Transhydrogenase Reaction Research Articles

Studies of the energy-transfer system of submitochondrial particles. I. Competition between oxidative phosphorylation and the energy-linked nicotinamide-adenine dinucleotide transhydrogenase reaction.

The relationship between oxidative phosphorylation and the energy‐linked nicotinamide‐adenine dinucleotide transhydrogenase reaction catalyzed by phosphorylating submitochondrial particles from beef heart has been investigated. The efficiency of the particles in exhibiting oxidative phosphorylation (P/O ratio) and NADH‐linked NADP+ reduction (NADP+/O ratio) was measured, using succinate as oxidizable substrate and rotenone as inhibitor of NADH oxidase, under conditions where the phosphorylating and transhydrogenating systems were operating one by one or in parallel. When succinate oxidation was limited by increasing concentrations of malonate, the P/O ratio remained unchanged in the absence of an active phosphorylating system, and decreased in its presence. The NADP+/O ratio increased with increasing concentrations of malonate, both in the absence and presence of an active phosphorylating system. The (P + NADP+)/O ratio in the combined system approached 2, i.e., the generally accepted maximal ∼/O ratio for succinate oxidase. Active transhydrogenation caused an increase in the apparent Km for Pi in the phosphorylating system from 1.7 to 4.5 mM, and active phosphorylation caused an increase in the apparent Km for NADH in the transhydrogenating system from 21 to 40 μM. It is concluded that oxidative phosphorylation and the energy‐linked transhydrogenase reaction are competing for a common nonphosphorylated high‐energy intermediate generated by the respiratory chain. The kinetic evidence presented further suggests that the two systems interact with two different moieties of this high‐energy intermediate.

Stoicheiometry of the energy-linked nicotinamide nucleotide transhydrogenase reaction in intact rat-liver mitochondria

Stoicheiometry of the energy-linked nicotinamide nucleotide transhydrogenase reaction in intact rat-liver mitochondria

Steroid hydroxylations by guinea-pig adrenal tissue fractions

The effect of oxidizable substrates, α-ketoglutarate and isocitrate on 11β-hydroxylation in guinea-pig adrenal mitochondria has been studied. These substrates supported the conversion of Compound S (17α,21-dihydroxy-pregnene-3,20-dione) into cortisol as well as exogenously added NADPH in Ca 2+-swollen mitochondria. Progesterone was also hydroxylated at the C-11β position but not at the C-21 position. Microsomes, on the other hand, when NADPH was added, converted pregnenolone, progesterone and 17α-hydroxyprogesterone into Compound S, but showed no steroid 11β-hydroxylating activity. Evidence obtained in incubations carried out with α-ketoglutarate and isocitrate in the presence of 2,4-dinitrophenol, oligomycin, amytal and antimycin A indicate that α-ketoglutarate utilization for steroid 11β-hydroxylation is dependent on activity of the classical electron chain. This activity can be related to high energy intermediates possibly needed for NADPH reduction arising from NADH oxidation via the energy-requiring transhydrogenase reaction. These reactions do not appear necessary for isocitrate utilization and isocitrate oxidation probably gives rise to intramitochondrial NADPH reduction as a result of a NADP +-linked isocitrate dehydrogenase. Data obtained in oxygen uptake studies with an antimycin A blocked system supplied with α-ketoglutarate, are in accordance with this conclusion. The high-speed supernatant fraction (103000 × g) could partially replace α-ketoglutarate, isocitrate or Ca 2+ + NADPH, indicating that it contains a factor(s) (the physiological substrate?) which brings about intramitochondrial NADPH.

Oxidative phosphorylation in Micrococcus denitrificans. II. The properties of pyridine nucleotide transhydrogenase

A pyridine nucleotide transhydrogenase activity, supported by ATP or by succinate oxidation, was demonstrated in phosphorylating membrane fragments from Micrococcus denitrificans. The ATP-supported reaction was inhibited by various energy-transfer inhibitors and uncouplers or by treatment with high concentrations of LiCl. P i and arsenate showed a stimulatory effect on the ATP-supported activity; half-maximal stimulation was attained by about 80 μM phosphate. The transhydrogenase reaction dependent on succinate oxidation was not appreciably inhibited by energy-transfer inhibitors, although oleate and pentachlorophenol were almost equally effective in both reactions. P i did not stimulate the succinate-supported activity. From the effects of thyroxine and its derivatives on the energy-dependent and independent reductions of NAD + by NADPH, the involvement of the same transhydrogenase enzyme in both reactions was suggested. These and other results indicated that the energy-transfer system of M. denitrificans was very similar to, though not identical with, that of mammalian mitochondria.

Inhibition of Rat Liver Glutathione Reductase by the Reduced B-Chain of Insulin

Rat liver glutathione reductase is markedly inhibited by the isolated reduced B-chain (phenylalanine chain) of insulin. The inhibition is progressive with time, of the irreversible type, and follows the characteristics of a bimolecular reaction. The reaction between enzyme and inhibitor is dependent upon the presence of small amounts of NADPH (>2.2x10-7 M). Reduced glutathione or cysteine completely and rapidly reactivates the enzyme, glutathione being more active than cysteine. Insulin, reduced or sulphonated A-chain of insulin has no effect on the enzyme. The sulphonated B-chain has a slight inhibitory effect. No inhibition is found on yeast glutathione reductase. It is possible that the inhibition of glutathione reductase by the B-chain may be of importance in regulating the amount of GSH available for the glutathione-insulin transhydrogenase reaction, thus providing a regulatory feed-back cycle.

Effect of ccupling factor 3 on oxidative phosphorylation

This chapter focuses on the mitochondrial coupling factors. Coupling factors are defined as being materials of mitochondrial origin that are essential for the process of energy conservation to function. The mitochondrial membrane loses its ability to synthesize ATP when the coupling factor is extracted from the membrane. This function can be restored when the coupling factor recombines with a membrane preparation specifically depleted of the coupling factor. Coupling factors are components of an uncharacterized multi-enzyme complex. Coupling factors with no known enzymatic activity are usually assayed by adding aliquots of solutions containing the coupling factor to suspensions of membranes deficient of an energy-linked function, for example, a complete or decreased ability to catalyze one or more of partial reactions of oxidative phosphorylation, ATPase, ATP-Pi exchange, ATP-dependent reduction of NAD by succinate, and the energy-driven transhydrogenase reactions. The ability of the added coupling factor to stimulate or restore the chosen reaction catalyzed by the membranes is used as a measure of coupling factor activity

Further studies on corticosteroidogenesis: IV. Inhibition of utilization of biolgical substrates for corticoid synthesis by high calcium concentrations possible role of transhydrogenase in corticosteroidogenesis

The biological substrates isocitrate, succinate, α-ketoglutarate and ß-hydroxybutyrate initiate the conversion of DOC into corticosterone in mitochondria incubated at pH 7.4. The bustrates are also utilized by the reconstituted homogenate (mitochondria + microsomes + supernatant fraction) and whole homogenate for the conversion of pregnenolone, progesterone or DOC into corticosterone. As is known to occur in liver mitochondria, low Ca 2+ concentrations in rat adrenal mitochondria are postulated to increase oxidation of biological substrates thereby effecting an increased transformation of DOC into corticosterone. At higher levels of Ca 2+ (1 mM) the biological substrates could no longer be utilized and this occurred when mitochondria were maximally swollen. When swelling of mitochondria was extensive, exogenous TPNH presumably penetrated into the mitochondria and was utilized for 11ß-hydroxylation of DOC. In the non-swollen mitochondria or at low Ca 2+ levels, TPNH was excluded. Whereas mitochondria alone could utilize the substrates, microsomes required the presence of the supernatant fraction. Data derived from mitochondrial, microsomal, particulate + supernatant fractions or whole homogenate incubations in the absence or presence of antimycin, ferricyanide, 2,4-dinitrophenol and Amytal indicated that the biological substrates are oxidized mainly via the classical electron or cytochrome chain by mitochondria. When exogenous TPNH was added to swollen mitochondria or when substrates were added to microsomes + supernatant fraction the flux of electrons initiated by TPNH oxidation appeared to occur through the chain designated as flavoprotein → non-heme iron-protein → cytochrome P 450. The level of transhydrogenase activity in mitochondria and microsomes was assessed by the amount of steroid substrates transformed into their hydroxylated derivatives when the particulate fractions were incubated in the presence of relatively high levels of DPNH plus TPN +. Ca 2+ was needed to demonstrate the activity in the mitochondria which indicated that non-swollen mitochondria are impermeable to pyridine nucleotides. In these instances, Ca 2+ had little effect on the microsomes whereas at high Ca 2+ plus low TPN + plus high DPNH concentrations this ion appeared to inhibit the transhydrogenase reaction. Although various effects of adenosine-3′,5′-monophosphate, ATP, thyroxine and ADP plus P 1 plus Mg 2+_ in mitochondrial and homogenate incubations were observed, these were of such a nature as to make impossible firm conclusions of a function of these substances in a possible control of the transhydrogenase enzyme. Incubations carried out with succinate in the absence or presence of thyroxine and ATP have yielded some data indicating that the transhydrogenase reaction might be implicated in bringing about an oxidation of biological substrates.

Energy-linked reactions in mitochondria: studies on the mechanism of the energy-linked transhydrogenase reaction.

Summary 1. The mechanism of both the energy-linked and non-energy-linked trans-hydrogenase reaction catalysed by submitochondrial particles from beef-heart mitochondria has been examined using 3 H- and 14 C-labelled pyridine nucleotides as substrates. 2. The energy-linked transhydrogenase reaction does not proceed via a net group transfer of either the adenine or nicotinamide moieties or of phosphate from one pyridine nucleotide to another. 3. Studies with 3 H-labelled pyridine nucleotides show conclusively that in the energy-linked transhydrogenase reaction hydrogen is transferred directly from the A locus of NADH to the B locus of NADP + , i.e . the energy-linked transhydrogenase is an A:B transhydrogenase involving direct hydrogen transfer. 4. Studies on the non-energy-linked transhydrogenase show conclusively that the stereochemistry of the non-energy-linked reaction is identical to that of the energy-linked transhydrogenase reaction. 5. When the reaction is carried out in tritiated water there is only a low incorporation of tritium into NADPH thus confirming that the reaction is one involving direct hydrogen transfer.

The Metabolism of Reduced Pyridine Nucleotides in Tumor Tissue

Abstract Tritiated reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate were generated in the intact tumor cell from specifically labeled substrates and their metabolism compared with patterns in normal liver. In three experimental hepatomas, differences between the metabolism of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate found in normal liver narrowed and tended to disappear. Of several possible explanations for the findings, activation of a transhydrogenase reaction appears most likely. Similar but less marked changes were obtained in regenerating rat liver, suggesting a regulatory role for transhydrogenase reactions in the metabolism of reduced pyridine nucleotides in the intact cell.

Some energy linked reactions in the Keilin-Hartree heart muscle preparation

Previously we ( Kettman and King, 1963) demonstrated an oligomycin-sensitive ATPase in the Keilin-Hartree heart muscle preparation (also cf. Huijing and Slater, 1961). The ATPase was also solubilized and the soluble enzyme was insensitive to the antibiotic. This note reports some energy linked reactions in the Keilin-Hartree preparation. A soluble extract from heart mitochondria stimulates not only the transhydrogenase reaction but also phosphorylation when the heart muscle preparation is used as the electron transport system for succinate oxidation.

Stereochemistry of hydrogen-transfer in the energy-linked pyridine nucleotide transhydrogenase and related reactions

The stereochemistry of hydrogen-transfer in the energy-linked and non-energy-linked pyridine nucleotide transhydrogenase, the respiratory chain-linked NADH dehydrogenase, and the DT diaphorase-catalyzed reactions have been investigated with tritiated pyridine nucleotides. Both the energy-linked and the non-energy-linked pyridine nucleotide transhydrogenase reactions, catalyzed by stibmitochondrial particles from beef heart, involve the 4A hydrogen atom of NADH, and the 4B hydrogen atom of NADPH. The reactions involve no intrinsic exchange of hydrogen atoms between the reduced pyridine nucleotides and water. The aerobic oxidation of NADH, and the energy-linked reduction of NAD+ by succinate, catalyzed by the same particles, involve the 4B hydrogen atom of NADH, and are connected with a rapid exchange of hydrogen atoms between the latter and water. The reaction catalyzed by purified rat-liver DT diaphorase involves the 4A hydrogen atom of either NADH or NADPH, and involves no exchange of hydrogen atoms between the reduced pyridine nucleotides and water. Studies with 14C-labelled NADH indicate that the energy-linked pyridine nucleotide transhydrogenase reaction proceeds without an exchange of the nicotinamide moieties of the two pyridine nucleotides. These results are discussed in relation to the mechanism of the energy-linked pyridine nucleotide transhydrogenase reaction.

On the mechanism of oxidative phosphorylation: VIII. Further evidence for distinct transhydrogenase reactions in submitochondrial particles

Reduction of exogenous NADP by NADH 2 in submitochondrial particles has been shown to proceed by an energy-unrelated and an energy-related transhydrogenase sequence. The latter is activated by Mg 2+ and ATP, and inhibited by oligomycin and uncouplers of oxidative phosphorylation. Energy-linked NADP reduction, in addition, appears related to the energy-coupled reactions of oxidative phosphorylation.

A nonspecific pyridine nucleotide diaphorase from spinach

A diaphorase which can employ either TPNH or DPNH as the reductant has been purified from spinach leaves. Preparations of the enzyme, which were estimated to be 90% pure by ultracentrifugal analysis, were unable to catalyze a transhydrogenase reaction between the pyridine nucleotides or either nucleotide and its analogs. The diaphorase which appears to be a flavoprotein catalyzes the reduction of menadione, indophenol dyes, and ferricyanide, but does not catalyze the reduction of flavin mononucleotide, flavin adenine dinucleotide, or cytochrome c.

Equilibrium studies of the energy-dependent and non-energy-dependent pyridine nucleotide transhydrogenase reactions

Equilibrium studies of the energy-dependent and non-energy-dependent pyridine nucleotide transhydrogenase reactions

INHIBITION OF PYRIDINE NUCLEOTIDE TRANSHYDROGENASE BY DICUMAROL.

WE have recently described the hormonal sensitivity of a transhydrogenase reaction in mitochondria (reaction I)1. Reaction I is stimulated by diethylstilbœstrol and œstradiol and thus differs from the transhydrogenase of Kaplan2. This may reflect a difference in the method of preparation which preserves its hormonal sensitivity3. In further studies of this reaction, the reverse direction (reaction II) has been examined. In this report, the inhibitory effect of dicumarol on reactions I and II will be described.

Demonstration of a mitochondrial energy-dependent pyridine nucleotide transhydrogenase reaction

Studies reported in the preceding communication (Danielson and Ernster, 1962) have led to the conclusion that pyridine nucleotide-linked dismutations in rat liver mitochondria require a supply of high-energy intermediates in order to proceed at maximal rate. The present paper describes direct evidence for an energy-dependent pyridine nucleotide transhydrogenase reaction, involving a reduction of TPN by DPNH, catalyzed by submitochondrial preparations from both rat liver and beef heart.

Pyridine nucleotide transhydrogenase from spinach. II. Requirement of enzyme for photochemical accumulation of reduced pyridine nucleotides

Experiments designed to elucidate the mechanism of photochemical reduction of pyridine nucleotides have yielded the following results: ( a) In the presence of excess PPNR, the rate of photochemical reduction of TPN is stimulated by the addition of transhydrogenase. ( b) Inclusion of antibody to the transhydrogenase enzyme in the reaction mixture inhibited completely the accumulation of either TPNH or DPNH in the presence of illuminated chloroplasts (or grana). This inhibition is reversed by the addition of the purified transhydrogenase enzyme, ( c) The photochemical reduction of DPN proceeds in the absence of exogenous or added TPN and requires both enzymes, ( d) The antibody to transhydrogenase is without effect on the photolytic activity of the chloroplasts (or grana). These results are explained by a proposed mechanism wherein the PPNR catalyzes the reduction of bound TPN, bound in the chloroplasts or grana, and the transhydrogenase catalyzes the transfer of hydrogen from the bound reduced TPN to free TPN. DPN is reduced in this system by substituting for TPN as the hydrogen acceptor in the transhydrogenase reaction. Consistent with this hypothesis is the earlier observation that the transhydrogenase from spinach is a TPN-specific enzyme.

Pathway of oxidation of isocitrate by mitochondria and the role of the transhydrogenase reaction

1. 1. The oxidation of isocitrate was studies in rat-heart and -liver mitochondria, depleted of their pyridine nucleotide by previous incubation, with four different acceptors: pyridine nucleotide, 2,6-dichlorophenolindophenol, cytochrome c and O 2. 2. 2. TPN + was reduced rapidly, but there was no reduction on DPN + unless TPN + was also added. 3. 3. The depleted mitochondria did not catalyse the reduction of 2,6-dichlorophenolindophenol by isocitrate unless TPN + was added. DPN + was completely ineffective. Maximum activity was obtained when both DPN + and TPN + were added. 4. 4. With cytochrome c as acceptor, only a slow reduction by isocitrate was found with either DPN + or TPN +. A rapid reduction required the addition of both pyridine nucleotides. Similar results were obtained with oxygen as acceptor, although the degree of stimulation obtained by adding TPN + to a system containing DPH + (3-fold) was much less than with cytochrome c (100-fold). 5. 5. TPNH was not oxidized by the mitochondria at an appreciable rate, unless DPN + was added. 6. 6. It is concluded that in depleted rat-heart or -liver mitochondria isocitrate is oxidized by the pathway: isocitrate → TPN + → DPN + → O 2. The enzymes catalysing these reactions are about four times as active in heart mitochondria as in liver.

Studies on the reaction mechanism of lipoyl dehydrogenase

A comparison of the rates of reaction of lipoyl dehydrogenase with dihydrolipoamide, reduced diphosphopyridine nucleotide and oxidized acetyl pyridine diphosphopyridine nucleotide have shown the involvement of the enzyme flavin in the transhydrogenase reaction catalyzed by this enzyme. The previously reported involvement of oxidized diphosphopyridine nucleotide in the catalytic oxidation of reduced diphosphopyridine nucleotide with lipoic acids as acceptors has been investigated by use of Neurospora DPNase, which by hydrolysing oxidized diphosphopyridine nucleotide has permitted a study of the effect of oxidized diphosphopyridine nucleotide on the oxidation-reduction state of the enzyme. It has been found that under certain conditions (which result in inhibition of enzyme activity) two molecules of reduced diphosphopyridine nucleotide will react to produce a fully reduced form of the enzyme, in which both the reactive disulphide and the flavin are fully reduced. Evidence is presented that the active form of the enzyme is a half-reduced form in which both flavin and the disulphide are only partially reduced. A reaction mechanism based on these findings is proposed.

On the mechanism of the transhydrogenase reaction catalyzed by a beef heart flavoprotein

On the mechanism of the transhydrogenase reaction catalyzed by a beef heart flavoprotein