxin. It reduces the pyridine nucleotides with reduced benzyl viologen. The enzyme also shows a pyridine nucleotide transhydrogenase activity. In these reactions, the type of pyridine nucleotide (NADP or NAD) which functions more efficiently with the enzyme varies with the concentration of the nucleotide used; at concentrations lower than approx. 1.0 mM, NADPH (or NADP +) is better electron donor (or acceptor), while NADH (or NAD +) is a better electron donor (or acceptor) at concentrations higher than approx. 1.0 mM. Reduction of dyes or cytochromes c catalysed by the enzyme is strongly inhibited by NADP +, 2′-AMP and and atebrin.

Energy-transducing adenosine triphosphatase from Escherichia coli: purification, properties, and inhibition by antibody.

The membrane adenosine triphosphatase (E.C. 3.6.1.3) from Escherichia coli has been solubilized with Triton X-100 and purified to near homogeneity. The purified enzyme has a sedimentation coefficient of 12.9S in a sucrose gradient, corresponding to a molecular weight of about 360,000. On electrophoresis in gels containing sodium dodecyl sulfate, it dissociates into subunits with apparent molecular weights of 60,000, 56,000, 35,000, and 13,000. The purified enzyme loses activity and breaks down into subunits when stored in the cold. Guanosine 5'-triphosphate and inosine 5'-triphosphate are alternative substrates. Ca(2+) and, to a small extent, Co(2+) or Ni(2+) will substitute for Mg(2+) in the reaction. The K(m) for Mg-adenosine triphosphate of the membrane-bound enzyme is 0.23 mM, and for the pure enzyme it is 0.29 mM. Azide is a noncompetitive inhibitor of both the membrane-bound enzyme and the pure enzyme. P(i) is a noncompetitive inhibitor of the solubilized enzyme. An antibody to the purified enzyme was obtained from rabbits. The antibody inhibits the solubilized enzyme and virtually all of the adenosine triphosphate hydrolysis by membranes from cells grown aerobically or anaerobically. The antibody also inhibits the adenosine triphosphate-stimulated pyridine nucleotide transhydrogenase (E.C. 1.6.1.1) of the E. coli membrane.