External NADH Research Articles

Substrate uptake mechanisms for yeast cells

This paper reports on experiments in which a fluorometer, which permits culture fluorescence to be measured in situ was coupled to a bioreactor. The fluorometer through external light excitement NADH can be made to fluoresce, thus allowing intracellular NADH profiles to be measured in response to extracellular step-changes in substrate concentration. Experiments with baker's yeast and Candida tropicalis as organisms and glucose and hexadecane as substrates are reported. The results suggest different uptake mechanisms depending on cell type and on substrate.

Effect of temperature on the pathways of NADH-oxidation in broad-bean mitochondria

1. Respiration rates of broad-bean (Vicia faba) mitochondria were studied as a function of temperature. Arrhenius plots of all membrane-bound enzymes, as obtained with saturating substrate concentrations, revealed a break in the lower temperature range. That break was considered to indicate a phase transition of membrane phospholipids, characteristic for chilling-sensitive plants. A second discontinuity at 30°C occurred only with activities linked to energy conservation. - 2. The activation energies for the oxidation of NAD(+)-linked substrates differ between states 3 and 4. State 3 respiration of NAD(+)-linked substrates is the result a superimposition of two branches of electron transport, which can be separated by different sensibilities to rotenone. A characteristic temperature dependency of the respiratory control, as well as a shift of the low temperature break in the Arrhenius plot toward a higher temperature after state 4 to state 3 transition, are calculated to be caused by the superimposition of the two branches. - 3. The temperature dependency of the oxidation of extra-mitochondrial NADH and of succinate differs remarkably from that of the oxidation of matrix-NADH. It has been concluded that the rotenone-resistant oxidation of matrix-NADH and the oxidation of external NADH are mediated via different pathways with individual regulation sites.

Mitochondrial Transport Processes and Oxidation of NADH by Hypotonically-Treated Boar Spermatozoa

1. After hypotonic treatment spermatozoa have metabolic characteristics of mitochondria isolated from other cells. Ejaculated boar spermatozoa treated in this way can oxidise external NADH via both a lactate-pyruvate shuttle and a malate-aspartate cycle; this oxidation is coupled to the phosphorylation of ADP. 2. The dicarboxylate transport inhibitors butylmalonate, phenylsuccinate and bathophenanthroline sulphonate inhibit NADH oxidation dependent on added malate, glutamate and aspartate. alpha-Cyanocinnamate, a strong inhibitor of pyruvate transport, inhibits lactate-dependent NADH oxidation. 3. NADH oxidation dependent on malate, glutamate and aspartate is inhibited by uncoupling agents, but lactate-dependent NADH oxidation is stimulated. 4. Lactate-dependent NADH oxidation is inhibited by oxamate, an inhibitor of lactate dehydrogenase. Aminooxyacetate, an aminotransferase inhibitor, inhibits glutamate, malate and aspartate-dependent NADH oxidation. 5. Hypotonically-treated spermatozoa retain radioactivity after incubation with L-[U-14C]malate, [1,5-14C]citrate or [2-14C]malonate. Exchanges of retained radioactivity with various substrates indicate that dicarboxylate and tricarboxylate exchange carriers exist in the mitochondrial membrane.

Nachweis von extramitochondrialer Sauerstoffaufnahme in Epidermis-Zellen von Allium cepa L.

By treatment of Allium cepa inner epidermal cells with a combination of amytal and cyanide oxygen uptake is not suppressed. This inhibitor combination, which blocks the respiratory chain completely, only diminishes the respiration in resting onions about 25 %, in sprouting onions respiration is even enhanced. Contrary to the normal respiration, oxygen uptake is stimulated by pyruvate and even ADP after adding amytal and cyanide. The oxidation of external NADH is inhibited by the inhibitor combination, but catalytic amounts of pyruvate make the oxidation possible. The inhibitor combination also induces a synthesis of lipids. The amytal-cyanide insensitive oxygen uptake is decreased in dormant onions, but increased in sprouting ones by additional catalase inhibition. This oxygen uptake is sensitive to inhibitors interacting with SH-groups. The results indicate that this oxygen consumption is based on enzyme systems located in microbodies and microsomes. The action of the inhibitor combination and of the additional catalase inhibition, which obviously depends on age, indicates also a shift to these extramitochondrial oxygen consuming pathways and also points to an activity increase of the microsomal fraction during the storage period.

The interaction of citrate and calcium in regulating the oxidation of exogenous NADH in plant mitochondria

Plant mitochondria oxidize exogenous NADH via an external NADH dehydrogenase; little is known about the regulation of this dehydrogenase. Data presented here show that citrate caused a non-competitive inhibition of NADH oxidation. This inhibition can be reversed by Ca 2+. It is postulated that citrate causes the inhibition by extracting Ca 2+ from the NADH-ubiquinone reductase. Citrate caused a more powerful effect if added before rather than after the NADH. This suggests that the Ca 2+ is less easily removed in the presence of NADH due to a conformational change in the membrane. Experimentals using a series of electron acceptors suggest that citrate prevents electron transfer between the flavoprotein and the ubiquinone. It is suggested that calcium facilitates electron flux between these components by binding the flavoprotein to the membrane.

Pyridine nucleotide interactions with isolated plant mitochondria

1. (1) Intact cauliflower mitochondria reduced added NAD, upon addition of malate, in the presence of antimycin A. The rate of NAD reduction was initially rapid but was greatly reduced within one or two minutes; over the same time period, disrupted mitochondria reduced NAD at a linear rate. Low levels of NADH in the reaction medium dramatically reduced the rate and extent of NAD reduction by intact mitochondria. Exogenous NADP was not reduced. 2. (2) External NAD reduction in the presence of malate was dependent on added P i and glutamate but was not affected by effectors of malic enzyme, again showing a need for malate to enter the mitochondria. 3. (3) Addition of isocitrate to intact mitochondria did not induce NAD reduction, although external NAD relieved rotenone inhibition of citrate oxidation. Disrupted mitochondria reduced NAD in the presence of isocitrate. 4. (4) Isolated beetroot mitochondria did not oxidize added NADH, but reduced external NAD in the presence of antimycin A and malate. External NAD relieved rotenone inhibition of FeCN reduction with malate as substrate. 5. (5) The results show that malate oxidation occurs within the mitochondrial matrix but that the reducing power appears as external NADH and are interpreted to provide support for the proposal of a transmembrane hydrogen transfer across the inner membrane of plant mitochondria.

Characteristics of rotenone-insensitive oxidation of matrix-NADH by broad bean mitochondria

Mitochondria from Vicia faba L. exhibit two pathways for the oxidation of endogenous NADH. One pathway is rotenone-sensitive and includes three phosphorylation sites, the other one is rotenone-insensitive and by-passes site I. This by-pass is located in the area of flavoproteins, but several lines of evidence suggest that the rotenone-insensitive oxidation of matrix-NADH is not mediated by the external NADH dehydrogenase. With saturating substrate concentrations the two pathways are superimposed in state 3 while in state 4 the electrons are transferred only via the by-pass. The rotenone-resistant electron flux is obviously regulated from the substrate side. We suggest that the function of the by-pass is to regulate the NADH/NAD(+) ratio of the matrix space.

Iron-sulphur centres in mitochondria from Arum maculatum spadix with very high rates of cyanide-resistant respiration.

X-band electron-paramagnetic-resonance spectroscopy at 4.2--77K combined with measurements of oxidation-reduction potential was used to identify iron--sulphur centres in Arum maculatum (cuckoo-pint) mitochondria. In the oxidized state a signal with a derivative maximum at g = 2.02 was assigned to succinate dehydrogenase centre S-3. Unreduced particles showed additional signals at g = 2.04 and 1.98 (at 9.2 GHz), which may be due to a spin-spin interaction. In the reduced state a prominent signal at g = 1.93 and 2.02 was resolved into at least three components that could be assigned to centres S-1 and S-2 of succinate dehydrogenase (midpoint potentials -7 and -240 mV respectively at pH 7.2) and a small amount of centre N-1b (e'o= -240 mV) of NADH-ubiquinone reductase. In addition, changes in line shape around -10 mV indicated the presence of a fourth component in this signal. The latter was more readily reduced by NADH than by succinate, suggesting that it might be associated with the external NADH dehydrogenase. The iron-sulphur centres of NADH-ubiquinone reductase were present in an unusually low concentration, indicating that the alternative, non-phosphorylating, NADH dehydrogenase containing a low number of iron-sulphur centres may be responsible for most of the high rate of oxidation of NADH.

Changes in the effects of Mg++-ions on mitochondrial respiration after wounding of potato tubers

Summary Mg ++ ions have two effects on mitochondrial NADH-consuming respiration: the first is a stimulation of state III respiration. This effect decreases after wounding and subsequent incubation of the tissue. The site of action of Mg ++ is at the external NADH dehydrogenase; the decrease in stimulation after wounding is probably not due to an increase in the Mg ++ concentration in the intermembrane space. The second effect of Mg ++ on NADH-consuming respiration is a reduction of the R.C. ratio to about 1, this effect being found only with mitochondria from wounded tissue. The same result is found with respect to succinate-consuming respiration when NAD is added to the reaction mixture. This effect of Mg ++ is not due to an uncoupling of respiration or a stimulation of ATP-ase activity. Following the addition of Mg ++ (and NAD), a net production of ADP and ATP was found to have taken place in state II prior to the addition of external ADP. This is the consequence of the formation of AMP in the preparation, whereby the presence of Mg ++ is necessary for the stimulation of the activity of adenylate kinase, which converts the AMP into ADP. The origin of the formation of AMP is located outside the true mitochondria, as the compound is not found following the purification of the mitochondria on a sucrose gradient. NAD-breakdown cannot account for the AMP formed. It is possible that AMP is produced as a result of the degradation of particulate, (poly)ribosomal RNA present in the preparations after wounding of the tissue.

Some properties of external NADH oxidation by human placental mitochondria

Isolated human term placenta mitochondria catalyse oxidation of external NADH in the presence of cytochrome c. This reaction is insensitive to the respiratory chain inhibitors such as rotenone and antimycin A, and is not coupled to phosphorylation. Comparison of the effect of Mg++ ion on NADH plus cytochrome c oxidation by human term placental, human skeletal muscle and rat skeletal mitochondria showed that Mg++ ion exerts an inhibitory effect in the case of human mitochondria and a stimulatory effect in the case of rat skeletal muscle mitochondria.

Characteristics of External NADH Oxidation by Beetroot Mitochondria

Mitochondria isolated from fresh red beetroot (Beta vulgaris L.) tissue do not oxidize external NADH with O(2) as the electron acceptor. These mitochondria have a rotenone- and antimycin-insensitive pathway of NADH oxidation associated with the outer membrane and are capable of reducing cytochrome c or potassium ferricyanide. They are also capable of oxidizing internal NADH via the inner membrane electron transport chain with normal rotenone and antimycin sensitivity and ADP/O ratios. They differ from other plant mitochondria in the apparent lack of the NADH dehydrogenase located on the outer surface of the inner membrane. It is shown that this activity develops during the aging of red beetroot slices in aerated dilute CaSO(4) solutions, and is present in the mitochondria isolated from aged tissue.Mitochondria isolated from fresh red beetroot tissue are capable of oxidizing external NADH via a malate-oxaloacetate shuttle system. It is suggested that these mitochondria possess a rapid oxaloacetate-transporting system.

Ziram, a sulfhydryl reagent and specific inhibitor of yeast mitochondrial dehydrogenases

The fungicide zinc dimethyldithiocarbamate (ziram) is a sulfhydryl reagent which inhibits specifically the growth of the yeast Saccharomyces cerevisiae on nonfermentable substrates. In isolated mitochondria, the uncoupled as well as the state 3 oxidations of succinate, α-ketoglutarate, ethanol, and malate plus pyruvate are sensitive to ziram concentrations of 10 to 30 μ m. The oxidations of isocitrate, of external NADH, of α-glycerophosphate, and of ascorbate plus tetramethylphenylenediamine exhibit a lower sensitivity to ziram. Succinate, α-ketoglutarate, and pyruvate dehydrogenases activities are 50% inhibited by concentration of ziram lower than 10 μ m. At the same concentrations, neither the mitochondrial transports of succinate, ADP, or phosphate nor oxidative phosphorylation and adenosine triphosphatase activities are modified. The kinetic study of the inhibition by ziram of succinate dehydrogenase activity shows that ziram is noncompetitive with succinate and produces sigmoidal inhibitions of state 3 and of uncoupled oxidation of succinate by intact mitochondria. Inhibition of succinate:phenazine methosulfate oxidoreductase activity yields exponential kinetics. However sigmoidal-type inhibition is observed when succinate dehydrogenase activity is stimulated by ATP.

Ruthenium red as a carrier of electrons between external NADH and cytochrome C in rat liver mitochondria

Exogenous NADH is oxidized by rat liver mitochondria when ruthenium red (2μM) is present in the medium. The oxidation is insensitive to rotenone and antimycin A, but sensitive to KCN, and is markedly reduced in a saline medium or in the presence of divalent cations. The same effect of ruthenium red can be seen in mitochondria depleted of the outer membrane. This suggests that ruthenium red acts as a mediator of electrons between NADH and cytochrome c. This is also supported by the observation that ruthenium red can replace N,N,N 1,N 1-tetramethyl-p-phenylenediamine (TMPD) in the oxidation of ascorbate by mitochondria. The effect of ruthenium red is abolished by LaCl 3.

Isolation and properties of the outer membrane of plant mitochondria

The outer membrane of turnip ( Brassica rapa L.) mitochondria was isolated by incubating the mitochondria with a dilute digitonin solution and differential centrifuging. The outer membrane fraction was not contaminated by inner membrane enzymes and lacked an NADPH-cytochrome c reductase. However it possessed very active NADH-cytochrome c, dichloroindophenol and ferricyanide reductases which were insensitive to antimycin A, Amytal and low (less than 10 μ m) concentrations of Dicumarol. p-Chloromercuribenzoate (ClHgBzO −) and high concentrations (greater than 10 μ m) of Dicumarol inhibited the reductases, ClHgBzO − almost completely. Preincubation of the outer membrane with NADH protected it from ClHgBzO − inhibition. An acid phosphatase and an NADPH-ferricyanide reductase were also detected, but the latter was only loosely bound to the membrane. The NADH dehydrogenase of the outer membrane was insensitive to ethylene glycol-bis(β-aminoethyl ether) N,N′-tetraacetate (1 m m) and was not stimulated by CaCl 2 (0.5 m m), thus differing from the external NADH oxidase of the inner membrane (Coleman, J. O. D., and Palmer, J. M. (1971) FEBS Lett., 17, 203–208). Respiratory-linked oxidation of exogenous NADH by intact mitochondria showed a similar pattern of inhibition by ClHgBzO − as did the outer membrane, but was inhibited strongly by low concentrations of Dicumarol (5 μ m inhibited by 70%).

Flexibility of coupling and stoichiometry of ATP formation in intact chloroplasts

Since coupling between phosphorylation and electron transport cannot be measured directly in intact chloroplasts capable of high rates of photosynthesis, attempts were made to determine ATP 2 e ratios from the quantum requirements of glycerate and phosphoglycerate reduction and from the extent of oxidation of added NADH via the malate shuttle during reduction of phosphoglycerate in light. These different approaches gave similar results. The quantum requirement of glycerate reduction, which needs 2 molecules of ATP per molecule of NADPH oxidized was found to be pH-dependent. 9–11 quanta were required at pH 7.6, and only about 6 at pH 7.0. The quantum requirement of phosphoglycerate reduction, which consumes ATP and NADPH in a 1 1 ratio, was about 4 both at pH 7.6 and at 7.0. ATP 2 e ratios calculated from the quantum requirements and the extent of phosphoglycerate accumulation during glycerate reduction were usually between 1.2 and 1.4, occasionally higher, but they never approached 2. Although the chloroplast envelope is impermeable to pyridine nucleotides, illuminated chloroplasts reduced added NAD via the malate shuttle in the absence of electron acceptors and also during the reduction of glycerate or CO 2. When phosphoglycerate was added as the substrate, reduction of pyridine-nucleotides was replaced by oxidation and hydrogen was shuttled into the chloroplasts to be used for phosphoglycerate reduction even under light which was rate-limiting for reduction. This indicated formation of more ATP than NADPH by the electron transport chain. From the rates of oxidation of external NADH and of phosphoglycerate reduction at very low light intensities ATP 2 e ratios were calculated to be between 1.1 and 1.4. Fully coupled chloroplasts reduced oxaloacetate in the light at rates reaching 80 and in some instances 130 μmoles · mg −1 chlorophyll · h −1 even though ATP is not consumed in this reaction. The energy transfer inhibitor phlorizin did not significantly suppress this reduction at concentrations which completely inhibited photosynthesis. Uncouplers stimulated oxaloacetate reduction by factors ranging from 1.5 to more than 10. Chloroplasts showing little uncoupler-induced stimulation of oxaloacetate reduction were highly active in photoreducing CO 2. Measurements of light intensity dependence of quantum requirements for oxaloacetate reduction gave no indication for the existence of uncoupled or basal electron flow in intact chloroplasts. Rather reduction is brought about by loosely coupled electron transport. It is concluded that coupling of phosphorylation to electron transport in intact chloroplasts is flexible, not tight. Calculated ATP 2 e ratios were obtained under conditions, where coupling should be expected to be optimal, i.e. at low phosphorylation potentials [ ATP] [ ADP] [ P i ] . Flexible coupling implies, that ATP 2 e ratios should decrease with increasing phosphorylation potentials inside the chloroplasts.

Stimulation of transport into Escherichia coli membrane vesicles by internally generated reduced nictotinamide adenine dinucleotide.

Membrane vesicles containing internal alcohol dehydrogenase and nicotinamide adenine dinucleotide (NAD) were prepared from Escherichia coli ML308-225. Upon the addition of ethanol, these vesicles respired and transported several amino acids. Transport rates driven by internally generated reduced NAD (NADH) were about 60 to 80% of that stimulated by d-lactate. This transport was inhibited by cyanide and anaerobiosis. Ferricyanide, a nonpermeable electron acceptor, inhibited transport stimulated by external NADH, but not that stimulated by internally generated NADH.

The oxidation of external NADH by adult and plerocercoid Diphyllobothrium latum.

1. The apparent Kmm's and relative rates of mitochondria NADH dehydrogenase and specific activity of NADH-CoQ6 oxidoreductase of adult and plerocercoid Diphyllobothrium latum were determined. 2. As electron acceptors for NADH dehydrogenase K-ferricyanide, 2,6-dichlorphenolindophenol and cytochrome c were used. 3. The Kmmferrocytochrome cferrocytochrome c of the adult enzyme is the same as that observed for the plerocercoid, where as the KmmNADHNADH of the adult enzymes is only one-twenty fifth of the plerocercoid enzymes. 4. The activity of the adult NADH-CoQ6 oxidoreductase was over twenty times greater than that of the pleocercoid enzymes.

The external NADH dehydrogenases of intact plant mitochondria

Evidence is given for the presence of two NADH dehydrogenase systems accessible to exogenous NADH in mitochondria from mung bean hypocotyls. One system, which is located on the outer membrane, contains a flavoprotein and cytochrome b 555, is insensitive to antimycin A and is specific for the 4α hydrogen atom of NADH. The second NADH dehydrogenase, which is located on the external face of the inner membrane, is linked to the respiratory chain and is specific for the 4β hydrogen atom of NADH. The intact mitochondria have a large antimycin A-insensitive NADH: ferricyanide oxidoreductase activity which is primarily due to the outer membrane system. The inner membrane dehydrogenase reacts with ferricyanide only after the mitochondria have been osmotically swollen. Such swelling seems to cause detachment of this flavoprotein from the inner membrane.

Pathways of hydrogen in mitochondria of Saccharomyces carlsbergensis.

With mitochondria from Saccharomyces carlsbergensis the functional relationship of the exogenous and endogenous NAD system has been investigated with respect to the membrane barrier, the localisation of NADH dehydrogenase and other NAD-and flavine-linked dehydrogenases. 1 Added NAD and NADH do not permeate through the inner membrane into the matrix space of mitochondria from S. carlsbergensis despite the high rate of oxidation. There is no exchange between exogenous and endogenous NADH and NAD. 2 A transhydrogenation between external and internal NADH and NAD across the inner membrane is not observed. 3 By applying the “ferricyanide method” two separate dehydrogenases can be identified: one for the oxidation of exogenous NADH, located towards the outer surface of the inner mitochondrial membrane; and a second for the oxidation of endogenous NADH directed towards the inner surface. Both dehydrogenases are connected to the cytochrome chain through the ubiquinone pool. 4 About 50% of the total NADH dehydrogenase activity of yeast mitochondria is solubilized by sonication. The solubilized NADH dehydrogenase is tentatively identified with the external enzyme. This agrees with the finding that on opening the membrane by sonication the total activity of NADH-ferricyanide reduction increases by 65%. 5 Applying both [4B-3H]NADH and [4A-3H]NADH the oxidation of both exogenous and endogenous NADH by ferricyanide and by oxygen are found to be B-specific. 6 By application of the “ferricyanide method” to other substrates it is concluded that the dehydrogenases for glycerolphosphate and possibly lactate are localized before the barrier for ferricyanide while the dehydrogenases for ethanol, isocitrate and succinate are localized behind it. This barrier is identical with the inner mitochondrial membrane. 7 The existence of a mitochondrial alcohol dehydrogenase, accounting for about 6% of the total cellular activity and located in the matrix space, is established. The dual localization of alcohol dehydrogenase is considered to facilitate the equilibration between the intra- and extra-mitochondrial NAD systems via an ethanol-acetaldehyde shuttle.

Glycerolphosphate oxidoreductases and the glycerophosphate cycle in ehrlich ascites tumor cells

Different strains of the Ehrlich ascites carcinoma were found to differ considerably in glycerophosphate dehydrogenase ( l-glycerol-3 phosphate:NAD oxidoreductase, EC 1.1.1.8) activity. Generally the activity of this enzyme amounted to 42–71 μmoles/hr/mg protein, but it was also found to approach zero. In the latter case the α-glycerophosphate cycle (GP cycle) would not be able to operate with sufficient capacity, while in the other strains the existence of low GP cycle activities was observed. Addition of NADH to the incubation medium, and elevation of the cellular triose phosphate level by the addition of glucose + iodoacetate stimulated the GP cycle. However, the increase in oxygen consumption induced by glucose and iodoacetate in the absence of external NADH could not be accounted for by an increased metabolization of glycolytically generated NADH via the GP cycle. Activities of glycerophosphate oxidase ( l-glycerol-3 phosphate:cytochrome c oxidoreductase EC 1.1.2.1) were in the range 0.27–0.55 μmoles/hr/mg mitochondrial protein. Administration of triiodothyronine to the tumor mice resulted in a significant elevation of the glycerophosphate oxidase activity of the tumor mitochondria. Concomitant with this elevation, a slight stimulation of NADH metabolization via the glycerophosphate cycle was observed.