Metabolic control over the oxygen consumption flux in intact skeletal muscle: in silico studies

It has been postulated previously that a direct activation of all oxidative phosphorylation complexes in parallel with the activation of ATP usage and substrate dehydrogenation (the so-called each-step activation) is the main mechanism responsible for adjusting the rate of ATP production by mitochondria to the current energy demand during rest-to-work transition in intact skeletal muscle in vivo. The present in silico study, using a computer model of oxidative phosphorylation developed previously, analyzes the impact of the each-step-activation mechanism on the distribution of control (defined within Metabolic Control Analysis) over the oxygen consumption flux among the components of the bioenergetic system in intact oxidative skeletal muscle at different energy demands. It is demonstrated that in the absence of each-step activation, the oxidative phosphorylation complexes take over from ATP usage most of the control over the respiration rate and oxidative ATP production at higher (but still physiological) energy demands. This leads to a saturation of oxidative phosphorylation, impossibility of a further acceleration of oxidative ATP synthesis, and dramatic drop in the phosphorylation potential. On the other hand, the each-step-activation mechanism allows maintenance of a high degree of the control exerted by ATP usage over the ATP turnover and oxygen consumption flux even at high energy demands and thus enables a potentially very large increase in ATP turnover. It is also shown that low oxygen concentration shifts the metabolic control from ATP usage to cytochrome oxidase and thus limits the oxidative ATP production. respiration rate; parallel activation; oxidative phosphorylation; metabolic control analysis; flux control coefficient; muscle contraction     

Substrate oxidation and ATP supply in AS-30D hepatoma cells

The oxidation of several metabolites in AS-30D tumor cells was determined. Glucose and glycogen consumption and lactic acid production showed high rates, indicating a high glycolytic activity. The utilization of ketone bodies, oxidation of endogenous glutamate, and oxidative phosphorylation were also very active: tumor cells showed a high respiration rate (100 ng atoms oxygen (min x 10(7) cells)(-1)), which was 90% oligomycin-sensitive. AS-30D tumor cells underwent significant intracellular volume changes, which preserved high concentrations of several metabolites. A high O(2) concentration, but a low glucose concentration were found in the cell-free ascites liquid. Glutamine was the oxidizable substrate found at the highest concentration in the ascites liquid. We estimated that cellular ATP was mainly provided by oxidative phosphorylation. These data indicated that AS-30D hepatoma cells had a predominantly oxidative and not a glycolytic type of metabolism. The NADH-ubiquinol oxido reductase and the enzyme block for ATP utilization were the sites that exerted most of the control of oxidative phosphorylation (flux control coefficient = 0.3-0.42).     

Influence of substrate activation (hydrolysis of ATP by first steps of glycolysis and beta-oxidation) on the effect of enzyme deficiencies,inhibitors, substrate shortage and energy demand on oxidative phosphorylation

In intact tissues respiratory substrates (glucose, fatty acids) must be activated with the use of ATP before they may be oxidised and used for energy (ATP) production. This activation by product constitutes an example of a typical positive feedback. In the present paper, the influence of substrate activation on the effect of inborn enzyme deficiencies, inhibitors, lowered oxygen tension, respiratory fuel shortage and increased energy demand on respiration and ATP synthesis is studied with the aid of the dynamic computer model of oxidative phosphorylation in isolated mitochondria developed previously. Computer simulations demonstrate that, in the case where oxidative phosphorylation in the whole organism is partially inhibited, the necessity of substrate activation can have significant impact on the relationship between the activity of (particular steps of) oxidative phosphorylation (or the value of energy demand) and the respiration rate. Depending on the sensitivity of ATP usage to ATP concentration, substrate activation may either slightly enhance the effect of the decrease in the oxidative phosphorylation activity (increase in energy demand) or may lead to a non-stability and sudden collapse of the respiration rate and phosphorylation potential below (above) a certain threshold value of oxidative phosphorylation activity (energy demand). This theoretical finding suggests a possible causal relationship between the affinity of ATP usage to [ATP] and the tissue specificity of mitochondrial diseases.     

Control of respiration and metabolism in growing Klebsiella aerogenes. The role of adenine nucleotides

1. A rapid-sampling technique was used to obtain perchloric acid extracts of cells growing in a chemostat culture, so that meaningful values for ATP content could be obtained in spite of the fact that the turnover time for the total ATP content was about 1sec. 2. For steady-state growth, it was found that, in a glucose-limited chemostat culture, the ATP/ADP concentration ratio was approximately constant with changes in dissolved-oxygen tensions above the critical value, but fell when the culture was grown under oxygen-limited conditions and was at a minimum in anaerobically grown cultures. The steady-state ATP content was lower in cells growing under nitrogen-limited conditions with glucose in excess than in glucose-limited cells. The steady-state ATP content was independent of growth rate at growth rates over 0.1hr.(-1). 3. When the respiration rate of the cells was stimulated by lowering the oxygen tension the ATP content did not increase, indicating either an increased turnover rate of ATP or a fall in the P/O ratio. The sudden addition of extra glucose or succinate to a glucose-limited culture increased the respiration rate of the cells, but the ATP content quickly returned to the steady-state value after initial perturbations. This control over ATP content is explained in terms of regulation by adenine nucleotides of the catabolism and anabolism of glucose. An exception to this control over ATP content was found when the respiration rate was stimulated by addition of an antifoam.     

Rate control of phosphorylation-coupled respiration by rat liver mitochondria

Liver mitochondria provided with an oxidizable substrate, ATP, oxygen, and an ADP-generating system (soluble F₁-ATPase) were used to reevaluate the rate-controlling step(s) intrinsic to all of the processes of mitochondrial oxidative phosphorylation. The quantity termed “control strength” (C), previously defined as the fractional change in flux through a (system) induced by a fractional change in the concentration of an individual enzyme in the system, has been used to evaluate rate-influencing steps in this overall process by carefully defining the dimensions of the “system” under analysis. If the system is defined by a suspension of mitochondria provided with substrates, plus an extrinsic ADP-generating process (ATPase), the value of C of the latter for the overall process of phosphorylation-linked respiration is near 1.0 until the capacity of the mitochondria to phosphorylate ADP is approached, after which C for the soluble ATPase becomes zero as the maximum capacity for phosphorylation is attained. Carboxyatractyloside was found only marginally to inhibit respiration stimulated by ATPase, even when a large percentage of adenine nucleotide translocase molecules were immobilized. The relative lack of effect of carboxyatractyloside on phosphorylating respiration is explained by the readjustment of the concentration of one of the substrates (ADP) and an inhibitor (ATP), which results from inhibition of adenine nucleotide translocase. The residual blunted inhibition of respiration is explained by product inhibition of the ADP-regenerating ATPase, and not necessarily to any intrinsically mitochondrial intermediate process. The system being evaluated can be redefined to include only the processes intrinsic to mitochondria. This can be achieved by providing exactly comparable substrate concentrations to the mitochondria under comparable incubation conditions. Under these conditions, the adenine nucleotide translocase is the principal, if not the only, rate-controlling step in the overall process of oxidative phosphorylation until a new rate-limitation is attained (ATP synthesis). These data are consistent with the conclusion that, at intermediate rates of phosphorylation-coupled respiration, the extramitochondrial $\text{ATP}\text{ADP}$ ratio regulates this process through its kinetic effects on the catalytic properties of the adenine nucleotide translocase.     

Control of mitochondrial respiration in the heart in vivo

The role of the hydrolysis products of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and inorganic phosphate (Pi), in the control of myocardial respiration was evaluated in vivo using ³¹P NMR. These studies were conducted to evaluate whether increases in the ATP hydrolysis products can be detected through the cardiac cycle or during increases in cardiac work. ³¹P NMR data acquisitions gated to various portions of the cardiac cycle (50 msec time resolution) revealed that cytosolic ATP, ADP and Pi did not change over the course of the cardiac cycle. These metabolites were also monitored during steady-state increases in cardiac work in conjunction with measurements of coronary blood flow and oxygen consumption. No changes were observed during 2 to 3 fold increases in myocardial oxygen consumption induced by various methods. These results demonstrate that the cytosolic ATP, ADP, and Pi concentrations remain relatively constant throughout the cardiac cycle and during physiological increases in cardiac work and oxygen consumption. Furthermore, it is shown that ADP and Pi cannot be solely responsible for the regulation of cardiac respiration in vivo based on the in vitro Km values of these compounds for oxidative phosphorylation. It is concluded that other mechanisms, working in concert with the simple kinetic feedback of ATP hydrolysis products, must be present in the cytosol to provide control of myocardial respiration in vivo.     

A model of oxidative phosphorylation in mammalian skeletal muscle

A dynamic computer model of oxidative phosphorylation in oxidative mammalian skeletal muscle was developed. The previously published model of oxidative phosphorylation in isolated skeletal muscle mitochondria was extended by incorporation of the creatine kinase system (creatine kinase plus phosphocreatine/creatine pair), cytosolic proton production/consumption system (proton production/consumption by the creatine kinase-catalysed reaction, efflux/influx of protons), physiological size of the adenine nucleotide pool and some additional minor changes. Theoretical studies performed by means of the extended model demonstrated that the CK system, which allows for large changes in P(i) in relation to isolated mitochondria system, has no significant influence on the kinetic properties of oxidative phosphorylation, as inorganic phosphate only slightly modifies the relationship between the respiration rate and [ADP]. Computer simulations also suggested that the second-order dependence of oxidative phosphorylation on [ADP] proposed in the literature refers only to the ATP synthesis flux, but not to the oxygen consumption flux (the difference between these two fluxes being due to the proton leak). Next, time courses of changes in fluxes and metabolite concentrations during transition between different steady-states were simulated. The model suggests, in accordance with previous theoretical predictions, that activation of oxidative phosphorylation by an increase in [ADP] can (roughly) explain the behaviour of the system only at low work intensities, while at higher work intensities parallel activation of different steps of oxidative phosphorylation is involved.     

Inhibition of adenine nucleotide transport in rat liver mitochondria by long-chain acyl-coenzyme A beta-oxidation intermediates

Long-chain acyl-coenzyme A esters (LCAC), which may accumulate under different pathological conditions and especially in patients with a mitochondrial fatty acid beta-oxidation defect, have long been known as potent inhibitors of several enzymes in multiple metabolic pathways, particularly the oxidative phosphorylation system (OXPHOS). To shed more light on the inhibitory mechanisms of acyl-CoA esters upon energy metabolism, the effect of palmitoyl-CoA and its beta-oxidation intermediates on OXPHOS was studied. We have recently shown that, using rat liver mitochondria, LCAC inhibit l-glutamate driven oxygen consumption in the presence of ADP whereas no effect is found when an uncoupler is used to stimulate respiration maximally. A similar inhibitory effect of these compounds is now reported upon the distribution of ATP for intra- and extra-mitochondrial utilization. Taken together these data strongly suggest that the inhibition of ADP-induced respiration with l-glutamate as substrate by LCAC is primarily due to inhibition of the mitochondrial ADP/ATP carrier.     

Rate-controlling steps of oxidative phosphorylation in rat liver mitochondria. A synoptic approach of model and experiment

The contribution of different steps to the control of oxidative phosphorylation in isolated rat liver mitochondria was investigated by a combination of experiments and computer simulations. The parameters of the mathematical model of phosphorylating mitochondria were derived from experimental data. The model correctly describes the competition between ATP utilization inside and outside mitochondria for the ATP generated in mitochondria. On the basis of the good agreement between experiments and simulations, the contribution of different steps to the control of respiration was estimated by computing their control strengths, i.e., the influence of their activities on the rate of respiration. The rate-controlling influences vary depending on the load of oxidative phosphorylation. The predominant steps are: in the fully active state (State 3) — the hydrogen supply to the respiratory chain; in the resting state (State 4) — the proton leak of the mitochondrial inner membrane; in states of non-maximum ATP export — the adenine nucleotide translocator. Titrations of respiration with phenylsuccinate, antimycin, oligomycin and carboxyatractyloside completely support these conclusions.     

Top-down control analysis of the effect of temperature on ectotherm oxidative phosphorylation

Top-down control and elasticity analysis was conducted on mitochondria isolated from the midgut of the tobacco hornworm (Manduca sexta) to assess how temperature affects oxidative phosphorylation in a eurythermic ectotherm. Oxygen consumption and protonmotive force (measured as membrane potential in the presence of nigericin) were monitored at 15, 25, and 35 degrees C. State 4 respiration displayed a Q(10) of 2.4-2.7 when measured over two temperature ranges (15-25 degrees C and 25-35 degrees C). In state 3, the Q(10)s for respiration were 2.0 and 1.7 for the lower and higher temperature ranges, respectively. The kinetic responses (oxygen consumption) of the substrate oxidation system, proton leak, and phosphorylation system increased as temperature rose, although the proton leak and substrate oxidation system showed the greatest thermal sensitivity. Whereas there were temperature-induced changes in the activities of the oxidative phosphorylation subsystems, there was no change in the state 4 membrane potential and little change in the state 3 membrane potential. Top-down control analysis revealed that control over respiration did not change with temperature. In state 4, control of respiration was shared nearly equally by the proton leak and the substrate oxidation system, whereas in state 3 the substrate oxidation system exerted over 90% of the control over respiration. The proton leak and phosphorylation system account for <10% of the temperature-induced change in the state 3 respiration rate. Therefore, when the temperature is changed, the state 3 respiration rate is altered primarily because of temperature's effect on the substrate oxidation system.     

Direct action of T3 on phosphorylation potential in the sheep heart in vivo

Thyroid acting through ligand binding to nuclear receptors modifies myocardial respiratory kinetics and oxidative phosphorylation in the heart. Direct nongenomic action of thyroid hormone on high-energy phosphate concentrations and respiratory kinetics has never been proven in vivo but might be responsible for observed changes in oxygen utilization efficiency immediately after triiodothyronine (T3) administration. We tested the hypothesis that T3 directly and rapidly modifies myocardial high-energy phosphate concentrations and phosphorylation potential in vivo. Anesthetized sheep (age 28-40 days) thyroidectomized shortly after birth (Thy) and euthyroid age-matched controls (Con) underwent median sternotomy and received T3 infusion (0.8 microg/kg), followed by epinephrine infusion to increase myocardial oxygen consumption (MVo2). 31P magnetic resonance spectra were monitored via a surface coil over the left ventricle. T3 increased phosphocreatine (PCr)/ATP and decreased ADP in Thy animals without causing a change in MVo2. T3 produced no changes in high-energy phosphates in Con animals. T3 did not modify the PCr/ATP or ADP response to epinephrine and elevation in MVo2 in either group. Cardiac mitochondria isolated from Thy and Con animals showed no change in respiratory rate or ADP/ATP exchange efficiency after T3 incubation. T3 infusion in a hypothyroid state decreases ADP concentration, thereby altering the equilibrium between phosphorylation potential and myocardial respiratory rate. These T3-induced effects are not due to changes in ADP/ATP exchange efficiency through action at the adenine nucleotide translocator but may be due to T3 mediation of substrate utilization, confirmed in other models.     

Theoretical studies on the regulation of oxidative phosphorylation in intact tissues

The theoretical studies on the regulation of oxidative phosphorylation that were performed with the aid of kinetic models of this process are overviewed. A definition of the regulation of the flux through a metabolic pathway is proposed and opposed to the control exerted by particular enzymes over this flux. Different kinetic models of oxidative phosphorylation proposed in the literature are presented, of which only the model proposed by myself and co-workers was extensively used in theoretical studies on the regulation and compensation in the oxidative phosphorylation system. These theoretical studies have led to the following conclusions: (1) in isolated mitochondria, an increase in the activity of an artificial ATP-using system stimulates mitochondria mainly via changes in [ADP], while changes in [ATP] and [P(i)] play only a minor role; (2) in non-excitable tissues (e.g. liver), hormones (acting via some cytosolic factor(s)) activate directly both ATP usage and at least some enzymes of the ATP-producing block; (3) in excitable tissues (e.g. skeletal muscle), neural signals stimulate (via some cytosolic factor(s)) in parallel all the steps of oxidative phosphorylation together with ATP usage and substrate dehydrogenation; (4) the decrease in the flux through cytochrome oxidase caused by a decrease in oxygen concentration is, at least partially, compensated by a decrease in Delta p and increase in the reduction level of cytochrome c. A theoretical prediction is formulated that there should exist and be observable a universal cytosolic factor/regulatory mechanism which directly activates (at least in excitable tissues) all complexes of oxidative phosphorylation during an increased energy demand.     

Simulation of state 4 --> state 3 transition in isolated mitochondria

The mathematical dynamic model of oxidative phosphorylation developed previously and in the accompanying paper was modified to involve isolated mitochondria conditions; it was also used to simulate state 4 --> state 3 transition in rat liver mitochondria incubated with succinate as respiratory substrate and glucose-hexokinase as an ADP-regenerating system. Changes in the respiration rate, protonmotive force and reduction level of ubiquinone and cytochrome c as well as the internal (i) and external (e) ATP/ADP ratio between state 4 and state 3 were calculated and compared with the experimental data. Flux control coefficients with respect to oxygen consumption flux for different reactions and processes of oxidative phosphorylation were simulated for different values of the respiration rate (state 4, state 3 and intermediate states). Flux control coefficients for the oxidation, phosphorylation and proton leak subsystems with respect to the oxidation, phosphorylation and proton leak fluxes for different values of the respiration rate were also calculated. These theoretical data were compared with the experimental results obtained in the frame of metabolic control analysis and the 'top-down' approach to this analysis. A good agreement was obtained. Simulated time courses of the respiration rate, the protonmotive force (Deltap) and other parameters after addition of a small amount of ADP to mitochondria in state 4 mimicked at least semiquantitatively the experimentally measured time courses of these parameters. It was concluded, therefore, that in the present stage, the model is able to reflect different properties of the oxidative phosphorylation system in a broad range of conditions fairly well, allows deeper insight into the mechanisms responsible for control and regulation of this process, and can be used for simulation of new experiments, thus inspiring experimental verification of the theoretical predictions.     

Relation between myocardial substrate utilization, oxygen consumption and regional oxygen balance in the dog heart in vivo

The interaction between myocardial function, oxygen consumption and energy production was examined in the left ventricular myocardium during various physiological conditions. Myocardial function was measured by both LV dP/dTmax and by local contractile tension. Coronary blood flow was measured from the coronary sinus; regional coronary blood supply was recorded using a thermistor placed on the epicardial surface. Intracellular oxygen balance was estimated using NADH fluorescence. Myocardial oxygen consumption and utilization of glucose, pyruvate, lactate and free fatty acids were calculated from their concentrations in the arterial and coronary sinus blood. The effects of tachycardia at 180 and 240 bpm, noradrenaline infusion (25 micrograms kg-1 min-1), and increased coronary blood flow caused by hypopneic respiration were examined. During pacing, contractile force, coronary flow and NADH fluorescence increased. At 240 bpm, the lactate/pyruvate ratio increased from 5.98 +/- 0.92 to 8.76 +/- 1.41 and NADH fluorescence increased from 50 to 71.7 +/- 3.73 (as compared to control), indicating impairment of myocardial oxygenation. Hypopneic respiration produced a marked elevation of coronary blood flow. Both noradrenaline infusion and hypopnea produced a decrease in both NADH fluorescence and the lactate/pyruvate ratio. No significant difference was found between the FORCE/ATP, FORCE/MVO2 and ATP/MVO2 ratios during pacing and noradrenaline. However, during hypopnea, the amount of ATP apparently formed (as calculated by substrate utilization assuming the formation of 3 ATP molecules per oxygen) was disproportionately greater than contractile force and oxygen consumption. It is suggested that this discrepancy may be due to the uncoupling of oxidative phosphorylation.     

A stage in glycolysis controls the metabolic adjustments of vertebrate rod photoreceptors upon illumination

The factors affecting the metabolic adjustments of toad rod photoreceptors were studied by monitoring the oxygen utilization of excised retinas and by measuring rod outer segment ATP and GTP concentrations. Respiratory adjustments upon illumination were observed when glucose or fructose was provided in the perfusate, but not when a glycolytic inhibitor was added to the perfusate containing glucose and pyruvate, or when a substrate beyond glycolysis or from a later stage of glycolysis was substituted for glucose. The amplitudes of the respiratory adjustments to illumination were dependent on the concentration of glucose in the perfusate. The ATP and GTP concentration changes were dependent on respiratory adjustments, including glycolytic effects, and on the levels of illumination. The data suggest a control point within glycolysis for light-induced adjustments of respiration, possibly at phosphofructokinase.     

Osteoclast precursors display dynamic metabolic shifts toward accelerated glucose metabolism at an early stage of RANKL-stimulated osteoclast differentiation.

Mature osteoclasts have an increased citric acid cycle and mitochondrial respiration to generate high ATP production and ultimately lead to bone resorption. However, changes in metabolic pathways during osteoclast differentiation have not been fully illustrated. We report that glycolysis and oxidative phosphorylation characterized by glucose and oxygen consumption as well as lactate production were increased during receptor activator of nuclear factor-kappaB ligand (RANKL)-induced osteoclastogenesis from RAW264.7 and bone marrow-derived macrophage cells. Cell proliferation and differentiation varied according to glucose concentrations (0 to 100 mM). Maximal cell growth occurred at 20 mM glucose concentration and differentiation occurred at 5 mM concentration. Despite the similar growth rates exhibited when cultured cells were exposed to either 5 mM or 40 mM glucose, their differentiation was markedly decreased in high glucose concentrations. This finding suggests the possibility that osteoclastogenesis could be regulated by changes in metabolic substrate concentrations. To further address the effect of metabolic shift on osteoclastogenesis, we exposed cultured cells to pyruvate, which is capable of promoting mitochondrial respiration. Treatment of pyruvate synergistically increased osteoclastogenesis through the activation of RANKL-stimulated signals (ERK and JNK). We also found that osteoclastogenesis was retarded by blocking ATP production with either the inhibitors of mitochondrial complexes, such as rotenone and antimycin A, or the inhibitor of ATP synthase, oligomycin. Taken together, these results indicate that glucose metabolism during osteoclast differentiation is accelerated and that a metabolic shift towards mitochondrial respiration allows high ATP production and induces enhanced osteoclast differentiation.     

Substrate utilization for lactate and energy production by heat-shocked L929 cells

The hypothesis that heat shock protein (HSP) induction depends on inhibition of respiration was tested by examining the effects of heat shock on tricarboxylic acid (TCA) cycle function. In control L929 cell cultures, glucose and exogenous pyruvate were converted primarily to lactate, and glutamine was extensively oxidized, accounting for more than one-half of the calculated ATP production. During heat shock at 42 degrees C, lactate production from all of the labeled substrates and total unlabeled lactate production increased significantly while oxygen consumption increased slightly. TCA cycle oxidation of pyruvate decreased during this period while that of glutamine increased. Uncoupling of oxidative phosphorylation caused large increases in oxygen consumption at both 37 degrees C and 42 degrees C, indicating that the capacity of the respiratory chain is not exceeded during heat shock. The net effect of these alterations in substrate utilization were decreased ATP generation and increased NADH utilization. Both 14CO2 and lactate production declined during the 24-h period after cultures were returned to 37 degrees C. On the basis of these data, we conclude that while inhibition of respiration plays no apparent role, other metabolic consequences of heat shock related to energy metabolism may be involved in HSP induction.     

Mitochondrial ATP-sensitive K+ channel opening decreases reactive oxygen species generation

Mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)) opening was shown previously to slightly increase respiration and decrease the membrane potential by stimulating K(+) cycling across the inner membrane. Here we show that mitoK(ATP) opening reduces reactive oxygen species generation in heart, liver and brain mitochondria. Decreased H(2)O(2) release is observed when mitoK(ATP) is active both with respiration stimulated by oxidative phosphorylation and when ATP synthesis is inhibited. In addition, decreased H(2)O(2) release is observed when mitochondrial Delta pH is enhanced, an effect expected to occur when mitoK(ATP) is open. We conclude that mitoK(ATP) is an effective pathway to trigger mild uncoupling, preventing reactive oxygen species release.     

Nitrogenase activity and regeneration of the cellular ATP pool in Azotobacter vinelandii adapted to different oxygen concentrations.

The in vivo activity of nitrogenase under aerobiosis was studied with diazotrophic chemostat cultures of Azotobacter vinelandii grown under glucose- or phosphate-limited conditions at different dilution rates (Ds, representing the growth rate mu) and different dissolved oxygen concentrations. Under steady-state conditions, the concentration as well as the cellular level of ATP increased in glucose-limited cultures when D was increased. Irrespective of the type of growth limitation or the dissolved oxygen concentration, the steady-state concentrations of ATP and of dinitrogen fixed by nitrogenase increased in direct proportion to each other. Specific rates of dinitrogen fixation as well as of the regeneration of the cellular ATP pool were compared with specific rates of cellular respiration. With glucose-limited cultures, the rate of regeneration of the ATP pool and the rate of respiration varied in direct proportion to each other. This relationship, however, was dependent on the dissolved oxygen concentration. As compared to the phosphate-sufficient control, phosphate-limited cultures exhibited the same nitrogenase activity but significantly increased respiratory activities. Rates of ATP regeneration and of cellular respiration of phosphate-limited cultures did not fit into the relationship characteristic of glucose-limited cultures. However, a linear relationship between the rates of dinitrogen fixation and ATP regeneration was identified irrespective of the type of growth limitation and the dissolved oxygen concentration. The results suggest that the ATP supply rather than cellular oxygen consumption is of primary importance in keeping nitrogenase activity in aerobic cultures of A. vinelandii.     

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.