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.     

Failure of atractyloside to inhibit synaptosomal mitochondrial energy transduction

Studies on synaptosome mitochondrial respiration are complicated by “free” mitochondria. Veratridine stimulation of synaptosomal respiration was due to increased Na⁺ cycling at the synaptosome membrane associated with increased oxidative phosphorylation of intraterminal ADP and was inhibited by oligomycin, ouabain or Na⁺ free medium. Atractylate or carboxyatractyloside failed to block veratridine-stimulated respiration but inhibited exogenous-ADP-stimulated respiration. Protein synthesis in the synaptosome fraction was inhibited by oligomycin, valinomycin or 2,4-dinitrophenol but was unaffected by excess atractylate. No change in synaptosomal adenine nucleotide content was found in the presence of atractylate, although a significant decrease in the [ATP]/[ADP] was found with oligomycin, veratridine or valinomycin. These findings show that atractylate does not modify intraterminal mitochondrial energy transduction and indirectly suggest an impermeability of the synaptosome membrane to atractylate.     

Effect of Escherichia coli endotoxin on adenine nucleotide translocation in canine heart mitochondria

The in vitro effect of Escherichia coli endotoxin on the translocation of adenine nucleotides in dog heart mitochondria was studied. Mitochondrial adenine nucleotides were labeled with ¹⁴C by incubating mitochondrial preparations in the presence of [¹⁴C]ADP. The exchange reaction was initiated by addition of unlabeled ADP, proceeded for 5 to 60 s at 4 °C, and was terminated by addition of atractyloside. The results showed that preincubation of mitochondria with endotoxin (50 μg/mg protein) for 10 min at 23 °C decreased the exchange reaction by 21.2% (P < 0.05). The inhibitory effect of endotoxin was increased with increasing concentrations of endotoxin with an I₅₀ value of 45 μg/mg protein. The initial rate and the total extent of exchange were both affected. Double reciprocal plots showed that only the V but not the K_m for ADP was affected by endotoxin, indicating that the inhibition was noncompetitive in nature. The exchange of adenine nucleotide remained depressed by endotoxin in the presence of either oligomycin or antimycin A, indicating that the inhibitory effect of endotoxin was independent of the action of endotoxin on oxidative phosphorylation. The leakage of labeled adenine nucleotides from mitochondria at 23 °C was increased by 100% by endotoxin (100 μg/mg protein) in the absence of added unlabeled ADP, and this increase in the leakage could not be blocked by atractyloside. The endotoxin-induced changes in adenine nucleotide exchange and leakage were either partially or completely prevented by hydrocortisone, heparin, dibucaine, or EDTA. Since most of these agents have in common an effect on lipid metabolism, it is suggested that endotoxin-induced alterations in the exchange and leakage of adenine nucleotides in heart mitochondria are protected through a mechanism involving membrane lipid reorganization.     

Comparison of the effect of temperature on kidney cortex mitochondria from rabbit,dog, pig,and human: Arrhenius plots of ADP-stimulated respiration

The effect of temperature on the rate of ADP-stimulated respiration of mitochondria from dog, rabbit, pig, and human kidney cortex mitochondria was plotted according to the Arrhenius relationship. The temperature at which the plot demonstrated a break was at 15 °C for mitochondria from dog, pig, and human kidneys. The discontinuity occurred at 10 °C or less for mitochondria from rabbit kidneys. This difference suggests that mitochondria from rabbit kidneys undergo a lipid-phase transition at lower temperatures than for other species commonly used in experimental renal preservation. The implications of this difference suggest caution in using results obtained with rabbit kidneys for comparison to results obtained from hypothermic renal preservation of other species kidneys. Apparent fluidization of dog kidney mitochondrial membranes with adamantine abolished the discontinuity in the Arrhenius plot.     

Adenine nucleotide translocation in plant mitochondria

The rapid translocation of external ADP-[^14C]by corn mitochondria is inhibited by high concentrations of atractyloside with enhanced inhibition occurring in the presence of Mg²⁺. This translocation is also inhibited by AMP or ATP but CDP, GDP, IDP or UDP have little effect. Backward exchange of internal ADP-[¹⁴C] occurs in the presence of AMP, ADP or ATP but is not promoted by other nucleoside diphosphates. It is suggested that the adenine nucleotide (AdN) carrier is specific for ADP and ATP and that apparent translocation of AMP is a result of adenylate kinase activity. The translocated ADP can be separated into 3 components: (1) atractyloside-insensitive binding; (2) carrier-bound ADP saturated at ca 30 μM external ADP; and (3) exchanged ADP saturated as ca 5 μM external ADP. It is suggested that the adenine nucleotide carrier of plant mitochondria possesses similar properties to the classical carrier of vertebrate mitochondria.     

Citrulline synthesis: Regulation by alterations in the total mitochondrial adenine nucleotide content

The total adenine nucleotide content of rat liver mitochondria was varied in vitro over a wide range in order to investigate a possible relationship between net changes in the total matrix ATP + ADP + AMP content and the overall rate of citrulline synthesis. Isolated mitochondria were specifically depleted of matrix adenine nucleotides by incubating with inorganic pyrophosphate (G. K. Asimakis and J. R. Aprille, 1980, Arch. Biochem. Biophys.203, 307–316); alternatively, matrix adenine nucleotides were increased by incubating mitochondria with 1 mm ATP at 30 °C. No exogenous ATP or ADP was included in the subsequent incubations for the determination of citrulline synthesis. Rates varied from 0.1 to 1.6 μmol citrulline/mg protein/h as a linear function of total adenine nucleotide content in the range 2–15 nmol (ATP + ADP + AMP)/mg protein. Further increases in the matrix ATP + ADP + AMP content caused no further increase in citrulline synthesis rates. Changes in the total adenine nucleotide content were reflected in proportional changes in both the ATP and ADP content of the matrix. The $\text{ATP}\text{ADP}$ ratio did not change significantly. Therefore, the variations in citrulline synthesis were most simply explained as the effect of different concentrations of ATP on the activity of carbamoyl-phosphate synthetase. It was concluded that net changes in the total adenine nucleotide content can contribute to the control of citrulline synthesis. These findings are significant in the context of recent evidence which shows that the matrix adenine nucleotide pool size is under hormonal control.     

Regulation of the mitochondrial adenine nucleotide pool size

A mechanism by which normal adult rat liver mitochondria may regulate the matrix adenine nucleotide content was studied in vitro. If mitochondria were incubated with 1 mm ATP at 30 ° C in 225 mm sucrose, 2 mm K₂HPO₄, 5 mm MgCl₂, and 10 mm Tris-Cl (pH 7.4), the adenine nucleotide pool size increased at a rate of 0.44 ± 0.02 nmol/mg mitochondrial protein/min. The rate of adenine nucleotide accumulation under these conditions was concentration dependent and specific for ATP or ADP; AMP was not taken up. The rate of net ADP uptake was 50–75% slower than that for ATP. The K_m values for net uptake of ATP and ADP were 2.08 and 0.36 mm, respectively. Adenine nucleotide uptake was stoichiometrically dependent on Mg²⁺ and stimulated by inorganic phosphate. Net uptake was inhibited by n-ethylmaleimide, or mersalyl, but not by n-butylmalonate. Nigericin inhibited net uptake, but valinomycin did not. In the presence of uncouplers, net uptake was not only inhibited, but adenine nucleotide efflux was observed instead. Like uptake, uncoupler-induced efflux of adenine nucleotides was inhibited by mersalyl, indicating that a protein was required for net flux in either direction. Carboxyatractyloside, bongkrekic acid, or respiratory substrates reduced the rate of adenine nucleotide accumulation, however, this did not appear to be a direct inhibition of the transport process, but rather was probably related indirectly to an increase in the matrix $\text{ATP}\text{ADP}$ ratio. The collective properties of the transport mechanism(s) for adenine uptake and efflux were different from those which characterize any of the known transport systems. It is proposed that uptake and efflux operate to regulate the total matrix adenine nucleotide pool size: a constant pool size is maintained if the rates of uptake and efflux are equal. Transient alterations in the relative rates of uptake and efflux may occur in response to hormones or other metabolic signals, to bring about net changes in the pool size.     

Regulation of mitochondrial energy production in cardiac cells of rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

In skinned rat cardiac fibres, mitochondrial affinity for endogenous ADP generated by creatine kinase and Ca²⁺-activated ATPases is higher than for exogenous ADP added to the surrounding medium, suggesting that mitochondria are functionally coupled to creatine kinase and ATPases. Such a coupling may be weaker or absent in ectothermic vertebrate cardiac cells, because they typically have less elaborate intracellular membrane structures, higher glycolytic capacity and lower working temperature. Therefore, we examined skinned cardiac fibres from rainbow trout at 10 °C. The apparent mitochondrial affinity for endogenous ADP was obtained by stimulation with ATP and recording of the release of ADP into the surrounding medium. The apparent affinity for endogenous ADP was much higher than for exogenous ADP suggesting a functional coupling between mitochondria and ATPases. The apparent affinity for exogenous ADP and ATP was increased by creatine or an increase in Ca²⁺-activity, which should increase intrafibrillar turnover of ATP to ADP. In conclusion, ADP seems to be channelled from creatine kinase and ATPases to mitochondria without being released to the surrounding medium. Thus, despite difference in structure, temperature and metabolic capacity, trout myocardium resembles that of rat with regard to the regulation of mitochondrial respiration.Abbreviations ACR acceptor control ratio - ANT adenine nucleotide translocase - K_{M ADP} apparent mitochondrial affinity for ADP - K_{M ATP} apparent mitochondrial affinity for ATP - LDH lactate dehydrogenase - V_ADP ADP-stimulated respiration rate - V_{ADP max} maximal ADP-stimulated respiration rate - V_ATP ATP-stimulated respiration rate - V_{ATP max} maximal ATP-stimulated respiration rate - V₀ basal respiration rate in the absence of ADPCommunicated by G. Heldmaier     

Control of adenine nucleotide metabolism in hepatic mitochondria from rats with ethanol-induced fatty liver

Male rats developed fatty liver after being fed on an ethanol-containing diet for 31 days. Liver mitochondria from these animals catalysed ATP synthesis at a slower rate when compared with mitochondria from pair-fed control rats (control mitochondria), and demonstrated lowered respiratory control with succinate as substrate, owing to a decrease in the State-3 respiratory rate. Respiration in the presence of uncoupler was comparable in mitochondria from both groups of rats. Translocation of both ATP and ADP was decreased in mitochondria from ethanol-fed rats, with ADP uptake being lowered more dramatically by ethanol feeding. Parameters influencing adenine nucleotide translocation were investigated in mitochondria from ethanol-fed rats. Experiments performed suggested that lowered adenine nucleotide translocation in these mitochondria is not the result of inhibition of the translocase by either long-chain acyl-CoA derivatives or unesterified fatty acids. Analysis of endogenous adenine nucleotides in these mitochondria revealed lowered ATP concentrations, but no decrease in total adenine nucleotides. In experiments where the endogenous ATP in these mitochondria was shifted to higher concentrations by incubation with oxidizable substrates or defatted bovine serum albumin, the rate of ADP translocation was increased, with a linear correlation being observed between endogenous ATP concentrations and the rate of ADP translocation. The depressed ATP concentration in mitochondria from ethanol-fed rats suggests that the ATP synthetase complex is replenishing endogenous ATP at a slower rate. The lowered ATPase activity of the ATP synthetase observed in submitochondrial particles from ethanol-fed animals suggests a decrease in the function of the synthetase complex. A decrease in the rate of ATP synthesis in mitochondria from ethanol-fed rats is sufficient to explain the decreased ADP translocation and State-3 respiration.     

Thryoid control over biomembranes. Rat liver mitochondrial inner membranes.

The effects of hypothyroidism and one injection of l-thyroxine on oxidative phosphorylation and the composition of proteins and phospholipids were examined in vesicles prepared from rat liver mitochondria by digitonin extractions. At 30 °C, the rates of ADP phosphorylation in sites I and II were below normal, and Mg²⁺-ATPase activity was greater than normal in vesicles from hypothyroid rats. At temperatures below 20 °C and above 30 °C, the Mg²⁺-ATPase was not accelerated above normal rates, a feature of temperature dependence shared by ADP phosphorylation (Chen, Y.-D. I., and Hoch, F. L., 1976, Arch. Biochem. Biophys.172, 741–744). Respiration at 30 °C was undiminished in hypothyroid vesicles, as were the flavin and cytochrome contents, and thyroxine administration corrected the phosphorylation rate at 30 °C in 3 days without changing either respiration or electron-carrier contents. The 30 °C phosphorylation defect comprised a decreased V and K_m for ADP and a decrease in the number of phosphorylating sites (measured with oligomycin) that accounted for most of the decreased phosphorylation rates, either dependent on or independent of the adenine nucleotide carrier. Vesicles from hypothyroid rats were not detectably depleted in major protein subunits, but were abnormal in phospholipid fatty acid contents. Thyroxine injection corrected the low unsaturation index of the fatty acids and the membrane contents of linoleic acid and its fatty acyl metabolites. Hypothyroidism appears to affect oxidative phosphorylation through the altered inner membrane lipid environment, which implies that previously reported direct, reversible effects of thyroxine may mimic repletion of the membranes with unsaturated fatty acyl groups.     

Effect of acetate on transport of organic acid (fluorescein) in renal proximal tubules of frog

The effect of acetate on active fluorescein transport in intact proximal tubules of surviving frog kidney was studied. When the kidneys were incubated in a 120 mM Na⁺ medium, 10 mM acetate stimulated fluorescein uptake in the tubules. The stimulation was more pronounced if the kidneys had been previously preincubated for 3 h in the substrate-free solution. Lowering of the Na⁺ concentration in the bathing medium to 10 mM resulted in the disappearance of the acetate effect. Preincubation of the kidneys with acetate at 2–4°C gave rise to stimulation of the fluorescein transport only in the 120 mM Na⁺ acetate-free medium. The acetate effect on the fluorescein uptake was partially prevented by ouabain. The stimulation of the uptake by acetate in the 120 mM Na⁺ medium correlated with an increase in the extent of reduction of pyridine nucleotides in the tubules. The pyridine nucleotides were reduced more markedly after incubation of the kidneys in the 10 mM Na⁺ medium, when acetate had no effect on the fluorescein transport. In both the 120 mM and the 10 mM Na⁺ media, the cold preincubation of the kidneys with 2.5 mM ADP or 2.5 mM ATP resulted in only slight stimulation of the fluorescein uptake. But in both media the uptake was significantly enhanced after cold preincubation of the kidneys with 2 mM NADH. After the cold precincubation with ADP, stimulation of the fluorescein transport by acetate was observed in the case of the 10 mM Na⁺ medium also. The absence of any stimulatory effect of acetate on the organic acid transport in the 10 mM Na⁺ medium is explained as the result of the transformation of mitochondria in the tubular cells into the inactive state 4 due to a decrease in the intracellular ADP level. Reducing equivalents are supposed to take part in energization and/or regulation of transport processes in plasma membranes of the renal proximal tubules.     

Approaches to the measurement of intracellular adenine and the discrimination between adenine transport and metabolism in L1210 leukemia cells

Incubation of L1210 leukemia cells with 10 μM [³H]adenine in the absence of energy substrate results in a very rapid accumulation of ³H within the cells. By 20 s intracellular adenine is near steady-state; beyond this the rate of accumulation of intracellular ³H reflects nucleotide synthesis, predominantly the rate of ATP accumulation within the cell as determined by liquid chromatography. Adenine incorporation into the nucleotides proceeds via adenine-phosphoribosyl transferase, which is rate-limiting to AMP formation and subsequently the formation of ADP and ATP. Acceleration of this pathway by the addition of glucose and phosphate decreases the intracellular adenine level far below equilibrium as metabolism is increased relative to transport. Assessment of methodology to evaluate intracellular adenine and its metabolites indicates that (i) a 4°C wash removes the major portion of intracellular adenine and (ii) at 4°C, transport of adenine remains rapid and while nucleotide synthesis is decreased, ATP still accumulates within the cell. Hence, measurement of cellular uptake of radioactive label at 4°C after cells are washed free of adenine cannot be used as a measurement of adenine surface binding since this radioactive label represents, at least in part, phosphorylated derivatives of adenine within the cell. Unlabeled adenine and structurally related compounds were found to inhibit [³H]adenine net uptake under conditions where metabolism of adenine was reduced, suggesting that base transport is mediated by a facilitated diffusion mechanism. This is consistent with other studies from this laboratory that demonstrate exchange diffusion between adenine and other bases.     

Increased levels of adenine nucleotides modify the interaction between starch synthesis and respiration when adenine is supplied to discs from growing potato tubers

To investigate the importance of the overall size of the total adenine nucleotide pool for the regulation of primary metabolism in growing potato tubers, freshly cut discs were provided with zero or 2 mM adenine in the presence of 1 or 100 mM [U-¹⁴C]glucose or 100 mM [U-¹⁴C]sucrose in the presence and absence of 20 mM orthophosphate (Pi). Adenine led to a 150–250% increase of the total adenine nucleotide pool, which included an increase of ADP, a larger increase of ATP and an increase of the ATP:ADP ratio. There was a 50–100% increase of ADP-glucose (ADPGlc), and starch synthesis was stimulated. Respiratory oxygen uptake was stimulated, and the levels of glycerate-3-phosphate, phosphoenolpyruvate and α-ketoglutarate decreased. The response to adenine was not modified by Pi. It is proposed that increased ATP stimulates ADPGlc pyrophosphorylase, leading to a higher rate of starch synthesis. The impact on starch synthesis is constrained, however, because increased ADP can lead to a stimulation of respiration and decline of glycerate-3-phosphate, which will inhibit ADPGlc pyrophosphorylase. The quantitative impact depends on the conditions. In the presence of 1 mM glucose, the levels of phosphorylated intermediates and the rate of starch synthesis were low. Adenine led to a relatively large stimulation of respiration, but only a small stimulation of starch synthesis. In the presence of 100 mM glucose, discs contained high levels of phosphorylated intermediates, low ATP:ADP ratios (<3) and low rates of starch synthesis (<20% of the metabolised glucose). Adenine led to marked increase of ATP and 2- to 4-fold stimulation of starch synthesis. Discs incubated with 100 mM sucrose already had high ATP:ADP ratios (>8) and high rates of starch synthesis (>50% of the metabolised sucrose). Adenine led to a further increase, but the stimulation was less marked than in high glucose. These results have implications for the function of nucleotide cofactors in segregating sucrose mobilisation and respiration, and the need for energy conservation during sugar-starch conversions. Received: 9 February 2000 / Accepted: 9 June 2000     

The effect of atractylate and oligomycin on the behaviour of mitochondria towards adenine nucleotides

1. Investigation of a number of reactions involving both internal and externally added adenine nucleotides of isolated liver mitochondria has revealed that atractylate and oligomycin differ markedly in the site of their inhibitory action. 2. Both atractylate and oligomycin inhibited the respiratory-chain-level phosphorylation of added ADP. Neither compound inhibited the substrate-level phosphorylation of internal (endogenous) ADP or the respiration-dependent accumulation of bivalent metal ions (Ca²⁺, Sr²⁺ or Mn²⁺). 3. Atractylate, but not oligomycin, inhibited the substrate-level phosphorylation of externally added ADP, the ATP- and carnitine-dependent reduction of nicotinamide nucleotide by palmitate and the ATP-induced activation of succinate oxidation. 4. Oligomycin, but not atractylate, inhibited the respiratory-chain-linked phosphorylation of internal ADP, and the dephosphorylation of internal ATP that occurred on the addition of antimycin. 5. The enhancement of arsenate-stimulated respiration by ADP was prevented by atractylate added either before or after the ADP. Oligomycin abolished both the arsenate and ADP stimulation. 6. It is suggested that atractylate prevents the passage of adenine nucleotides across the mitochondrial membrane, whereas oligomycin interferes with the formation of a `high-energy' phosphorylated intermediate.     

In vitro alteration of the size of the liver mitochondrial adenine nucleotide pool: Correlation with respiratory functions

Mitochondrial respiration was studied as a function of the total adenine nucleotide content of rat liver mitochondria. The adenine nucleotide content was varied by treating isolated mitochondria with pyrophosphate or by incubating pyrophosphate-treated mitochondria with ATP. Mitochondria with at least 4 nmol adenine nucleotides/mg protein maintained at least 80% of the State 3 activity of control mitochondria, which had approximately 10 nmol/mg protein. However, State 3 decreased rapidly once the adenine nucleotide content fell below 4 nmol/mg protein. Between 2 and 4 nmol adenine nucleotides/mg, State 3 was not limited by the maximal capacity of electron flow as measured by the uncoupled respiration. However, at very low adenine nucleotide levels (<2 nmol/mg), the uncoupled rates of respiration were markedly depressed. State 4 was not affected by changes in the mitochondrial adenine nucleotide content. Adenine translocase activity varied in almost direct correlation with changes in the adenine nucleotide content. Therefore, adenine translocase activity was more sensitive than State 3 to changes in total adenine nucleotides over the range of 4 to 10 nmol/mg protein. The results suggest that (i) State 3 is dependent on the level of intramitochondrial adenine nucleotides, particularly in the range below 4 nmol/mg protein, (ii) adenine translocase activity is not rate-limiting for oxidative phosphorylation in mitochondria with the normal complement of adenine nucleotides, however, at low adenine nucleotide levels, depressed State 3 rates may be explained in part by the low rate of ADP translocation, and (iii) a mechanism of net ATP uptake exists in mitochondria with low internal adenine nucleotides.     

Interdependence of mitochondrial ATP production and extramitochondrial ATP utilization in intact spermatozoa

The dependence of both respiration and total activity of ATP-consuming reactions on the cellular adenine nucleotide pattern was investigated in intact bovine spermatozoa. ATP consumption was manipulated by inhibition with vanadate and activation with caffeine, leading to a decrease or increase in the rate of respiration up to 70% or 20%, respectively. Oligomycin blocked the respiration to the same extent as did vanadate, suggesting that the total extramitochondrial ATP-consuming activity is vanadate-sensitive. The major part of ATP utilization must be linked to dynein ATPase, since inhibition of (Na⁺, K⁺) ATPase by ouabain showed only a small effect on respiration (−17%). Being a potent inhibitor of dynein ATPase, vanadate drastically reduced the amount of motile cells, whereas caffeine tended to increase the intensity of motion. The effects of vanadate or caffeine on respiration were paralleled by changes in cellular ATP, reflecting the response of mitochondrial respiration on the cellular ATP/ADP ratio. Respiration was found to depend on changes in the ATP/ADP ratio in the range from about 3 (+ caffeine) to 9 (+ vanadate). The range of response of ATP consumption to the ATP/ADP ratio was determined by varying the mitochondrial ATP production via the concentration of lactate which was used as substrate. The measured effects on both respiratory rate and ATP/ADP ratio suggested that ATP consumption was markedly dependent on ATP/ADP ratios below 5. It is concluded that lactate concentrations above 1 mM sufficiently supply bovine spermatozoa with substrate and the energy turnover is mainly limited by the activity of dynein ATPase rather than by the capacity of mitochondrial oxidative phosphorylation.     

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.     

The stimulation of the mitochondrial respiration by citrulline synthesis.

1. The influence of ammonia and ornithine on the oxygen uptake and the formation of citrulline was investigated with isolated rat liver mitochondria. The experiments were performed in a cytosol-like saline medium at 38 degrees C. 2. Under these conditions an increase of the respiration rate by ammonia and ornithine was observed, but a small response to external ADP, only. The missing stimulation by ADP was due to a partial inhibition of the respiratory chain by traces of zinc (approximately 1 microM) present in the medium. This inhibition was only detected at low concentrations of mitochondria. 3. For activation of respiration by ammonia plus ornithine two different processes were responsible: (i) chelation of the inhibiting zinc by ornithine, which could be prevented by EDTA; (ii) ADP production in the matrix space during formation of carbamoyl phosphate, which could be prevented by oligomycin but not by carboxyatractyloside. 4. This stimulus of the carbamoyl phosphate formation and of the equivalent citrulline synthesis on the mitochondrial respiration ran to 12% of that increase caused by phosphorylation of external ADP. The maximum rate of citrulline formation was limited by the activity of carbamoyl phosphate synthetase. 5. Added ADP suppresses the production of citrulline probably by the exchange of extramitochondrial ADP versus intramitochondrial ATP. The data suggest a common adenine nucleotide pool delivering ATP to the adenine nucleotide translocase as well as to the carbamoyl phosphate synthetase.     

The stimulation of diffusion of adenine nucleotides across bimolecular lipid membranes by divalent metal ions

The diffusion rates of [³H] adenine nucleotides across bimolecular lipid membranes were shown to be directly related to their organic/water partition coefficients, the order being ATP > ADP > AMP. Nucleotide diffusion was stimulated by divalent metal ions with the order of stimulation being Cu⁺² ? Zn⁺² > Mg⁺². The ability of a divalent metal ion to stimulate diffusion appears to be related to its ability to bind to the N-7 of the adenine ring. The divalent metal ions increase adenine nucleotide diffusion both by complexing with the nucleotide thus decreasing the charge on the nucleotide and by increasing the permeability of the lipid bilayer.     

The interaction of Ga2+ with mitochondria from human myometrium.

Mitochondria prepared from human myometrium contain large amounts of endogenous Ca²⁺ (up to 200 nmol/mg of protein) even if isolated in media containing ethylene glycol-bis(β-aminoethylether)-N,N′-tetraacetic acid. The endogenous Ca⁺², however, is not irreversibly sequestered, since it can be rapidly and quantitatively discharged by uncouplers. Human myometrial mitochondria are capable of efficient energy-linked Ca²⁺ transport. In the absence of phosphate, the amount of Ca²⁺ accumulated is reduced to insignificant levels. Mg²⁺ has a strong inhibitory effect, which has been exploited to develop an inhibitor-stop method which has permitted the determination of the affinity of myometrial mitochondria for Ca²⁺ (K_m, ~5 μM) and of the maximal velocity of uptake (0.55 nmol/mg of protein/s). The respiration of human myometrial mitochondria is stimulated by Ca²⁺, with respiratory control indexes of the order of 4–5. In contrast, ADP induces an insignificant stimulation, or no stimulation at all. The response of respiration to ADP is somewhat improved if mitochondria are preincubated under conditions which decrease their endogenous Ca²⁺ content. The adenine nucleotide exchange in human myometrial mitochondria is deficient with respect to liver mitochondria.