Adenine nucleotide translocation in yeast mitochondria. Effect of inhibitors of mitochondrial biogenesis on the ADP translocase

1. Optimal test conditions for adenine nucleotide translocation in Candida utilis mitochondria are a standard medium, consisting of 0.63 M mannitol, 2 mM EDTA (or ethylene glycol tetraacetic acid, EGTA), 10 mM morpholinopropane sulfonic acid (pH 6.8), and a temperature of 0 °C.

2. Adenine nucleotide translocation in C. utilis mitochondria is an exchange-diffusion process. The whole pool of internal adenine nucleotides is exchangeable, ADP being the most readily exchangeable nucleotide. The rate of mitochondrial ADP exchange, but not the K_m value, depends on growth conditions. At 0 °C, the rate is about 3 to 4 nmoles ADP/min per mg protein for mitochondria obtained from yeast grown in the presence of 1.5% glucose; it rises to 11.5 nmoles when glucose is replaced by 3% ethanol in the growth medium. The K_m value for ADP is 2 μM. The Q₁₀ is about 2 between 0 and 20 °C. Among other exchangeable adenine nucleotides are ATP, dADP and the methylene and the hypophosphate analogues of ADP. Unlike mammalian mitochondria, C. utilis mitochondria are able to transport external UDP by a carboxyatractyloside-sensitive process.

3. Under conditions of oxidative phosphorylation (phosphate and substrate present in an aerated medium), added ADP is exchanged with internal ATP. A higher ATP/ADP ratio was found in the extramitochondrial space than in the intramito-chondrial space. The difference between the calculated phosphate potentials in the two spaces was 0.9–1.7 kcal/mole.

4. Atractyloside, carboxyatractyloside, bongkrekic acid and palmityl-CoA inhibit mitochondrial adenine nucleotide translocation in C. utilis as they do in mammalian mitochondria, but 2 to 4 times less efficiently. The inhibition due to atractyloside or palmityl-CoA is competitive with respect to ADP whereas that due to bongkrekic acid and carboxyatractyloside is non-competitive. Carboxyatractyloside and atractyloside inhibitions are additive. The apparent K_d for the binding of [³⁵S]-carboxyatractyloside and [¹⁴C]bongkrekic acid is 10–15 nM and the concentration of sites 0.4–0.6 nmole/mg protein in both cases. [³⁵S]Carboxyatractyloside binding is competitively displaced by atractyloside and vice versa.

5. Binding of [¹⁴C]ADP has been carried out with mitochondria depleted of their endogenous adenine nucleotides by incubation with phosphate and Mg²⁺ at 20 °C. The amount of bound [¹⁴C]ADP which is atractyloside removable is 0.08–0.16 nmole/mg protein.

6. The rate of ADP transport is quite different in mitochondria isolated from C. utilis, according to whether it is grown on glucose, or on ethanol or in the presence of chloramphenicol; for instance, it decreases by 10 times when 3% ethanol in the growth medium is replaced by 10% glucose and by 5 times when chloramphenicol is added to the medium. These variations are accompanied by parallel variations in cytochrome aa₃. The number of atractyloside-sensitive ADP binding sites is not modified by the above conditions of culture, nor the number of [³⁵S]carboxyatractyloside binding sites. The affinity for ADP is apparently not significantly modified, nor the size of the endogenous adenine nucleotide pool. In contrast to glucose repression or chloramphenicol inhibition, semi-anaerobiosis in C. utilis lowers significantly the mitochondrial binding capacity for carboxyatractyloside. Strict anaerobiosis in S. cerevisiae results in a practical loss of the cytochrome oxidase activity, and also of the carboxyatractyloside and ADP binding capacity. Transition from anaerobiosis to aerobiosis restores the cytochrome oxidase activity and the ADP and carboxyatractyloside binding capacities.     

Adenine nucleotides in thrombocytes of birds

Analysis of free nucleotide composition of both avian thrombocytes and pig platelets showed quantitative differences in the level of adenine nucleotides. 3H-adenine taken up by turkey thrombocytes was metabolized mainly to adenine nucleotides was not released after thrombin action. Thrombin liberated non-radioactive adenine nucleotides (18.2 +/- 1.5%, 20.6 +/- 1.9%) of the total, probably localized in a storage pool. Malonyldialdehyhyde (MDA) production due to thrombin was observed in both platelets and thrombocytes.     

Adenine and guanine nucleotide content of Triton-extracted cytoskeletal fractions of nonmuscle cells.

Determination of the adenine and guanine nucleotides in Triton X-100-extracted cytoskeletal fractions was utilized to estimate the actin and tubulin content of the assembled cytoskeletons in nonmuscle cells. Results with stable cell lines (i.e., rat pheochromocytoma PC12 and neuroblastoma NB41A3) and with primary cultures (i.e., human foreskin fibroblasts and chick embryonic dorsal root ganglion neurons) exhibited levels of cytoskeletal fraction ADP and GDP consistent with their assembly-induced nucleoside-5'-triphosphatase activities only previously analyzed in vitro. Likewise, estimates of actin and tubulin content fall in the range of values obtained by other experimental approaches. In contrast, analysis of whole cell nucleotides showed high [ATP]/[ADP] and [GTP]/[GDP] ratios, suggesting there is little, if any, contamination of the cytoskeletal nucleotide pool by other cellular nucleotides.     

The interaction of thrombin and nucleotides: Effects of glycolysis in human platelets

Thrombin stimulated lactate formation in intact, but not disrupted, platelets, an effect inhibited by ADP and ATP. ADP and ATP stimulated lactate formation in disrupted, but not intact, platelets, an effect inhibited by thrombin. Both nucleotides altered the electrophoretic mobility of thrombin in polyacrylamide gel without affecting its molecular weight. Binding of thrombin to nucleotides could not be demonstrated by gel filtration, equilibrium dialysis, or affinity chromatography.     

Oxidative stress and adenine nucleotide control of mitochondrial permeability transition

Mitochondria can initiate apoptosis by releasing cytochrome c after undergoing a calcium-dependent permeability transition (MPT). Although the MPT is enhanced by oxidative stress and prevented by adenine nucleotides such as adenosine 5'-diphosphate (ADP), the hypothesis has not been tested that oxidants regulate the effects of exogenous adenine nucleotides on the MPT and cytochrome c release. We found that cytochrome c release from intact rat liver mitochondria depended strictly on pore opening and not on membrane potential, and that MPT-enhancing oxidative stress also augmented cytochrome c release. At low oxidative stress, micromolar (ADP) and low adenosine 5'-triphosphate (ATP)/ADP ratio inhibited the MPT and cytochrome c release, whereas ATP or high ATP/ADP had only a slight effect. In freshly isolated mitochondria, the time to half-maximal MPT was related to the log of the ATP/ADP ratio. This function was shifted to shorter times by oxidative stress which decreased ADP protection and caused ATP to accelerate the calcium-dependent MPT. By comparison, mitochondria treated with reducing agents and those isolated from septic rats were protected from the MPT by both nucleotides. These results indicate that oxidation-sensitive site(s) in the membrane regulate the effects of adenine nucleotides on the MPT. The oxidant-based differences in the effects of ADP and ATP on the pore support the novel hypothesis that failure of the cell to consume ATP and provide adequate ADP at the adenine nucleotide transporter during oxidative stress predisposes to cytochrome c release and initiation of apoptosis.     

Synthesis of new polymer-bound adenine nucleotides using starburst PAMAM dendrimers

Two types of new polymer-bound adenine nucleotides were synthesized by coupling adenine nucleotides (ATP and ADP) with starburst polyamidoamine (PAMAM) dendrimers. The first type was obtained by coupling native adenine nucleotides directly with a carboxy-terminated PAMAM dendrimer. In the second type, the nucleotides were modified by introducing a spacer arm containing a carboxylic end group (N(6)-R-ATP and N(6)-R-ADP) and coupled with an amine-terminated PAMAM dendrimer. Both types of the dendrimers were coupled with native or the modified nucleotides using the well-known carbodiimide activation technique. The optimum coupling pH and temperature were 4 and 30 degrees C, respectively, for preparing the carboxy-terminated PAMAM-bound ATP or ADP, and were 9 and 50 degrees C, respectively, for preparing the amine-terminated PAMAM-bound N(6)-R-ATP or N(6)-R-ADP. The ATP or ADP contents in the synthesized polymers were found to be 4 mol of ATP or of ADP/mol of carboxy-terminated PAMAM-bound ATP or ADP and 25 mol of ATP or of ADP/mol of amine-terminated PAMAM-bound N(6)-R-ATP or N(6)-R-ADP. The coenzymatic activities relative to the native ATP of the carboxy-terminated PAMAM-bound ATP against glucokinase and hexokinase were 16 and 7%, respectively, and those of the amine-terminated PAMAM-bound N(6)-R-ATP 2 and 1%, respectively. The coenzymatic activities relative to the native ADP of the carboxy-terminated PAMAM-bound ADP and the amine-terminated PAMAM-bound N(6)-R-ADP against acetate kinase were 24 and 3.5%, respectively.     

Effect of adenine nucleotide pool size in mitochondria on intramitochondrial ATP levels.

Net adenine nucleotide transport into and out of the mitochondrial matrix via the ATP-Mg/Pi carrier is activated by micromolar calcium concentrations in rat liver mitochondria. The purpose of this study was to induce net adenine nucleotide transport by varying the substrate supply and/or extramitochondrial ATP consumption in order to evaluate the effect of the mitochondrial adenine nucleotide pool size on intramitochondrial adenine nucleotide patterns under phosphorylating conditions. Above 12 nmol/mg protein, intramitochondrial ATP/ADP increased with an increase in the mitochondrial adenine nucleotide pool. The relationship between the rate of respiration and the mitochondrial ADP concentration did not depend on the mitochondrial adenine nucleotide pool size up to 9 nmol ADP/mg mitochondrial protein. The results are compatible with the notion that net uptake of adenine nucleotides at low energy states supports intramitochondrial ATP consuming processes and energized mitochondria may lose adenine nucleotides. The decrease of the mitochondrial adenine nucleotide content below 9 nmol/mg protein inhibits oxidative phosphorylation. In particular, this could be the case within the postischemic phase which is characterized by low cytosolic adenine nucleotide concentrations and energized mitochondria.     

Compartmentalized ATP pools produced from adenosine are nuclear pools

Incubation of African green monkey kidney (BS-C-1) cells and mouse fibroblasts (3T6) in the presence of adenosine for 4 hours resulted in increases in the nuclear compartment pools of adenosine 5'-triphosphate (ATP) and nuclear ATP/adenosine 5'-diphosphate (ADP) ratios. Adenine and inosine, which yield increases in total cellular ATP pools and ATP/ADP ratios similar to those promoted by adenosine, do not produce similar increases in the nuclear compartment. Adenosine-promoted increases in nuclear ATP pools were higher in the untransformed, serially propagated, BS-C-1 cells than in the spontaneously transformed 3T6 cells. Adenosine-promoted compartmentalized ATP pools in primary chick embryo fibroblasts were reduced upon transformation of these cells with Rous sarcoma virus, resulting in free mixing of all of the ATP pools synthesized from various salvage precursors. The growth regulatory properties of the nuclear compartment pools of adenine nucleotides is suggested by the big increases in nuclear ATPase and adenosine 5'-monophosphate (AMP) deaminase activities upon the entry of 3T6 cells into the S phase of their cycle. These enzymatic activities would tend to lower the nuclear ATP/ADP ratios and reduce the total adenine nucleotide pools in these nuclei respectively--conditions which were shown by earlier in vitro studies to be favorable to DNA replication.     

Energy-dependent exchange of adenine nucleotides on chloroplast coupling factor (CF1)

1. [¹⁴C]ADP is incorporated into washed broken chloroplasts in the light. The bound labelled nucleotides which cannot be removed by washing are almost exclusively related to coupling factor CF₁. [¹⁴C]ADP binding exhibits a monophasic concentration curve with a K_m of 2 μM.2. By illumination of the chloroplasts, previously incorporated labelled nucleotides are released. A fast release is obtained in the presence of unlabelled ADP and ATP, indicating an energy-dependent exchange. A slow and incomplete release is induced by light in the absence of unlabelled adenine nucleotides. Obviously, under those conditions, an adenine nucleotide depleted CF₁ conformation is established.3. Re-binding of [¹⁴C]ADP by depleted membranes is an energy-independent process. Even after solubilization of adenylate-depleted CF₁, [¹⁴C]ADP is incorporated into the protein. By re-binding of ADP in the dark, CF₁ is converted to a non-exchangeable form.4. Energy-dependent adenine nucleotide exchange on CF₁ is suggested to include three different conformational states of the enzyme: (1) a stable, non-exchangeable form which contains firmly bound nucleotides, is converted to (2), an unstable form containing loosely bound adenine nucleotides. This conformation allows adenylate exchange; it is in equilibrium with (3) a metastable, adenylate-depleted form. The transition from state (1) to state (2) is the energy-requiring step.     

Effect of bed rest on the adenine nucleotides concentration in human blood platelets.

The effect of immobilization in bed on metabolism and function of human blood platelet was studied. Blood platelets taken from patients with bone fractures after long term bed rest (14 days and 28 days) demonstrated significantly reduced concentration of total adenine nucleotides (after 28 days reduction about 30%). This decrease of total platelet adenine nucleotides after immobilization in bed is probably caused by stimulation of platelet secretory process. Thrombin which released from control platelets 58.2% +/- 1.5% of total adenine nucleotides liberated decreased amounts (only 23.1% +/- 3.3% of total) of nucleotides from patient platelets isolated after 28 days of immobilization in bed. Loss of nucleotides from platelets was accompanied by slightly increased extent of platelet aggregation. It is concluded that during bed rest the reactivity of blood platelets (aggregation and release reaction) is stimulated.     

Changes in the energy state of tissues in spontaneously hypertensive rats]

The contents of adenine nucleotides (ATP, ADP, AMP), phosphocreatine (PCr) and creatine (Cr) in the heart, skeletal muscle, liver and spleen in spontaneously hypertensive (SHR) and normotensive (WKY) rats. The ATP/ADP ratio in cardiac tissue was lower in SHR compared with WKY, while myocardial contents of adenine nucleotides, PCr and Cr did not differ significantly between the groups. A lower ATP/ADP ratio in the skeletal muscle SHR of was accompanied by a reduction of PCr content comparing with these indices in WKY rats. The liver and spleen of SHR exhibited lower ATP contents and higher ADP and AMP levels compared with those ones in WKY rats, despite of the close values of adenine nucleotide pools (sigma AN = ATP + ADP + AMP). This redistribution of tissue adenine nucleotides was corresponded to lower energy charges (EC = (ATP + 0.5 ADP)/sigma AN) and ATP/ADP ratios in SHR group. The reduction of the energy state of tissues in SHR rats increased in the following rank: heart > skeletal muscle > liver > spleen, thus, reflecting progressive decrease of intensity of oxidative metabolism. The results suggest changes in the balance of rates of ATP formation and hydrolysis occur at the system level in primary hypertension. Probably, consequences of such rearrangement in energy metabolism are functional disturbances of plasma membrane and sacroplasmic reticulum well-documented in a number of experimental and clinical studies.     

Inhibition of 2,4-dinitrophenol-induced potassium efflux by adenine nucleotides in mitochondria

The influence of nucleotides on 2,4-dinitrophenol (DNP)-induced K+ efflux from intact rat liver mitochondria has been studied. ATP and ADP at micromolar concentrations were found to inhibit mitochondrial potassium transport, whereas GTP, GDP, CTP, and UTP did not show tha same effect. The values of half-maximal inhibition (IC50) were approximately 20 microM for ATP and approximately 60 microM for ADP. It is suggested that adenine nucleotides exert their inhibitory action at the matrix side of the inner mitochondrial membrane since the inhibitor of adenine nucleotide translocase atractyloside at concentration of 1 microM completely removed the inhibitory effect of ATP and ADP. The mitochondrial ATPase inhibitor oligomycin (2 microg/ml) was found to reduce slightly the rate of DNP-induced K+ efflux and had no effect on inhibition by adenine nucleotides; the latter was insensitive to Mg2+ and the changes in pH. It seems likely that the regulation of potassium transport is not due to phosphorylation of the channel-forming protein but to binding of the nucleotides in specific regulatory sites. The possibility of potassium efflux from mitochondria in the presence of uncoupler via the ATP-dependent potassium channel is discussed.     

Some thoughts on the evolutionary basis for the prominent role of ATP and ADP in cellular energy metabolism

The predominance of the adenosine triphosphate/adenosine diphosphate (ATP/ADP) couple in cellular phosphorylation reactions, including those that form the basis for cellular energy metabolism, cannot be explained on thermodynamic grounds since a variety of "high energy phosphate" compounds (including ADP itself) found in the cell would, based on thermodynamic considerations, be at least as effective as ATP in serving as a phosphoryl donor. How then did present-day organisms come to rely on the ATP/ADP couple as the principal mediator of phosphorylation reactions? The early appearance of adenine compounds in the prebiotic environment is suggested by experiments indicating that, relative to other purine or pyridimine compounds, adenine derivatives are preferentially synthesized under simulated prebiotic conditions (Ponnamperuma et al., 1963). In addition to the roles of adenine nucleotides in phosphorylation reactions, other adenine derivatives (e.g. Coenzyme A, flavin adenine dinucleotide, puridine nucleotides) are employed in a variety of metabolic roles. The principal function of the adenine moiety in these latter cases is in the binding of these derivatives to the relevant enzyme. The capability for binding of the adenine moiety appears to have arisen early in evolution and been exploited in a multitude of contexts, a suggestion consistent with observed similarities between the binding sites of several enzymes employing adenine derivatives as substrate. The early availability of suitable adenine compounds in the biosphere and development of complementary binding sites on cellular proteins, coupled with the expected advantages in having a limited number of metabolites as central mediators of endergonic and exergonic metabolism could readily have led to the observed pre-eminence of adenine nucleotides in cellular energy metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane Biochemistry : by E. Sim Chapman and Hall; London, 1982

Adenine, cytidine and guanosine nucleotides were supplied to cultures of Rhodopseudomonas capsulata under aerobic heterotrophic and phototrophic growth conditions. Aerobic growth is not affected by exogenous nucleotides (up to 10 mM) whereas phototrophic growth is strongly inhibited by adenine but not by guanosine or cytidine nucleotides. During phototrophic growth there is an inverse relationship between the concentration of exogenous adenine nucleotides and photopigment synthesis. There are no statistically significant differences between the inhibitory effect of AMP, ADP and ATP on the growth rate and bacteriochlorophyll synthesis since adenine nucleotides are incorporated into the cell as AMP by means of the phosphoribosyl transferase system.     

A study of spatial and temporal variability in the adenine nucleotides of lake phytoplankton during a diel cycle (Lac Pavin — France)

A study of diel variations in the adenine nucleotide concentrations of natural phytoplanktonic populations led to the following conclusions:There are significant diel variations in cellular ATP concentrations. However, this temporal variability is not as great as the spatial variability between the various sampling depths, characterized by different phytoplankton populations. Within a globally stable pool of adenine nucleotides, diel variations inside the algal cell mainly take place at the expense of ATP and AMP, while ADP concentrations remain relatively stable.The statistical relationship between algal biomass obtained from cell counts and from adenine nucleotides also confirms maximum stability for ADP and (ATP + ADP + AMP). The diel variations in adenine nucleotide cell concentrations gradually become smaller with depth. This probably reflects the importance of light in causing such variability.     

Characteristics of adenine nucleotide fluxes and transport in human tumor mitochondria

The efflux of adenine nucleotides from three human tumor mitochondria has been investigated with mitochondria prelabeled with radioactive ATP. Uncouplers induce a large efflux of adenine nucleotides from mitochondria from human hepatoma and oat cell carcinoma while efflux from astrocytoma mitochondria is less. This efflux does not require exchangeable anions, i.e., adenine nucleotides or pyrophosphate, in the extramitochondrial medium, and is not sensitive to atractyloside. The efflux is more extensive with dinitrophenol and CCCP than with valinomycin-K+, and may account for the differential effects of the two types of uncouplers on uncoupler-stimulated ATPase of tumor mitochondria previously reported by us. Dinitrophenol and CCCP do not elicit any efflux of adenine nucleotides from normal liver mitochondria. Efflux of orthophosphate from tumor mitochondria is also greater with dinitrophenol and CCCP; however, the more interesting finding is that the concentration of orthophosphate in these mitochondria is unusually high, i.e., 10-40-times greater than the intramitochondrial phosphate concentration of liver mitochondria. Atractyloside sensitive transport of ATP and ADP in human tumor mitochondria has also been determined. Vmax values for both ADP and ATP transport are lower than those obtained with liver mitochondria, especially with ADP transport. ATP transport in tumor mitochondria is not affected by CCCP in contrast to the 4-5-fold stimulation observed in liver mitochondria.     

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.     

The effects of octylglucoside on the interactions of chloroplast coupling factor 1 (CF1) with adenine nucleotides

The effects of octylglucoside (OcGlc) micelles, which stimulate a Mg-specific ATPase activity in chloroplast coupling factor 1 [Pick, U. and Bassilian, S. (1982) Biochemistry, 21, 6144-6152], on the interactions of the enzyme with adenine nucleotides have been studied. 1. OcGlc specifically accelerates the binding and the release of ADP but not of ATP or adenosine 5'[beta, gamma-imido]triphosphate (AdoPP[NH]P) from the tight-sites. The binding affinity for ADP and for ATP is only slightly decreased (twofold) by the detergent. ATP competitively inhibits the binding of ADP and vice versa in the presence or absence of OcGlc. 2.OcGlc-induced inactivation of CF1-ATPase is correlated with the release of bound nucleotides. In the absence of medium nucleotides ADP X CF1 is rapidly inactivated while ATP X CF1 and AdoPP[NH]P X CF1 are slowly inactivated by OcGlc in parallel with the release of bound nucleotide. In contrast, low concentrations of either ATP or ADP in the medium effectively protect against OcGlc inactivation while AdoPP[NH]P, whose binding to CF1 is inhibited by OcGlc, is ineffective even at millimolar concentrations. The results suggest that the occupancy of the tight-sites protects the enzyme against OcGlc-induced inactivation. 3. Mg ions specifically inhibit the release of bound ADP and the OcGlc-induced inactivation of CF1. High concentrations of medium ATP and ADP (K50 = 100 microM) also inhibit the OcGlc-induced release of bound nucleotides in an EDTA medium. In contrast, in the absence of OcGlc, medium ADP and ATP accelerate the release of bound adenine nucleotides. 4. Mg-ATP in the presence of OcGlc stimulates the release of bound ADP from CF1. Bound ATP is neither released nor hydrolyzed at the tight-sites under these conditions where medium ATP is rapidly hydrolyzed. Mg-ADP stimulates the release of bound ADP only in the presence of inorganic phosphate or of phosphate analogs, e.g. arsenate, pyrophosphate or selenate. 5. It is suggested that: (a) ATP and ADP bind to the same tight-sites, but OcGlc activation specifically accelerates the exchange of bound ADP at the site. (b) CF1 contains low affinity adenine nucleotide binding sites which may be the catalytical sites and which influence the tight-sites by cooperative interactions. (c) Mg-ATP in the presence of OcGlc induces a conformational change at the catalytical site which accelerates the release of ADP from the tight-site. The implications of these results to the role of adenine nucleotides in the regulation and mechanism of ATP hydrolysis by CF1 are discussed.     

The effect of resveratrol on the platelet secretory process induced by endotoxin and thrombin

The effect of resveratrol (trans-3,4',5-trihydroxystilbene) on the release of adenine nucleotides and proteins from blood platelets activated by lipopolysaccharide (LPS), from Proteus mirabilis and by thrombin, were studied. Thrombin stimulated the release of adenine nucleotides from dense granules and proteins from alpha-granules. The LPS (0.3 microg/10(8) platelets, 5 min, 37 degrees C), like thrombin (2.5 U/10(8) platelets, 5 min, 37 degrees C) was found to cause a release of adenine nucleotides and proteins (p <0.05). Resveratrol (6.25-100 microg/ml, 30 min, 37 degrees C) had a different effect on the platelet release reaction caused by either LPS or thrombin. The results indicated that resveratrol inhibited, in dose-dependent manner, the secretory process (release of adenine nucleotides and proteins) induced by thrombin (p <0.05), but it significantly stimulated the liberation of proteins from blood platelets activated by LPS (p <0.05).     

Net uptake of adenine nucleotides by newborn rat liver mitochondria

In newborn rat liver, the adenine nucleotide content (ATP + ADP + AMP) of mitochondria increases severalfold within 2 to 3 h of birth. The net increase in mitochondrial adenines suggests a novel mechanism by which mitochondria are able to accumulate adenine nucleotides from the cytosol (J. R. Aprille and G. K. Asimakis, 1980, Arch. Biochem. Biophys.201, 564.). This was investigated further in vitro. Isolated newborn liver mitochondria incubated with 1 mM ATP for 10 min at 30 °C doubled their adenine nucleotide content with effects on respiratory functions similar to those observed in vivo: State 3 respiration and adenine translocase activity increased, but uncoupled respiration was unchanged. The mechanism for net uptake of adenine nucleotides was found to be specific for ATP or ADP, but not AMP. Uptake was concentration dependent and saturable. The apparent K_m′s for ATP and ADP were 0.85 ± 0.27 mM and 0.41 ± 0.20 mM, respectively, measured by net uptake of [¹⁴C]ATP or [¹⁴C]ADP. The specific activities of net ATP and ADP uptake averaged 0.332 ± 0.062 and 0.103 ± 0.002 nmol/min/mg protein, respectively. ADP was a competitive inhibitor of net ATP uptake. If P_i was omitted from the incubations, net uptake of ATP or ADP was reduced by 51%. Either mersalyl or N-ethylmaleimide severely inhibited the accumulation of adenine nucleotides. Net ATP uptake was stoichiometrically dependent on MgCl₂, suggesting that Mg²⁺ is accumulated along with ATP (or ADP). Uptake was energy dependent as indicated by the following results: Net AdN uptake (especially ADP uptake) was stimulated by the addition of an oxidizable substrate (glutamate) and inhibited by FCCP (an uncoupler). Antimycin A had no effect on net ATP uptake but inhibited net ADP uptake, suggesting that ATP was able to serve as an energy source for its own accumulation. If carboxyatractyloside was added to inhibit the exchange translocase, thereby preventing rapid access of exogenous ATP to the matrix, net ATP uptake was inhibited; carboxyatractyloside had no effect on ADP uptake. It was concluded that the net uptake of adenine nucleotides from the extramitochondrial space occurs by a specific transport process distinct from the classic adenine nucleotide exchange translocase. The accumulation of adenine nucleotides may regulate matrix reactions which are allosterically affected by adenines or which require adenines as a substrate.