Competition for energy between phosphoenolpyruvate and citrulline synthesis in guinea-pig liver mitochondria

1. The interrelationship between citrulline synthesis and phosphoenolpyruvate formation has been studied in guinea-pig liver mitochondria incubated with glutamate in State 3 and in the presence of uncoupler and oligomycin.

2. In coupled mitochondria the rate of phosphoenolpyruvate production was limited by a higher capacity of aspartate aminotransferase than that of phosphopyruvate carboxylase for the intramitochondrial oxalacetate. Citrulline formation was low due to the small production of NH₃ since glutamate oxidation in State 3 proceeds via the transamination pathway.

3. Inhibition of aspartate aminotransferase by aminooxyacetate in State 3 resulted in increases in both phosphoenolpyruvate and citrulline synthesis. Under uncoupled conditions, however, an increase of phosphoenolpyruvate formation was accompanied by a decrease of both citrulline production and the ATP content of the incubation medium. Restoration of the citrulline production was observed on the addition of exogenous ATP.

4. The results indicate that when energy is generated via substrate-level phosphorylation, the inhibition of citrulline production is probably due to a higher availability of GTP to the phosphopyruvate carboxylase than to the nucleoside diphosphate kinase.     

The regulation of phosphoenolpyruvate synthesis in pigeon liver

1. The intracellular location and maximal activities of enzymes involved in phosphoenolpyruvate synthesis have been investigated in pigeon liver. Enolase and pyruvate kinase were cytoplasmic, and the activities were 50–60 and 180–210μmoles/min./g. dry wt. at 25° respectively. Phosphoenolpyruvate carboxykinase was present exclusively, and nucleoside diphosphokinase predominantly, in the mitochondria; the particles had to be disrupted to elicit maximal activities, which were 27–33 and 400–600μmoles/min./g. dry wt. at 25° respectively. The activities of all four enzymes did not change significantly during 48hr. of starvation. 2. Conditions for incubation of washed isolated mitochondria were established, to give high rates of synthesis of phosphoenolpyruvate, linear with time and proportional to mitochondrial concentration. Inorganic phosphate and added adenine nucleotides were stimulatory, whereas added Mg²⁺ inhibited, partly owing to activation of contaminant pyruvate kinase. Phosphoenolpyruvate formation occurred from oxaloacetate, malate, fumarate, succinate, α-oxoglutarate and citrate, in decreasing order of effectiveness. 3. The steady-state ATP/ADP ratio of mitochondrial suspensions was decreased in the presence of added 2·5mm-Mg²⁺ (owing to stimulation of adenylate kinase and possibly of an adenosine triphosphatase), 0·5mm-Ca²⁺ or 0·4mm-dinitrophenol. In each case the rate of substrate removal and oxygen uptake was increased, whereas phosphoenolpyruvate synthesis was inhibited. Citrate formation was enhanced, owing to de-inhibition of citrate synthase. These effects were not primarily related to changes in the oxaloacetate concentration. 4. Both phosphoenolpyruvate carboxykinase and nucleoside diphosphokinase were active within the atractylosidesensitive barrier to the mitochondrial metabolism of added adenine nucleotides. There was no correlation between the rate of substrate-level phosphorylation associated with the oxidation of α-oxoglutarate, and the synthesis of phosphoenolpyruvate. 5. The results suggest that phosphoenolpyruvate formation in pigeon-liver mitochondria is regulated partly by the phosphorylation state of the adenine and guanine nucleotides, and partly by variations in the oxaloacetate concentration, all in the mitochondrial matrix. 6. Phosphoenolpyruvate is assumed to be the metabolite transported from the mitochondria to the cytoplasm during gluconeogenesis from oxaloacetate in pigeon liver.     

Phenethylbiguanide and the inhibition of hepatic gluconeogenesis in the guinea pig

1. Phenethylbiguanide inhibits the synthesis of phosphoenolpyruvate from malate or 2-oxoglutarate by isolated guinea-pig liver mitochondria. This inhibition is time- and concentration-dependent, with the maximum decrease in the rate of phosphoenolpyruvate synthesis (80%) evident after 10min of incubation with 1mm-phenethylbiguanide. 2. The phosphorylation of ADP by these mitochondria is also inhibited at increasing concentrations of phenethylbiguanide and there is a progressive increase in AMP formation. Guinea-pig liver mitochondria are more sensitive to this inhibition in oxidative phosphorylation caused by phenethylbiguanide than are rat liver mitochondria. 3. Simultaneous measurements of O(2) consumption and ADP phosphorylation with guinea-pig liver mitochondria oxidizing malate plus glutamate in State 3 indicated that phenethylbiguanide at low concentrations (0.1mm) inhibits respiration at Site 1. At higher phenethylbiguanide concentrations Site 2 is also inhibited. 4. Gluconeogenesis from lactate, pyruvate, alanine and glycerol by isolated perfused guinea-pig liver is inhibited to various degrees by phenethylbiguanide. Alanine is the most sensitive to inhibition (60% inhibition of the maximum rate by 0.1mm-phenethylbiguanide), whereas glycerol is relatively insensitive (25% inhibition at 4mm). 5. Gluconeogenesis from lactate and pyruvate by perfused rat liver was also inhibited by phenethylbiguanide, but only at high concentrations (8mm). Unlike guinea-pig liver, the inhibitory effect of phenethylbiguanide on rat liver was reversible after the termination of phenethylbiguanide infusion. 6. The time-course of inhibition of gluconeogenesis from the various substrates used in this study indicated a time-dependency which was related in part to the concentration of infused phenethylbiguanidine. This time-course closely paralleled that noted for the inhibition by phenethylbiguanide of phosphoenolpyruvate synthesis in isolated guinea-pig liver mitochondria.     

The effect of inhibitors on the formation of phosphoenolpyruvate by rat liver mitochondria

1. Rat liver mitochondria oxidizing malate produce PEP (phosphoenolpyruvate) without the addition of ATP or other nucleotides. 2. The addition of oligomycin in the presence of 2,4-dinitrophenol did not abolish PEP formation and in some instances stimulated its formation. 3. Formation of PEP was inhibited by arsenate. 4. Arsenite decreased PEP formation and caused accumulation of pyruvate. 5. Added GTP and ITP had no effect on PEP formation. 6. PEP formed from malate in the presence of GTP and labelled P(i) had a specific radioactivity approximately the same as the P(i) with no contribution from the phosphate of the added GTP. 7. There was no parallelism between the effects of inhibitors on PEP formation from malate and their effects on the assayed activity of PEP carboxykinase. 8. In a direct comparison it was shown that the PEP carboxykinase content of mitochondria was insufficient to account for the PEP formation from malate. 9. Consideration of the kinetic characteristics of PEP carboxykinase and mitochondrial content of oxaloacetate and GTP show that this enzyme cannot account for the PEP formed from malate by mitochondria.     

Inhibition of nucleoside diphosphate kinase in rat liver mitochondria by added 3'-azido-3'-deoxythymidine

The effect of 3'-azido-3'-deoxythymidine on nucleoside diphosphate kinase of isolated rat liver mitochondria has been studied. This is done by monitoring the increase in the rate of oxygen uptake by nucleoside diphosphate (TDP, UDP, CDP or GDP) addition to mitochondria in state 4. It is shown that 3'-azido-3'-deoxythymidine inhibits the mitochondrial nucleoside diphosphate kinase in a competitive manner, with a Ki value of about 10 microM as measured for each tested nucleoside diphosphate. It is also shown that high concentrations of GDP prevent 3'-azido-3'-deoxythymidine inhibition of the nucleoside diphosphate kinase.     

Functional Coupling between Nucleoside Diphosphate Kinase of the Outer Mitochondrial Compartment and Oxidative Phosphorylation

In rat liver mitochondria all nucleoside diphosphate kinase of the outer compartment is associated with the outer surface of the outer membrane (Lipskaya, T. Yu., and Plakida, K. N. (2003) Biochemistry (Moscow), 68, 1136-1144). In the present study, three systems operating as ADP donors for oxidative phosphorylation have been investigated. The outer membrane bound nucleoside diphosphate kinase was the first system tested. Two others employed yeast hexokinase and yeast nucleoside diphosphate kinase. The two enzymes exhibited the same activity but could not bind to mitochondrial membranes. In all three systems, muscle creatine phosphokinase was the external agent competing with the oxidative phosphorylation system for ADP. Determination of mitochondrial respiration rate in the presence of increasing quantities of creatine phosphokinase revealed that at large excess of creatine phosphokinase activity over other kinase activities (of the three systems tested) and oxidative phosphorylation the creatine phosphokinase reaction reached a quasi-equilibrium state. Under these conditions equilibrium concentrations of all creatine phosphokinase substrates were determined and K(eq)app of this reaction was calculated for the system with yeast hexokinase. In samples containing active mitochondrial nucleoside diphosphate kinase the concentrations of ATP, creatine, and phosphocreatine were determined and the quasi-equilibrium concentration of ADP was calculated using the K(eq)app value. At balance of quasi-equilibrium concentrations of ADP and ATP/ADP ratio the mitochondrial respiration rate in the system containing nucleoside diphosphate kinase was 21% of the respiration rate assayed in the absence of creatine phosphokinase; in the system containing yeast hexokinase this parameter was only 7% of the respiration rate assayed in the absence of creatine phosphokinase. Substitution of mitochondrial nucleoside diphosphate kinase with yeast nucleoside diphosphate kinase abolished this difference. It is concluded that oxidative phosphorylation is accompanied by appearance of functional coupling between mitochondrial nucleoside diphosphate kinase and the oxidative phosphorylation system. Possible mechanisms of this coupling are discussed.     

Relationship of the intra- and extramitochondrial adenine nucleotide ratios during synthesis of phosphoenolpyruvate using extramitochondrial ATP

Mitochondria prepared from the livers of guinea pig, chicken, and pigeon all actively synthesize phosphoenolpyruvate from oxalacetate and GTP, utilizing phosphoenolpyruvate carboxykinase. It was previously shown (Wilson, D. F., Erecińska, M., and Schramm, V. L. (1983). J. Biol. Chem. 258, 10464-10473) that phosphoenolpyruvate carboxykinase is freely reversible and that, in conjunction with nucleoside diphosphate kinase and malate dehydrogenase, which are also present in the mitochondria, it can be used to measure the intramitochondrial [ATPfree]/[ADPfree]. In this study, synthesis of phosphoenolpyruvate by guinea pig liver mitochondria was studied under conditions for which the only source of GTP was extramitochondrial ATP via adenine nucleotide translocase and nucleoside diphosphate kinase (the mitochondria were treated with rotenone, oligomycin, uncoupler, and fluorocitrate). When the extramitochondrial [ATP]/[ADP] was greater than the intramitochondrial [ATPfree]/[ADPfree] calculated from the phosphoenolpyruvate carboxykinase reaction, there was net synthesis of phosphoenolpyruvate, but when it was less, there was net disappearance of phosphoenolpyruvate. Thus, the intramitochondrial [ATPfree]/[ADPfree] was equal to the extramitochondrial value at the point of reversal of the phosphoenolpyruvate carboxykinase reaction. This equality of the intra- and extramitochondrial adenine nucleotide ratios occurred with a measured mitochondrial membrane potential of approximately -36 mV, whereas in the previous experiments, equality was observed for conditions in which the measured membrane potential was -111 to -125 mV. Thus, adenine nucleotide translocation was not dependent on the transmembrane electrical potential and must, therefore, have occurred by electroneutral exchange.     

Apparent ATP-linked succinate thiokinase activity and its relation to nucleoside diphosphate kinase in mitochondrial matrix preparations from rabbit.

The relative abilities of ATP and GTP to support succinyl-CoA synthesis by mitochondrial matrix fractions prepared from rabbit heart and liver mitoplasts were investigated. The activity supported by ATP in rabbit heart preparations was less than 15% of that obtained with GTP, while no ATP-supported activity was observed in rabbit liver preparations. However, the addition of 30 micromolar GDP to matrix fractions from either heart or liver stimulated the ATP-supported activity to 40% of that observed with GTP, and the further addition of bovine liver nucleoside diphosphate kinase in the presence of 8 microM added GDP increased the activity to near that observed with GTP. The specific activity of nucleoside diphosphate kinase assayed directly in mitochondrial matrix from heart was about 15% of the specific activity of ATP-supported succinate thiokinase induced upon adding GDP. Evidence for a complex between nucleoside diphosphate kinase and succinate thiokinase in mitochondrial matrix from rabbit heart was obtained by glycerol density gradient centrifugation. It is proposed that binding of nucleoside diphosphate kinase to succinate thiokinase activates the former enzyme, accounts for the ATP-supported succinyl-CoA synthetase activity observed, and is involved in the channeling of high energy phosphate from GTP produced in the Krebs cycle to the ATP pool.     

The metabolism of deoxyguanosine in mitochondria: relationship of the uptake of deoxyguanosine to the electron transport chain and adenosine triphosphate

That deoxyguanosine is taken up by isolated rat liver mitochondria has been shown. This report describes the relationship between that uptake and oxidative phosphorylation. By measuring this process in the presence of standard inhibitors of oxidative phosphorylation it was determined that a functional electron transport chain, but not the phosphorylation of ADP, was essential for uptake. ATP analogs adenyl(beta,gamma-methylene)diphosphate and adenylimidodiphosphate blocked uptake, indicating that ATP hydrolysis was required. ADP also proved to be an inhibitor. Exogenous UTP slightly stimulated deoxyguanosine uptake, as did added ATP, but several other nucleotides (dGTP, dATP, UDP, dGDP) were inhibitory. A sulfhydryl group was important for deoxyguanosine uptake and inhibition of uptake by N-ethylmaleimide was protected by both deoxyguanosine and ATP. These data show that deoxyguanosine uptake by mitochondria is a process which is coordinated and, perhaps, regulated by other events which take place in the organelle.     

Protective role of pinacidil against adrenaline-induced myocardium injury in guinea pig liver mitochondria

We investigated the role of the ATP-sensitive potassium channel opener pinacidil and blocker glibenclamide on guinea pig liver mitochondrial function, and a possible significance of pinacidil in the pharmacological treatment during myocardium dystrophy. First, a series of experiments was performed to determine the effect of pinacidil and glibenclamide on mitochondrial oxygen consumption. We found that pinacidil increased the rate of mitochondrial respiration for FAD-generated substrate (succinate oxidation), but was most effective for α-ketoglutarate oxidation with enhancement of respiratory control ratio. Oxidation of FAD-generated substrate inhibited efficiency of phosphorylation for α-ketoglutarate oxidation in pinacidil-treated animals. Glibenclamide decreased the rate of respiration with the lowest value of efficiency of phosphorylation, especially for α-ketoglutarate oxidation. A second series of experiments was performed to determine the effects of pinacidil and glibenclamide on oxidative phosphorylation during adrenaline-induced myocardium dystrophy. The increase in respiratory control ratio and efficiency of phosphorylation for α-ketoglutarate oxidation was greater than for succinate oxidation in mitochondria of pinacidil-pretreated animals during myocardium dystrophy. Inhibitory analysis with malonate suggested that endogenous succinate increased oxidation of NADH-generated substrates in mitochondria. Pinacidil is mainly involved in the adrenaline-induced alterations of mitochondrial function due to elevation of phosphorylation efficiency for α-ketoglutarate oxidation and a decreased level of lipid peroxidation.     

Photosynthesis in phosphoenolpyruvate carboxykinase-type C4 plants: photosynthetic activities of isolated bundle sheath cells from Urochloa panicoides

A method has been developed for rapidly preparing bundle sheath cell strands from Urochloa panicoides, a phosphoenolpyruvate (PEP) carboxykinase-type C4 plant. These cells catalyzed both HCO3(-)- and oxaloacetate-dependent oxygen evolution; oxaloacetate-dependent oxygen evolution was stimulated by ATP. For this activity oxaloacetate could be replaced by aspartate plus 2-oxoglutarate. Both oxaloacetate- and aspartate plus 2-oxoglutarate-dependent oxygen evolution were accompanied by PEP production and both were inhibited by 3-mercaptopicolinic acid, an inhibitor of PEP carboxykinase. The ATP requirement for oxaloacetate- and aspartate plus 2-oxoglutarate-dependent oxygen evolution could be replaced by ADP plus malate. The increased oxygen evolution observed when malate plus ADP was added with oxaloacetate was accompanied by pyruvate production. These results are consistent with oxaloacetate being decarboxylated via PEP carboxykinase. We suggest that the ATP required for oxaloacetate decarboxylation via PEP carboxykinase may be derived by phosphorylation coupled to malate oxidation in mitochondria. These bundle sheath cells apparently contain diffusion paths for the rapid transfer of compounds as large as adenine nucleotides.     

Mitochondrial nucleoside diphosphate kinase: Mode of interaction with the outer mitochondrial membrane and proportion of catalytic activity functionally coupled to oxidative phosphorylation

In the present study, we found that ionic interactions are not essential for the binding of nucleoside diphosphate kinase of liver mitochondria outer compartment to outer mitochondrial membrane and that the proportion of the enzyme activity involved in functional coupling with oxidative phosphorylation (we demonstrated the existence of functional coupling earlier) is only 17%. Additional evidence was obtained that functionally coupled activity of nucleoside diphosphate kinase is associated with the outer surface of mitochondria. Dextran (10%) did not increase functional coupling. The physological importance of these effects is discussed. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 3, pp. 395–407.     

Amaranth seed oil: Effect of oral administration on energetic functions of rat liver mitochondria activated with adrenaline

Respiration parameters of liver mitochondria (MCh) in rats fed with amaranth seed oil for 3 weeks have been evaluated. Thirty minutes before decapitation, adrenaline was injected intraperitoneally at a low dose (350 μg/kg body weight) to both control and experimental animals. It was shown that in animals that were injected with adrenaline and did not receive oil, the rate of phosphorylating respiration increased by 32% and phosphorylation time decreased by 22% upon oxidation of succinate; upon oxidation of α-ketoglutarate in the presence of the succinate dehydrogenase inhibitor malonate, phosphorylating respiration was activated by 23%. The respiration of MCh upon oxidation of succinate + glutamate and α-ketoglutarate in the absence of malonate was not affected by adrenaline. The intake of oil markedly activated almost all parameters of mitochondrial respiration in experimental rats upon oxidation of all above-listed substrates in both coupled and uncoupled MCh. However, phosphorylation time was close to the control value (upon oxidation of succinate) or increased (upon oxidation of α-ketoglutarate in the presence and absence of malonate). The injection of adrenaline to animals receiving oil did not affect the oil-activated respiration of MCh oxidizing the substrates used; however, phosphorylation time in all groups of animals decreased. Ca²⁺ capacity of MCh in rats receiving amaranth oil did not change. Thus, our data show that feeding of rats with amaranth oil activates mitochondrial respiration and prevents MCh hyperactivation induced by adrenaline.     

Regulation of gluconeogenesis and lipogenesis. The regulation of mitochondrial pyruvate metabolism in guinea-pig liver synthesizing precursors for gluconeogenesis

1. The carboxylation of pyruvate to oxaloacetate by pyruvate carboxylase in guinea-pig liver mitochondria was determined by measuring the amount of (14)C from H(14)CO(3) (-) fixed into organic acids in the presence of pyruvate, ATP, Mg(2+) and P(i). The main products of pyruvate carboxylation were malate, fumarate and citrate. Pyruvate utilization, metabolite formation and incorporation of (14)C from H(14)CO(3) (-) into these metabolites in the presence and the absence of ATP were examined. The synthesis of phosphoenolpyruvate from pyruvate and bicarbonate is minimal during continued oxidation of pyruvate. Larger amounts of phosphoenolpyruvate are formed from alpha-oxoglutarate than from pyruvate. Addition of glutamate, alpha-oxoglutarate or fumarate did not appreciably increase formation of phosphoenolpyruvate when pyruvate was used as substrate. With alpha-oxoglutarate as substrate addition of fumarate resulted in increased formation of phosphoenolpyruvate, whereas addition of succinate inhibited phosphoenolpyruvate formation. In the presence of added oxaloacetate guinea-pig liver mitochondria synthesized phosphoenolpyruvate in amount sufficiently high to play an appreciable role in gluconeogenesis. 2. Addition of fatty acids of increasing carbon chain length caused a strong inhibition of pyruvate oxidation and phosphoenolpyruvate formation, and greatly promoted carbon dioxide fixation and malate, citrate and acetoacetate accumulation. The incorporation of (14)C from H(14)CO(3) (-), [1-(14)C]pyruvate and [2-(14)C]pyruvate into organic acids formed was examined. 3. It is concluded that guinea-pig liver pyruvate carboxylase contributes significantly to gluconeogenesis and that fatty acids and metabolites play an important role in its regulation.     

Electron transport phosphorylation coupled to fumarate reduction by H2- and Mg2+-dependent adenosine triphosphatase activity in extracts of the rumen anaerobe Vibrio succinogenes.

Vibrio succinogenes, an anaerobic bacterium, obtains its energy for growth from H2 or formate oxidation coupled to the reduction of fumarate to succinate. Membrane preparations have been obtained from this organism that catalyze the synthesis of ATP during H2 oxidation coupled to fumarate reduction. Esterification of orthophosphate is dependent on electron transfer, as evidenced by the requirement for both H2 and fumarate. Phosphorylation is also dependent on ADP and is destroyed by boiling the membrane preparations. H2 utilized for fumarate reduction and succinate formed are stoichiometric. The phosphorylation is markedly uncoupled by pentachlorophenol and gramicidin, but to a lesser extent by dinitrophenol and methyl viologen. 2-n-Heptyl-4-hydroxyquinoline-N-oxide causes severe inhibition of H2 oxidation as well as phosphorylation, but oligomycin or antimycin A has no demonstrable effect. Among several electron acceptors tested, significant phosphorylation is observed only with fumarate. A Mg2+-dependent adenosine triphosphatase activity is present in both the membrane and soluble protein fractions. Highest activity is obtained with ATP as the substrate, and considerably less activity is obtained with other nucleoside triphosphates. The possibility that phosphorylation during "fumarate respiration" may play an important physiological role in the growth of many anaerobic and facultatively anaerobic bacteria is discussed.     

RNA synthesis during morphogenesis of the fungusMucor racemosus

Bacteroides succinogenes produces acetate and succinate as major products of carbohydrate fermentation. An investigation of the enzymes involved indicated that pyruvate is oxidized by a flavin-dependent pyruvate cleavage enzyme to acetyl-CoA and CO₂. Active CO₂ exchange is associated with the pyruvate oxidation system. Reduction of flavin nucleotides is CoASH-dependent and does not require ferredoxin. Acetyl-CoA is further metabolized via acetyl phosphate to acetate and ATP. Reduced flavin nucleotide is used to reduce fumarate to succinate by a particulate flavin-specific fumarate reductase reaction which may involve cytochrome b. Phosphoenolpyruvate (PEP) is carboxylated to oxalacetate by a GDP-specific PEP carboxykinase. Oxalacetate, in turn, is converted to malate by a pyridine nucleotide-dependent malate dehydrogenase. The organism has a NAD-dependent glyceraldehyde-3-phosphate dehydrogenase. The data suggest that reduced pyridine nucleotides generated during glycolysis are oxidized in malate formation and that the electrons generated during pyruvate oxidation are used to reduce fumarate to succinate.     

Investigation of the dependence of the intramitochondrial [ATP]/[ADP] ratio on the respiration rate

The relationship between the respiration rate and the intra- and extramito-chondrial adenine nucleotides was investigated in isolated rat liver mitochondria.

For the determination of adenine nucleotide patterns in both compartments a new procedure was developed, based on the evaluation of these metabolites from incubation of various amounts of mitochondria under identical stationary states of oxidative phosphorylation. These identical states were adjusted by addition of appropriate amounts of hexokinase to a glucose-containing incubation mixture.

Adenine nucleotides were measured in aliquots of the total extract of the incubation mixture without any separation. The concentrations of the adenine nucleotides in both compartments were obtained from a plot of the total concentration of these species versus mitochondrial protein. Disturbances of this method by unspecific efflux of adenine nucleotides could be excluded.

The results obtained for the total adenine nucleotide content (12 nmol · mg⁻¹ protein) and the intramitochondrial [ATP]/[ADP] ratio (about 4 in the resting state) are in good agreement with data obtained by other methods.

Strong evidence is provided for a decrease of the intramitochondrial [ATP]/[ADP] ratio with increasing rate of oxygen consumption. Therefore it is not necessary to assume a microcompartmentation of the intramitochondrial adenine nucleotide pool in respect to the ATPase reaction and the adenine nucleotide translocation.     

Synthesis and antiviral activity of 3′-azido-3′-deoxythymidine triphosphate distearoylglycerol: a novel phospholipid conjugate of the anti-HIV agent AZT

Phospholipid conjugates of 3′-azido-3′-deoxythymidine (AZT) show activity against the human immunodeficiency virus (HIV) in vitro. In a previous report (K.Y. Hostetler, L.M. Stuhmiller, B.H.M. Lenting, H. van den Bosch and D.D. Richman (1991), J. Biol. Chem. 265, 6112–6117) the syntheses and anti-HIV activities of AZT mono- and diphosphate diglyceride have been described. We now report on the synthesis, characterization and biological activity of 3′-azido-3′-deoxythymidine triphosphate distearoylglycerol (AZTTP-DSG). The compound was prepared by the condensation of AZT diphosphate with distearoylphosphatidic acid morpholidate in anhydrous pyridine at room temperature and purified by means of high-performance liquid chromatography using a silica column. Characterization was performed with ³¹P-NMR and IR analyses and determination of the fatty acid, phosphorus and nucleoside content of the product. AZTTP-DSG inhibited HIV-1 replication in both CEM and HT4-6C cells at a level intermediate in potency between its mono- and diphosphate analogs. The IC₅₀ values of AZTTP-DSG were 0.33 and 0.79 μM in these two cell lines, respectively. In addition, AZTTP-DSG was less toxic to CEM cells in vitro than the other AZT liponucleotides and reduced viable cell numbers in this cell type by 50% at 1000 μM. Initial studies on the metabolism of AZTTP-DSG revealed that both AZT and AZT monophosphate were liberated from the lipid pro-drug by a rat liver mitochondrial enzyme preparation. These phospholipid derivatives of AZT nucleotides represent pro-drugs for the intracellular delivery of phosphorylated antiviral nucleoside analogs.     

Phytochrome-mediated control of respiratory activities of mitochondria in cotyledons of dark-grown cucumber seedlings

Mitochondria isolated from cotyledons of dark-grown cucumber ( Cucumber sativus L., cv. Shimotsuki-Aonaga) seedlings after illumination with continuous far-red light showed an increased capacity for oxidation of malate or α-ketoglutarate, as compared with those from cotyledons of non-illuminated seedlings. This increase is supposed to be caused by phytochrome action (high irradiance response). Exogenous NAD⁺ had no effect on the rate of the oxidation of α-ketoglutarate or malate by mitochondria isolated from far-red light-treated cotyledons, but it enhanced the oxidation rate of mitochondria from control cotyledons to the level of mitochondria from light-treated ones. The NAD (NAD⁺+ NADH) content was higher in mitochondria isolated from continuously far-red light-treated cotyledons than in mitochondria from controls. The NAD content was also increased by the treatment with a red light pulse and this response was reversed by a subsequent far-red light pulse. It is proposed that phytochrome controls respiratory activities of cucumber mitochondria by changing the size of the NAD pool in the mitochondria.     

Mitochondria from the lamprey (Lampetra fluviatilis). Oxidative phosphorylation and related processes.

1. High efficiency of oxidative phosphorylation and a good respiratory control in liver, heart and somatic muscle mitochondria of the lamprey (Lampetra fluviatilis) were observed when the particles were isolated in a complex sucrose medium containing EDTA, heparin and nicotinamide. The coupling properties of these mitochondria were further improved by including serum albumin in the incubation medium. 2. The content of total adenine nucleotides in lamprey mitochondria was between 4 and 6 nmoles/mg protein. The translocation of these nucleotides across mitochondrial membrane was stimulated by serum albumin. 3. Lamprey mitochondrial phospholipids contain a large proportion (64-72%) of polyunsaturated fatty acids. 4. Electron micrographs of mitochondria from lamprey liver, heart and somatic muscle are presented.