The Accumulation of Calcium Ions by Sarcotubular Vesicles

The accumulation of Ca⁺⁺ by microsomal (sarcotubular) preparations of rabbit skeletal muscle in the presence of oxalate, and the concurrent splitting of nucleoside triphosphate, displayed moderate nucleotide specificity in the sequence ATP > GTP, CTP, ITP > UTP > (ADP) > ATetraP for the former, ATP > (ADP) > ITP > GTP > CTP > UTP > ATetraP for the latter process. The "calcium pump" was weakly inhibited by caffeine, and was inhibited together with the ATPase by pyridoxalphosphate. Carnosine had no effect as such nor in the presence of pyridoxalphosphate except at high concentration; thiourea and p-chloromercuribenzoate were inhibiting while iodoacetate was inactive. Ca⁺⁺ accumulation and ATPase were inhibited by atabrine (not tested on ATPase), dinitrophenol, and amytal. High concentrations of oligomycin and rutamycin inhibited Ca⁺⁺ uptake while slightly stimulating ATPase. Antimycin A stimulated the Ca⁺⁺ uptake. These results are discussed in the light of their possible relation to partial reactions in oxidative phosphorylation. The Ca⁺⁺ uptake and relaxing factor activities did not behave identically throughout. This is in part ascribed to changes in reactivity of actomyosin in the relaxation test, in part to the participation of relaxing substances other than the calcium pump.     

Some biochemical properties of mitochondria isolated from Euglena gracilis.

Mitochondria were isolated from Euglena gracilis strain Z by pressure-breakage of the cells and sucrose-cushion centrifugation. Multiple peaks (2-4) were observed in the rate of phosphorylation with Mg-ADP-phosphate concentration curves. The phosphorylative and oxidative activities were highest with NADH as the substrate, moderate with succinate, and lowest with glutamate. Inhibition of phosphorylation with 2,4-dinitrophenol and carbonyl cyanide, m-chlorophenylhydrazone gave sigmoidal concentration curves, with the extent of inhibition by DNP depending on the substrate used. Inhibition of phosphorylation by valinomycin, atractyloside, or carboxyatractyloside was only approximately 60%. Oligomycin inhibited phosphorylation in 2 phases at low and high concentrations; it inhibited Mg-ATPase in a sigmoidal fashion. Both phosphorylation and oxidation had discontinuities in Arrhenius plots at 34 C and 18 C. The relative Mg2+-dependent nucleoside triphosphatase activity was: 1 for ATP and GTP, 0.6 for ITP, 0.15 for CTP and UTP; with Ca2+ in place pf Mg2+ this activity was 0.35. Both DNP and CCCP stimulated the Mg-ATPase 50-200%. The optimal pH for the stimulation was approximately 7 regardless of the uncoupler used, and approximately 8 without the uncouplers. The few differences observed between mitochodria from Euglena and those from other sources are probably due to the fragmentation of the reticular mitochondrial structure during isolation and not to unique characteristics of these mitochondria.     

The relationship between energy generation and cholesterol side-chain cleavage reaction in the mitochondria from human term placenta

1. The interrelationship between progesterone (from cholesterol) biosynthesis and oxidative phosphorylation in human placental mitochondria was examined. 2. ADP and ATP stimulated the malate, succinate and alpha-ketoglutarate-supported progesterone biosynthesis probably via the energy-dependent pyridine nucleotide transhydrogenase activation. The effect of ADP was abolished by rotenone and antimycin in the presence of malate or alpha-ketoglutarate. 3. In the non-energized state of mitochondria malate may supported progesterone biosynthesis by the malic enzyme-dependent pathway. 4. The inhibitory effects of antimycin or cyanide, and the stimulatory effect of rotenone on the succinate-supported progesterone biosynthesis indicate that the succinate to malate conversion is a necessary condition for the stimulation of progesterone biosynthesis from cholesterol. 5. alpha-Ketoglutarate plus malonate did support progesterone biosynthesis also in the presence of ADP or ATP and to a lesser degree in the presence of DNP and rotenone. Arsenate in the presence of alpha-ketoglutarate, malonate, dinitrophenol and rotenone did not affect significantly progesterone biosynthesis. These results indicate that NADPH may be generated also by a non-energy-dependent transhydrogenation in placental mitochondria.     

Effect of nucleotide structure on cardiac myosin subfragment 1 transient kinetics

Transient kinetic data of the hydrolysis of several nucleotides (TTP, CTP, UTP, GTP) by cardiac myosin subfragment 1 (S1) were analyzed to obtain values for the equilibrium constant for nucleotide binding and rate constants for the S1-nucleotide isomerization and the subsequent nucleotide hydrolysis as well as the magnitudes of the relative fluorescence enhancements of the myosin that occur upon isomerization and hydrolysis. These data are compared with data from a previous study with ATP. Nucleotide binding is found to be relatively insensitive to nucleotide ring structure, being affected most by the group at position C6. Isomerization and hydrolysis are more sensitive to nucleotide structure, being inhibited by the presence of a bulky group at position C2. Kinetic parameters decrease as follows: for binding, GTP greater than UTP approximately TTP greater than ATP greater than CTP; for isomerization, ATP greater than UTP approximately TTP approximately CTP greater than GTP; for hydrolysis, ATP greater than TTP greater than CTP approximately UTP greater than GTP. Fluorescence enhancements appear to be most dependent upon the relative values of the individual rate constants.     

Crystal structures of CTP synthetase reveal ATP, UTP, and glutamine binding sites

CTP synthetase (CTPs) catalyzes the last step in CTP biosynthesis, in which ammonia generated at the glutaminase domain reacts with the ATP-phosphorylated UTP at the synthetase domain to give CTP. Glutamine hydrolysis is active in the presence of ATP and UTP and is stimulated by the addition of GTP. We report the crystal structures of Thermus thermophilus HB8 CTPs alone, CTPs with 3SO4(2-), and CTPs with glutamine. The enzyme is folded into a homotetramer with a cross-shaped structure. Based on the binding mode of sulfate anions to the synthetase site, ATP and UTP are computer modeled into CTPs with a geometry favorable for the reaction. Glutamine bound to the glutaminase domain is situated next to the triad of Glu-His-Cys as a catalyst and a water molecule. Structural information provides an insight into the conformational changes associated with the binding of ATP and UTP and the formation of the GTP binding site.     

Some Biochemical Properties of Mitochondria Isolated from Euglena gracilis*

SYNOPSIS. Mitochondria were isolated from Euglena gracilis strain Z by pressure-breakage of the cells and sucrose-cushion centrifugation. Multiple peaks (2-4) were observed in the rate of phosphorylation with Mg-ADP-phosphate concentration curves. The phosphorylative and oxidative activities were highest with NADH as the substrate, moderate with succinate, and lowest with glutamate. Inhibition of phosphorylation with 2,4-dinitrophenol and carbonyl cyanide, m-chlorophenylhydrazone gave sigmoidal concentration curves, with the extent of inhibition by DNP depending on the substrate used. Inhibition of phosphorylation by valinomycin, atractyloside, or carboxyatractyloside was only ～ 60%. Oligomycin inhibited phosphorylation in 2 phases at low and high concentrations; it inhibited Mg-ATPase in a sigmoidal fashion. Both phosphorylation and oxidation had discontinuities in Arrhenius plots at 34 C and 18 C. The relative Mg²⁺-dependent nucleoside triphosphatase activity was: 1 for ATP and GTP, 0.6 for ITP, 0.15 for CTP and and UTP; with Ca²⁺ in place of Mg²⁺ this activity was 0.35. Both DNP and CCCP stimulated the Mg-ATPase 50-200%. The optimal pH for the stimulation was ～ 7 regardless of the uncoupler used, and ～ 8 without the uncouplers. The few differences observed between mitochondria from Euglena and those from other sources are probably due to the fragmentation of the reticular mitochondrial structure during isolation and not to unique characteristics of these mitochondria.     

Na+, K+ -ATPase: causes of activation of K+ -phosphatase reaction by nucleotides

Hydrolysis of umbelliferone phosphate is inhibited by sodium ions and ATP added to the incubation medium together with Na+ activates it, CTP, ITP, UTP and GTP produce the same effect. Acetyl phosphate, p-nitrophenyl phosphate, ADP and adenylyl imidodiphosphate beta, gamma-NH-ATP, ATP nonhydrolyzable analog, have no activating effect. ATP produces a mixed inhibition of umbelliferone phosphate hydrolysis both in the presence and absence of Na+. A scheme is suggested which explains the mechanism of the nucleotide activating effect.     

Conversion of the guanine nucleotide binding sites of ras protein resulting in the reduction of base specificity

A gene coding for the novel ras protein, p21X, in which the domains of guanine binding and phosphate binding were exchanged, was constructed and expressed in Escherichia coli. The gene product, p21X, showed GTP binding activity, but no GPTase activity. In addition, p21X revealed binding activity toward ATP and CTP. In a competitive binding assay, [3H]GTP binding to p21X was inhibited in the presence of ATP, CTP and UTP, ITP as well as GDP, GTP and dGTP.     

The final step of aldosterone biosynthesis is catalyzed by an NADPH-dependent and molecular oxygen-requiring enzyme

Using sonicated mitochondria fraction prepared from bovine adrenal glomerulosa cells, aldosterone biosynthesis from 18-hydroxycorticosterone was examined as its final step, as production of [³H]-aldosterone from [³H]-corticosterone was strongly reduced by addition of non-radioactive 18-hydroxycorticosterone during the incubation. Significant conversion of 18-hydroxycorticosterone to aldosterone by the mitochondria sonicate was observed in the presence of NADPH, but not NADP⁺. This reaction was almost completely inhibited in the atmosphere of 100% carbon monoxide in the presence of either NADP⁺, or NAD⁺, and significantly reduced in the mixture of carbon monoxide and oxygen (90:10) in the presence of NADPH. Several drugs, such as SU compounds, spironolactone, amphenone B and SKF 525A which affect cytochrome P-450 blocked production of aldosterone from 18-hydroxycorticosterone. From these results, we conclude that a mixed function oxidase involving a cytochrome P-450 is engaged in the final course of aldosterone biosynthesis.     

Distinct interactions of GTP,UTP, and CTP with G(s) proteins

Early studies showed that in addition to GTP, the pyrimidine nucleotides UTP and CTP support activation of the adenylyl cyclase (AC)-stimulating G(s) protein. The aim of this study was to elucidate the mechanism by which UTP and CTP support G(s) activation. As models, we used S49 wild-type lymphoma cells, representing a physiologically relevant system in which the beta(2)-adrenoreceptor (beta(2)AR) couples to G(s), and Sf9 insect cell membranes expressing beta(2)AR-Galpha(s) fusion proteins. Fusion proteins provide a higher sensitivity for the analysis of beta(2)AR-G(s) coupling than native systems. Nucleoside 5'-triphosphates (NTPs) supported agonist-stimulated AC activity in the two systems and basal AC activity in membranes from cholera toxin-treated S49 cells in the order of efficacy GTP > or = UTP > CTP > ATP (ineffective). NTPs disrupted high affinity agonist binding in beta(2)AR-Galpha(s) in the order of efficacy GTP > UTP > CTP > ATP (ineffective). In contrast, the order of efficacy of NTPs as substrates for nucleoside diphosphokinase, catalyzing the formation of GTP from GDP and NTP was ATP > or = UTP > or = CTP > or = GTP. NTPs inhibited beta(2)AR-Galpha(s)-catalyzed [gamma-(32)P]GTP hydrolysis in the order of potency GTP > UTP > CTP. Molecular dynamics simulations revealed that UTP is accommodated more easily within the binding pocket of Galpha(s) than CTP. Collectively, our data indicate that GTP, UTP, and CTP interact differentially with G(s) proteins and that transphosphorylation of GDP to GTP is not involved in this G protein activation. In certain cell systems, intracellular UTP and CTP concentrations reach approximately 10 nmol/mg of protein and are higher than intracellular GTP concentrations, indicating that G protein activation by UTP and CTP can occur physiologically. G protein activation by UTP and CTP could be of particular importance in pathological conditions such as cholera and Lesch-Nyhan syndrome.     

Inactivation and covalent modification of CTP synthetase by thiourea dioxide.

Thiourea dioxide was used in chemical modification studies to identify functionally important amino acids in Escherichia coli CTP synthetase. Incubation at pH 8.0 in the absence of substrates led to rapid, time dependent, and irreversible inactivation of the enzyme. The second-order rate constant for inactivation was 0.18 M-1 s-1. Inactivation also occurred in the absence of oxygen and in the presence of catalase, thereby ruling out mixed-function oxidation/reduction as the mode of amino acid modification. Saturating concentrations of the substrates ATP and UTP, and the allosteric activator GTP prevented inactivation by thiourea dioxide, whereas saturating concentrations of glutamine (a substrate) did not. The concentration dependence of nucleotide protection revealed cooperative behavior with respect to individual nucleotides and with respect to various combinations of nucleotides. Mixtures of nucleotides afforded greater protection against inactivation than single nucleotides alone, and a combination of the substrates ATP and UTP provided the most protection. The Hill coefficient for nucleotide protection was approximately 2 for ATP, UTP, and GTP. In the presence of 1:1 ratios of ATP:UTP, ATP:GTP, and UTP:GTP, the Hill coefficient was approximately 4 in each case. Fluorescence and circular dichroism measurements indicated that modification by thiourea dioxide causes detectable changes in the structure of the protein. Modification with [14C]thiourea dioxide demonstrated that complete inactivation correlates with incorporation of 3 mol of [14C]thiourea dioxide per mole of CTP synthetase monomer. The specificity of thiourea dioxide for lysine residues indicates that one or more lysines are most likely involved in CTP synthetase activity. The data further indicate that nucleotide binding prevents access to these functionally important residues.     

The reoxidation of cytochrome P-450 by paraquat inhibits aldosterone biosynthesis from 18-hydroxycorticosterone

Paraquat is an artificial electron carrier that captures electrons from reduced cytochrome P-450 instead of the natural acceptors, thus decreasing the concentration of reduced mitochondrial cytochrome P-450. In the present study, paraquat inhibited the biosynthesis of aldosterone from 18-hydroxycorticosterone by mitochondria from duck adult adrenal gland, under aerobic conditions. Since paraquat did not induce any change in the absorption spectrum of highly purified cytochrome P-450 11 beta, the possibility of a displacement of steroid by the drug is ruled out. Moreover, paraquat did not affect oxidative phosphorylating chain nor did it alter by itself the chemical structure of 18-hydroxycorticosterone. In our conditions, the inhibitory role of paraquat seems restricted to a capture of electrons from reduced cytochrome P-450. Under the same conditions metopirone and spironolactone, known to bind cytochrome P-450 11 beta at the steroid binding site, also inhibited the reaction. Altogether these results show that for aldosterone synthesis from 18-hydroxycorticosterone to take place, the steroid binding site on cytochrome P-450 must be accessible to 18-hydroxycorticosterone and that the cytochrome P-450 must be the direct donor of reducing equivalents. Hence, cytochrome P-450 appears as the final linking point between 18-hydroxycorticosterone and the reducing equivalents provided by NADPH.     

The effect of adenosine triphosphate on porphyrin excretion and on glycine metabolism in Rhodopseudomonas spheroides

1. ATP, GTP, CTP and UTP at concentrations of 1mm markedly decrease the amount of coproporphyrin excreted by Rhodopseudomonas spheroides illuminated in a medium containing glycine, succinate and fumarate. 2. The effect of ATP is decreased if ethionine is also added to the medium. 3. Evidence is presented showing that ATP is taken up by the organisms from the medium. 4. ATP is shown to have a marked effect on the utilization of glycine. In the presence of ATP the incorporation of the methylene carbon atom of glycine into the fatty acid moieties of the phospholipids is greatly increased, and more of the carboxyl carbon atom is lost, probably as carbon dioxide. 5. ATP has little effect on the utilization of succinate or fumarate. 6. The possible significance of these results with regard to the control by ATP of porphyrin synthesis and excretion and glycine metabolism is discussed.     

Biochemistry of terminal deoxynucleotidyltransferase: mechanism of inhibition by adenosine 5'-triphosphate

The polymerization of deoxyribunucleoside triphosphate catalyzed by terminal deoxyribonucleotidyltransferase (TdT, EC 2.7.7.31) is severely inhibited by the addition of ribonucleoside triphosphates, ATP being the most potent inhibitor. Examination of the inhibitory effect of ATP using oligo(dA)12-18 as well as activated DNA as primers revealed that (a) ATP inhibition is not due to its addition onto a 3'-OH primer terminus ad judged by the lack of incorporation of labeled ATP, although under similar conditions incorporation of GTP can be demonstrated, (b) a consistent degree of inhibition was noted independent of primer or enzyme concentration; (c) addition of ATP to an ongoing reaction promptly reduces the rate of polymerization; (d) kinetic studies indicate a competitive (with respect to substrate deoxy triphosphate) pattern of inhibition; (e) addition of excess deoxyribotriphosphate promptly relieves the inhibition. Unlike ATP, other ribotriphosphates yield a mixed pattern of inhibition partly mediated by competitive mechanisms. GTP and CTP and to a minor extent UTP are incorporated into DNA in the presence or absence of deoxy triphosphate. Furthermore, addition of ATP also inhibits incorporation of GTP and CTP.     

Oxidative phosphorylation in right-side-out membrane vesicles from Escherichia coli.

Oxidative phosphorylation in Escherichia coli membrane vesicles with a right-side-out orientation and loaded with ADP was investigated. Substrates of the electron transport chain could energize the phosphorylation of ADP, with the order of effectiveness being D-lactate greater than reduced phenazinemethosulfate greater than succinate greater than reduced nicotinamide adenine dinucleotide. Inhibitors of D-lactate oxidation, proton conductors, and inhibitor of the Mg2+ATPase (EC 3.6.1.3) all inhibited oxidative phosphorylation when coupled to D-lactate oxidation. ATP synthesis was absent in membrane vesicles prepared from a mutant strain lacking the Mg2+ATPase. Valinomycin or nigericin partially inhibited oxidative phosphorylation in the presence of potassium. Valinomycin plus nigericin completely inhibited ATP synthesis. The effect of various agents on the respiration-dependent establishment of a transmembrane pH gradient was also examined. NaCN and carbonyl cyanide p-trifluoromethoxyphenylhydrazone inhibited the establishment of a pH gradient while dicyclohexylcarbodiimide had no effect. These results are in good agreement with a chemiosmotic model for oxidative phosphorylation.     

Relation between energy metabolism in mitochondria and conversion of 18 hydroxycorticosterone to aldosterone in adrenals

The purpose of this study was to see whether there was any link between conversion of 18 hydroxycorticosterone to aldosterone and mitochondrial energy metabolism. In vitro incubations of duck adrenal mitochondria with 18 OH B were used in this study. Results show that 18 oxidation is inhibited by compounds blocking electron transport (antimycin A, cyanide, rotenone, amytal). Inhibition induced by cyanide and antimycin A is reversed with ATP. 2,4 dinitrophenol, oligomycin and DCCD inhibit 18 oxidation but guanidine stimulate this reaction. Thus aldosterone synthesis from 18 OH B depends on energy metabolism in mitochondria. This is a very new aspect related to the last step of aldosterone synthesis.     

Cardiac sarcolemmal and sarcoplasmic reticulum membrane vesicles exhibit distinctive (Ca-Mg)-ATPase substrate specificities

The nucleoside 5'-triphosphate (NTP) substrate specificities for Ca-stimulated ATPase and ATP-dependent Ca2+ uptake activities have been examined in cardiac sarcolemma (SL) and sarcoplasmic (SR) membrane vesicles. The results indicate that SL membrane vesicles exhibit a much narrower range of NTP substrate specificities than SR membranes. In SR membrane vesicles, the Ca-stimulated Mg-dependent hydrolysis of ATP and dATP occurred at nearly equivalent rates, whereas the rates of hydrolysis of GTP, ITP, CTP, and UTP ranged from 16-33% of that for ATP. All of the above nucleotides also supported Ca2+ transport into SR vesicles; dATP was somewhat more effective than ATP while GTP, ITP, CTP, and UTP ranged from 28-30% of the activity for ATP. In the presence of oxalate, the initial rate of Ca accumulation with dATP was 4-fold higher than for ATP, whereas the activity for GTP, ITP, CTP, and UTP ranged from 35-45% of that for ATP. For the SL membranes, Ca-activated dATP hydrolysis occurred at 60% of the rate for ATP; GTP, ITP, CTP, and UTP were hydrolyzed by the SL preparations at only 7-9% of the rate for ATP. NTP-dependent Ca2+ uptake in SL membranes was supported only by ATP and dATP, with dATP 60% as effective as ATP. GTP, ITP, CTP, and UTP did not support the transport of Ca2+ by SL vesicles. The results indicate that the SL and SR membranes contain distinctly different ATP-dependent Ca2+ transport systems.     

Respiration-department uncoupler-stimulated ATPase activity in castor bean endosperm mitochondria and submitochondrial particles.

1. The uncoupler-stimulated ATPase activity of castor bean endosperm mitochondria and submitchondrial particles has been studied. The rate of ATP hydrolysis catalyzed by intact mitochondria was slow and little enhanced by addition of uncouplers at the concentration required for uncoupling the oxidative phosphorylation. ATP-ase activity was stimulated at higher concentrations of uncouplers. 2. 1-Anilinonaphthalene 8-sulfonate fluorescence was decreased when the mitochondria were oxidizing succinate. Carbonylcyanide-p-trifluoromethoxyphenylhydrazone and antimycin reversed the succinate-induced fluorescence diminution. ATP did not induce the fluorescence response. 3. The addition of succinate, NADH or ascorbate/N,N,N'-N'-tetramethyl-p-phenylenediamine as electron donor induced high ATPase activity in the presence of low concentrations of uncouplers. Stimulating effect of uncouplers was completely abolished by further addition of antimycin. 4. Submitochondrial particles were prepared by sonication. The particles catalyzed a rapid hydrolysis of ATP and carbonylcyanide-p-trifluoromethoxyphenylhydrazone at 10-8 M did not stimulate the ATPase activity. Addition of succinate induced uncoupler-stimulated ATPase activity. The effect of succinate was completely abolished by further addition of antimycin. 5. The treatment of submitochondrial particles by trypsin or high pH also induced uncoupler-stimulated ATPase activity. 6. The above results were interpreted to indicate that ATPase inhibitor regulated the back-flow reaction of mitochondrial oxidative phosphorylation.     

Mitochondria from human term placenta. II. Characterization of respiratory pathways and coupling mechanisms.

Pathways of electron transport utilized for respiration in human term placental mitochondrial preparations were differentiated and characterized through the use of classical respiratory chain inhibitors and multiple sources of reducing equivalents. Mechanisms of associated energy conservation and utilization were examined in the preparations with uncouplers and inhibitors of phosphorylation. Inhibition by rotenone, antimycin A and cyanide established the classical electron transport chain as the major pathway of respiration with glutamate and succinate as substrates. Approximately 20% of glutamate-supported respiration was insensitive to inhibitors and may proceed by the cytochrome P-450 linked pathway of electron transport. Approximately 50% of ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine supported respiration was insensitive to 10-3 M cycanide and must utilize an undefined by-pass of cytochrome oxidase. A rotenone- and antimycin-insensitive, exterior pathway for NADH oxidation was demonstrated which could be artificially linked by exogenous cytochrome c to the cytochrome oxidase region of the classical electron transport system. Glycerol 3-phosphate also supported oxidative phosphorylation yielding ADP/O ratios of 2. Respiration of placental mitochondria was stimulated by 2,4-dinitrophenol and gramicidin. With succinate, dinitrophenol-stimulated respiration exceeded that obtained in the presence of ADP. Oligomycin and atractyloside prevented the stimulation of respiration by ADP. Thus, respiration appeared coupled through normal mechanisms to ATP formation and ion transport. A preferential coupling of respiration to the energy-utilizing processes of steroid hormone biosynthesis may exist.     

Electrogenic function of submitochondrial particles at the water-octane interphases]

Studies on submitochondrial particles (SMP) preparation showed that in the sourse of the redox reactions at the octane-water interface, catalyzed by SMP enzymes, the charges are transferred from the aqueous to the octane phase. The effects were detected by a shift of the Volta potential, using the vibrating electrode method. In the presence of 2-N-methyl-amino-1,4-naphthoquinone in octane, acting as electron acceptor, the negative charges were transferred from water to octane following the oxidation of NADH, succinate and ascorbate. The charging of the octane phase was sensitive to the inhibitors of the respiratory chain, e. g. rotenone, antimycin and cyanide. In the presence of 2,4-DNP in octane, acting as a proton acceptor, the oxidation of NADH and succinate by ferricyanide, catalyzed by CMP in the presence of antimycin and cyanide correspondingly, was followed by a transfer of positive charges from water to octane. The positive charging of the octane phase, coupled with NADH oxidation, was found insensitive to rotenone, and that coupled with succinate oxidation, was completely inhibited by antimycin. The positive charging of the octane phase was also observed during the reverse transhydrogenase reaction, catalyzed by SMP at the division of the phases. The effect was inhibited by palmitoyl-CoA.