Preparation and some properties of submitochondrial particles from tightly coupled mung bean mitochondria

Osmotic shock was found to be better than freezing and thawing, a French press, or sonic oscillation for the preparation of submitochondrial particles from mung bean (Phaseolus aureus) hypocotyl mitochondria. Particles prepared by osmotic shock rapidly oxidize reduced nicotinamide adenine dinucleotide and succinate, but they oxidize malate slowly. NADH oxidation was slightly stimulated by cytochrome c, ATP, and ADP; succinate oxidation was markedly increased by ATP, slightly by ADP and cytochrome c; and malate oxidation required the addition of NAD⁺ NADH oxidation is inhibited weakly by amytal, completely by antimycin A and KCN, but not by rotenone. Chlorsuccinate, malonate, antimycin A, and KCN inhibit succinate oxidation. The action of antimycin A and KCN is incomplete, while chlorsuccinate and malonate were competitive inhibitors. Antimycin A combined stoichiometrically with particle protein in the ratio of 0.23 millimicromole per milligram of protein.     

Malic enzyme activity and cyanide-insensitive electron transport in plant mitochondria.

In the presence of exogenous NAD⁺, malate oxidation by cauliflower mitochondria takes place essentially via an electron transport pathway that is insensitive to rotenone, antimycin and cyanide but is strongly sensitive to salicyl hydroxamic acid. It bypasses all phosphorylation sites. NAD⁺ is reduced by an enzyme identified as malic enzyme (L-malate:NAD oxidoreductase (decarboxylating), EC 1.1.1.39). The NADH produced is reoxidized by an internal rotenone-insensitive NADH dehydrogenase that yields electrons directly to the cyanide-insensitive pathway.     

Reduction of Nitrate via a Dicarboxylate Shuttle in a Reconstituted System of Supernatant and Mitochondria from Spinach Leaves

Substantial rates of nitrate reduction could be achieved with a reconstituted system from spinach leaves containing supernatant, mitochondria, NAD⁺, oxaloacetate (OAA), and an oxidizable substrate. Appropriate substrates were glycine, pyruvate, citrate, isocitrate, fumarate, or glutamate. The reduction of NO₃⁻ with any of the substrates could be inhibited by n-butyl malonate, showing that the transfer of reducing power from the mitochondria to the supernatant involved the malate exchange carrier. The addition of ADP to the reconstituted system decreased NO₃⁻ reduction and this decrease could be reversed by the addition of rotenone or antimycin A. The operation of the OAA/malate shuttle was achieved most quickly in the system when low concentrations (≤0.1 millimolar) of OAA were added. A corresponding increase in the lag time for the operation of the OAA/malate shuttle was observed when the OAA concentration was increased. Concentrations for half-maximal activity of OAA, glycine, NAD⁺, and NO₃⁻ in the reconstituted system were 42 micromolar, 0.5 millimolar, 0.25 millimolar, and 26 micromolar, respectively. The transfer of reducing power from the mitochondria to the soluble phase via the OAA/malate shuttle can not only provide NADH for cytoplasmic reduction but can also sustain oxidation of tricarboxylic cycle acids and the generation of α-ketoglutarate independently of the respiratory electron transport chain.     

Exogenous NAD Effects on Plant Mitochondria: A Reinvestigation of the Transhydrogenase Hypothesis

Addition of NAD⁺ to purified potato (Solanum tuberosum L.) mitochondria respiring α-ketoglutarate and malate in the presence of the electron transport inhibitor rotenone, stimulated O₂ uptake. This stimulation was prevented by incubating mitochondria with N-4-azido-2-nitrophenyl-aminobutyryl-NAD⁺ (NAP₄-NAD⁺), an inhibitor of NAD⁺ uptake, but not by 1 mm EGTA, an inhibitor of external NADH oxidation. NAD⁺-stimulated malate-cytochrome c reductase activity, and reduction of added NAD⁺ by intact mitochondria, could be duplicated by rupturing the mitochondria and adding a small quantity to the cuvette. The extent of external NAD⁺ reduction was correlated with the amount of extra mitochondrial malate dehydrogenase present. Malate oxidation by potato mitochondria depleted of endogenous NAD⁺ by storing on ice for 72 hours, was completely dependent on added NAD⁺, and the effect of NAD⁺ on these mitochondria was prevented by incubating them with NAP₄-NAD⁺. External NAD⁺ reduction by these mitochondria was not affected by NAP₄-NAD⁺. We conclude that all effects of exogenous NAD⁺ on plant mitochondrial respiration can be attributed to net uptake of the NAD⁺ into the matrix space.     

The Effect of Exogenous Nicotinamide Adenine Dinucleotide on the Oxidation of Nicotinamide Adenine Dinucleotide-linked Substrates by Isolated Plant Mitochondria

The oxidation of malate, citrate, and α-ketoglutarate by cauliflower (Brassica oleacea L.) bud mitochondria was inhibited by rotenone. This inhibition was relieved upon addition of NAD⁺ to the medium, and ADP/O values were lowered to less than 2 when both rotenone and NAD⁺ were present. Dinitrophenol did not affect the relief of rotenone inhibition by exogenous NAD⁺.     

Malate Oxidation in Plant Mitochondria via Malic Enzyme and the Cyanide-insensitive Electron Transport Pathway

Malate oxidation in plant mitochondria proceeds through the activities of two enzymes: a malate dehydrogenase and a NAD⁺-dependent malic enzyme. In cauliflower, mitochondria malate oxidation via malate dehydrogenase is rotenone- and cyanide-sensitive. Addition of exogenous NAD⁺ stimulates the oxidation of malate via malic enzyme and generates an electron flux that is both rotenone- and cyanide-insensitive. The same effects of exogenous NAD⁺ are also observed with highly cyanide-sensitive mitochondria from white potato tubers or with mitochondria from spinach leaves. Both enzymes are located in the matrix, but some experimental data also suggest that part of malate dehydrogenase activity is also present outside the matrix compartment (adsorbed cytosolic malate dehydrogenase?). It is concluded that malic enzyme and a specific pool of NAD⁺/NADH are connected to the cyanide-insensitive alternative pathway by a specific rotenone-insensitive NADH dehydrogenase located on the inner face of the inner membrane. Similarly, malate dehydrogenase and another specific pool of NAD⁺/NADH are connected to the cyanide- (and antimycin-) sensitive pathway by a rotenone-sensitive NADH dehydrogenase located on the inner face of the inner membrane. A general scheme of electron transport in plant mitochondria for the oxidation of malate and NADH can be given, assuming that different pools of ubiquinone act as a branch point between various dehydrogenases, the cyanide-sensitive cytochrome pathway and the cyanide-insensitive alternative pathway.     

Effect of phosphate and uncouplers on substrate transport and oxidation by isolated corn mitochondria

A study was made to determine conditions under which malate oxidation rates in corn (Zea mays L.) mitochondria are limited by transport processes. In the absence of added ADP, inorganic phosphate increased malate oxidation rates by processes inhibited by mersalyl and oligomycin, but phosphate did not stimulate uncoupled respiration. However, the uncoupled oxidation rates were inhibited by butylmalonate and mersalyl. When uncoupler was added prior to substrate, subsequent O₂ uptake rates were reduced when malate and succinate, but not exogenous NADH, were used. Uncoupler and butylmalonate also inhibited swelling in malate solutions and malate accumulation by these mitochondria, which were found to have a high endogenous phosphate content. Addition of uncoupler after malate or succinate produced an initial rapid oxidation which declined as the mitochondria lost solute and contracted. This decline was not affected by addition of ADP or AMP, and was not observed when exogenous NADH was substrate. Increasing K⁺ permeability with valinomycin increased the P-trifluoromethoxy (carboxylcyanide)phenyl hydrazone inhibition. Kinetic studies showed the slow rate of malate oxidation in the presence of uncoupler to be characterized by a high Km and a low V_max, probably reflecting a diffusion-limited process.     

Effects of rotenoids on isolated plant mitochondria

The effects of several rotenoids have been studied on potato (Solanum tuberosum L.) tuber and etiolated mung bean (Phaseolus aureus Roxb.) hypocotyls mitochondria. The selective inhibition of mitochondrial complex I is characterized by several tests: (a) no effect can be observed on exogenous NADH or succinate oxidation; (b) malate oxidation is inhibited at pH 7.5; (c) one-third decrease of ADP/O ratio appears during malate oxidation at pH 6.5 or during α-ketoglutarate, citrate, or pyruvate oxidation at a pH about 7; (d) during malate oxidation at pH 6.5, a transient inhibition appears which can be maintained by addition of exogenous oxaloacetate; (e) in potato mitochondria, the inhibition of malate oxidation disappears at pH 6.5 when NAD⁺ is added. Then, a one-third decrease of the ADP/O ratio can be measured.

Such a selective inhibition of complex I is obtained with deguelin, tephrosin, elliptone, OH-12 rotenone, and almost all the rotenoids extracted from Derris roots. The presence of the rings A, B, C, D, E seems to be necessary for the selective inhibition. Opening of the E ring and hydroxylation of the 9 position (rot-2′-enoic acid) give a rotenoid derivative with multisite inhibitory activities on flavoproteins, which are quite comparable to those of common flavonoids such as kaempferol (Ravanel et al. 1982 Plant Physiol 69: 375-378).

     

The effect of 4-hydroxy-2, 3-trans-pent-en-1-al and maleylaldehyde on some mitochondrial functions

Low concentrations of HPE and MLA inhibited state 3 respiration of rat liver mitochondria in the presence of different NAD⁺-dependent substrates. MLA appeared to be more active than HPE. High aldehyde concentrations inhibited the state 3 respiration with succinate. The restraint of succinate oxidation by HPE and MLA and of glutamate plus malate oxidation by MLA correlated with the inhibition of succinate and glutamate dehydrogenase activites, respectively. HPE inhibited glutamate dehydrogenase at concentrations higher than those affecting glutamate oxidation. Malate dehydrogenase activity was slightly sensitive to HPE and MLA. Both aldehydes inhibited NADH oxidation by freeze-thawed mitochondria. These results suggest the existence of a site particularly sensitive to aldehydes in the electron transport chain between the specific NAD⁺-linked dehydrogenases and ubiquinone.     

Effect of bicarbonate and oxaloacetate on malate oxidation by spinach leaf mitochondria

Mitochondria isolated from spinach leaves oxidized malate by both a NAD⁺-linked malic enzyme and malate dehydrogenase. In the presence of sodium arsenite the accumulation of oxaloacetate and pyruvate during malate oxidation was strongly dependent on the malate concentration, the pH in the reaction medium and the metabolic state condition.Bicarbonate, especially at alkaline pH, inhibited the decarboxylation of malate by the NAD⁺-linked malic enzyme in vitro and in vivo. Analysis of the reaction products showed that with 15 mM bicarbonate, spinach leaf mitochondria excreted almost exclusively oxaloacetate.The inhibition by oxaloacetate of malate oxidation by spinach leaf mitochondria was strongly dependent on malate concentration, the pH in the reaction medium and on the metabolic state condition.The data were interpreted as indicating that: (a) the concentration of oxaloacetate on both sides of the inner mitochondrial membrane governed the efflux and influx of oxaloacetate; (b) the NAD⁺/NADH ratio played an important role in regulating malate oxidation in plant mitochondria; (c) both enzymes (malate dehydrogenase and NAD⁺-linked malic enzyme) were competing at the level of the pyridine nucleotide pool, and (d) the NAD⁺-linked malic enzyme provided NADH for the reversal of the reaction catalyzed by the malate dehydrogenase.     

Stoichiometry of proton translocation coupled to substrate oxidation in plant mitochondria

The proton translocation coupled to the electron flux from succinate, exogenous NADH, and NAD⁺-linked substrates (malate and isocitrate) to cytochrome c and to oxygen was studied in purified potato (Solanum tuberosum) mitochondria using oxygen and ferricyanide pulse techniques. In the presence of valinomycin plus K⁺ (used as a charge compensating cation), optimum values of H⁺/2 e⁻ were obtained when low amounts of electron acceptors (oxygen or ferricyanide) were added to the mitochondria (1-2 nanogram [2 e⁻] equivalents per milligram protein). The stoichiometry of proton translocation to electron flux was unaffected in the presence of N-ethylmaleimide, an inhibitor of the Pi/H⁺ symport. With succinate as substrate, H⁺/2 e⁻ ratios were 4.0 ± 0.2 and 3.7 ± 0.3 with oxygen and ferricyanide as electron acceptors, respectively. With exogenous NADH, H⁺/2e⁻ ratios were 4.1 ± 0.9 and 3.4 ± 0.2, respectively. The proton translocation coupled to the oxidation of NAD⁺-linked substrates (malate, isocitrate) was dependent upon the presence of adenylates (ADP, AMP, or ATP). For malate (+ glutamate) oxidation the observed H⁺/2 e⁻ ratios were increased from 3.6 ± 2.2 to 6.5 ± 0.5 in the presence of 20 micromolar ADP.     

Impaired substrate utilization in mitochondria from strain 129 dystrophic mice

Mitochondria from skeletal muscle, heart and liver of strain 129/ReJ-dy dystrophic mice and their littermate controls were characterized with respect to their respiratory and phosphorylating activities. Skeletal muscle mitochondria from dystrophic mice showed significantly lower state 3 respiratory rates than controls with both pyruvate + malate and succinate as substrates (P < 0.01). ADP/O and Ca²⁺/O ratios were found to be normal. A decreased rate of NADH oxidation (0.01 <P < 0.05) by sonicated mitochondrial suspensions from dystrophic mice was also seen. High respiratory rates with ascorbate + phenazine methosulfate as substrates indicated that cytochrome oxidase was not rate limiting in the oxidation of either pyruvate + malate or succinate. Skeletal muscle mitochondria from dystrophic mice showed no deficiency in any of the cytochromes or coenzyme Q. Mg²⁺-stimulated ATPase activity was higher in dystrophic muscle mitochondria than in controls, but basal and oligomycin-insensitive activities were virtually identical to those of controls. A significant reduction in the intramitochondrial NAD⁺ content (0.01 <P < 0.02) was seen in dystrophic skeletal muscle as compared to controls. Heart mitochondria from dystrophic mice showed similar, though less extensive abnormalities while liver mitochondria were essentially normal. We concluded from these results that skeletal muscle mitochondria from strain 129 dystrophic mice possess impairments in substrate utilization which may result from (1) an abnormality in the transfer of electrons on the substrate side of coenzyme Q in the case of succinate oxidation; (2) a defect on the path of electron flow from NADH to cytochrome c, and (3) a deficiency of NAD⁺ in the case of NAD⁺-linked substrates.     

Effect of NAD on Malate Oxidation in Intact Plant Mitochondria

Potato tuber mitochondria oxidizing malate respond to NAD⁺ addition with increased oxidation rates, whereas mung bean hypocotyl mitochondria do not. This is traced to a low endogenous content of NAD⁺ in potato mitochondria, which prove to take up added NAD⁺. This mechanism concentrates NAD⁺ in the matrix space. Analyses for oxaloacetate and pyruvate (with pyruvate dehydrogenase blocked) are consistent with regulation of malate oxidation by the internal NAD⁺/NADH ratio.     

Mechanisms of citrate oxidation by percoll-purified mitochondria from potato tuber

The mechanisms and accurate control of citrate oxidation by Percoll-purified potato (Solanum tuberosum) tuber mitochondria were characterized in various metabolic conditions by recording time course evolution of the citric acid cycle related intermediates and O₂ consumption. Intact potato tuber mitochondria showed good rates of citrate oxidation, provided that nonlimiting amounts of NAD⁺ and thiamine pyrophosphate were present in the matrix space. Addition of ATP increased initial oxidation rates, by activation of the energy-dependent net citrate uptake, and stimulated succinate and malate formation. When the intramitochondrial NADH to NAD⁺ ratio was high, α-ketoglutarate only was excreted from the matrix space. After addition of ADP, aspartate, or oxaloacetate, which decreased the NADH to NAD⁺ ratio, flux rates through the Krebs cycle dehydrogenases were strongly increased and α-ketoglutarate, succinate, and malate accumulated up to steady-state concentrations in the reaction medium. It was concluded that NADH to NAD⁺ ratio could be the primary signal for coordination of fluxes through electron transport chain or malate dehydrogenase and NAD⁺-linked Krebs cycle dehydrogenases. In addition, these results clearly showed that the tricarboxylic acid cycle could serve as an important source of carbon skeletons for extra-mitochondrial synthetic processes, according to supply and demand of metabolites.     

Vitamin E deficiency in the rat. IV. Alteration in mitochondrial membrane and its relation to respiratory decline

The respiratory control and rate of oxidation of exogenous NADH in vitro by liver mitochondria from vitamin E deficient rats were studied as a means of providing information concerning possible mitochondrial membrane alterations due to the deficiency.When mitochondria were aged at different temperatures for various periods of time, half-maximal inhibition of respiratory control occurred at lower temperatures and shorter aging periods in deficient mitochondria than in normal ones. Also, respiratory control was lost more rapidly in deficient mitochondria than in normal ones in the presence of either digitonin or low (hypotonic) concentrations of mannitol.Microsomes, both freshly prepared and boiled, dramatically lowered respiratory control and the effect was greater in the deficient mitochondria. Bovine serum albumin overcame the suppressed respiratory control, and exogenously added fatty acids mimiced the action of the microsomes.NADH oxidation by normal mitochondria proceeded slowly in isotonic media, while mitochondria of vitamin E deficient rats oxidized NADH much more rapidly. When mitochondria were subjected to ultrasonic disruption or incubated in hypotonic media, the rates of NADH oxidation by both types of mitochondria were similar.Respiratory decline associated with oxidation of β-hydroxybutyrate by the deficient mitochondria was decreased by including in the medium either a high concentration of NAD⁺, 0.5 mm oxalacetate, or 2 mm aspartate plus 1 mm α-ketoglutarate. This observation, plus the finding of similar activities of malate dehydrogenase and glutamic-oxalacetic transminase in normal and deficient livers, suggests that the action of each was due to an elevation of the mitochondrial NAD⁺/NADH ratio via a malate shuttle and cytoplasmic and mitochondrial glutamic-oxalacetate transaminase. It is postulated that the marked mitochondrial respiratory decline in the deficient rats is attributed to a limiting availability of NAD⁺ and a low ratio of NAD⁺ to NADH.     

Oxidative phosphorylation and the electron transport system of castor bean mitochondria

The difference spectrum (reduced minus oxidized) of castor bean(Ricinus communis L.) mitochondria showed the presence of cytochromeoxidase (cytochromes a+a₃), b-type cytochromes and cytochromec. The mitochondria actively oxidized succinate, -ketoglutarate,pyruvate and exogenous NADH, and oxidations of these substrateswere stimulated by added ADP, as in mammalian mitochondria.Values for the P/O ratio obtained for succinate, pyruvate and-ketoglutarate were the same as those reported for mammalianmitochondria, indicating that theoretical values are 2, 3 and4, respectively. The theoretical P/O ratio for exogenous NADHseemed to be 2. Oxidations of succinate and exogenous NADH instate 3 were almost completely inhibited by 0.3 mM cyanide and10 µM its antimycin A, while those of NAD⁺-linked substratesin state 3 were not completely suppressed even by excess concentrationsof these inhibitors. There seem to be two types of pathway forelectron transfer in the oxidation of NAD⁺-linked substratesin castor bean mitochondria, i.e. pathways which are sensitiveand insensitive to these inhibitors. Oxidation of exogenousNADH in state 3 was not inhibited by rotenone. Transitions of redox levels of the respiratory components fromstate 4 to state 3 on addition of ADP and from state 3 to state4 on exhaustion of added ADP were observed with a dual-wavelengthspectrophotometer. Effects of inhibitors on redox levels ofthe respiratory components in state 3 were investigated. Cytochromesof b-type and cytochrome c were fully reduced on addition ofcyanide. Cytochromes of b-type were also fully reduced on additionof antimycin A, but cytochrome oxidase (cytochromes a + a₃)and cytochrome c changed to the oxidized forms. The redox levelof the component(s) with an absorption maximum at 465 mµshifted further, but not completely, to the reduced side onaddition of antimycin A. However, this component(s) was oxidizedon addition of cyanide. Cyanide-, or antimycin A-resistant oxidationof NAD⁺-linked substrates seems to occur via an alternate electrontransfer pathway branching from NAD⁺-linked flavoprotein(s)in the mitochondria, not via the normal pathway through thecytochromes-cytochrome oxidase system. (Received June 8, 1970; )     

Respiratory Properties of Mitochondria from Rice Seedlings Germinated under Water and Their Changes during Air Adaptation

Respiratory activities were compared among rice seedlings germinated in air for 6 days (aerobic seedlings), those germinated under water for 5 days (submerged seedlings), and those grown in air for 1 day after 5 days' submerged germination (air-adapted seedlings). The respiratory activity of the submerged seedlings increased rapidly on transfer to air and reached a plateau at 16 hours in air. Respiration of the submerged seedlings was as sensitive to cyanide as those of aerobic and air-adapted seedlings. 2,4-Dinitrophenol had no effect on the respiration of the submerged seedlings, but stimulated those of the other two types of seedlings. Mitochondria from three types of seedlings did not differ in the ADP/O ratio and the respiratory control ratio (RCR) when succinate was oxidized. However, mitochondria from submerged seedlings (submerged mitochondria) showed poor RCR of about unity when malate was oxidized. Both the rate of succinate oxidation and succinate dehydrogenase activity were low in submerged mitochondria, but increased during air adaptation. Although submerged mitochondria oxidized malate very slowly, this activity increased after exposure to air without any increase in malate dehydrogenase activity. When NAD⁺ was added to submerged mitochondria, oxidation of malate was restored to the level of the aerobic controls. Addition of NAD⁺ enhanced the state 3 rate in submerged mitochondria, and RCR recovered to nearly the same value as that of the aerobic controls. Similar effects of NAD⁺ on 2-oxoglutarate oxidation were observed. All these defects in submerged mitochondria were repaired during air adaptation. These results suggest that NAD⁺-linked substrate oxidation was low in submerged mitochondria because of NAD⁺ deficiency, and that the oxidation increased with an increasing level of NAD⁺ during air adaptation.     

Metabolism of N6,O2'-[3H]dibutyryl cyclic adenosine 3',5'-monophosphate and macromolecular interactions of the products perfused rat liver.

Mitochondria from liver, kidney, brain, and skeletal muscle metabolized acetaldehyde. Acetaldehyde oxidation by liver and kidney mitochondria was maximal at low levels of acetaldehyde and was sensitive to rotenone, suggesting the involvement of a NAD⁺-dependent aldehyde dehydrogenase with a high affinity for acetaldehyde. Acetaldehyde oxidation was stimulated 50% by ADP, suggesting that, in state 4, reoxidation of NADH is rate limiting for acetaldehyde oxidation. In state 4, acetaldehyde oxidation was decreased by NAD⁺-dependent substrates, as well as by succinate and ascorbate. The inhibition by the latter two substrates was prevented by ADP, dinitrophenol, valinomycin, and gramicidin, but not by oligomycin. Since these compounds are linked to energy transduction and utilization, the data suggest that the inhibition is mediated via energy-dependent reversed electron transport. In state 3, all of these substrates caused considerably less inhibition of acetaldehyde oxidation, suggesting that the activity of aldehyde dehydrogenase, and not of NADH reoxidation, is probably rate limiting for acetaldehyde oxidation. The ionophores valinomycin and gramicidin stimulated acetaldehyde oxidation to a greater extent than ADP. These ionophores also stimulated acetaldehyde oxidation in the presence of ADP. Stimulation by valinomycin occurred in the presence of monovalent cations transported by this ionophore, e.g., K⁺, Rb⁺, Cs⁺. Stimulation by gramicidin also occurred in the presence of these cations, but did not occur with Na⁺ or Li⁺. Na⁺ prevents the stimulation of acetaldehyde oxidation, which occurs in the presence of gramicidin and K⁺. The stimulation by valinomycin and gramicidin was energy dependent and required the presence of a permeant anion. In the absence of an ionophore, potassium phosphate had no effect on acetaldehyde oxidation. These data suggest that the oxidation of acetaldehyde by rat liver and kidney mitochondria is influenced by the oxidation-reduction state of the mitochondria and by the cationic environment. With brain and muscle mitochondria, the rate of acetaldehyde oxidation increased two- to threefold as the concentration of acetaldehyde was raised from 0.167 to 0.50 mm. Acetaldehyde oxidation in these mitochondria was also sensitive; to rotenone, indicating dependence on NAD⁺. ADP, valinomycin, gramicidin, and succinate, compounds which either increased or decreased the rate of acetaldehyde oxidation by liver and kidney mitochondria, had no effect on acetaldehyde oxidation by muscle or brain mitochondria. In state 4, mitochondria from Becker-transplantable hepatocellular carcinoma HC-252 oxidized acetaldehyde at the same rate as liver mitochondria. However, in the presence of ADP, dinitrophenol, valinomycin and gramicidin, the rate of acetaldehyde oxidation by the tumor mitochondria was two to three times greater than that of liver mitochondria, suggesting the presence of a more active; acetaldehyde-oxidizing system in tumor than in liver mitochondria.     

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.     

The Influence of Exogenous Nicotinamide Adenine Dinucleotide on the Oxidation of Malate by Jerusalem Artichoke Mitochondria

Mitochond$$$a isolated from Jerusalem artichoke tubers oxidizedendogenous NADH by both a piericidin A-sensitive and -resistantdehydrogenase if the level of oxaloacetate was kept low. Inwashed mitochondria the addition of NAD⁺ stimulated respirationin the presence of a variety of NAD⁺ -linked substrates. Inmitochondria purified through a sucrose density gradient exogenousNAD⁺ caused a substantial stimulation of respiration only inthe presence of malate. The apparent K_m for malate was 20 mMin the absence of NAD⁺ and 2 mM in the presence of exogenousNAD⁺ The products of malate oxidation were altered by the additionof exogenous NAD⁺. Oxaloacetate and pyruvate were produced inequal amounts in the absence of added NAD⁺, but in the presenceof exogenous NAD⁺ more pyruvate was formed. The rapid oxidationof malate in the absence of added NAD⁺ required phosphate whilethe NAD⁺-stimulated component was not affected by the absenceof phosphate. Phenylsuccinate inhibited the reduction of exogenousNAD⁺ by malate; this compound was found to inhibit isolatedma!ate dehydrogenase and mahe enzyme. Several lines of evidencesuggest that electron flux through one of the NADH dehydrogenasesystems may directly affect the flow through the other dehydrogenases.