Micromolar concentrations of Al3+ induce phase separation, aggregation and dye release in phosphatidylserine-containing lipid vesicles

It has been proposed that hexokinase bound to mitochondria occupies a preferred site to which ATP from oxidative phosphorylation is channeled directly (Bessman, S. (1966) Am. J. Medicine 40, 740-749). We have investigated this problem in isolated Zajdela hepatoma mitochondria. Addition of ADP to well-coupled mitochondria in the presence of an oxidizable substrate initiates the synthesis of glucose 6-phosphate via bound hexokinase. This reaction is only partially inhibited by oligomycin, carboxyatractyloside, carbonyl cyanide m-chlorophenylhydrazone (CCCP) or any combination of these, suggesting a source of ATP in addition to oxidative phosPhorylation. This source appears to be adenylate kinase, since Ado2P5, an inhibitor of the enzyme, suppresses hexokinase activity by about 50% when added alone or suppresses activity completely when added together with any of the inhibitors of oxidative phosphorylation. Ado2P5 does not uncouple oxidative phosphorylation nor does it inhibit ADP transport (state 3 respiration) or hexokinase. The relative amount of ATP contributed by adenylate kinase is dependent upon the ADP concentration. At low ADP concentrations, glucose phosphorylation is supported by oxidative phosphorylation, but as the adenine nucleotide translocator becomes saturated the ATP contributed by adenylate kinase increases due to the higher apparent Km of the enzyme. Under conditions of our standard experiment ([ADP] = 0.5 mM), adenylate kinase provides about 50% of the ATP used by hexokinase in well-coupled mitochondria. In spite of this, externally added ATP supported higher initial rates of hexokinase activity than ADP. Our findings demonstrate that oxidative phosphorylation is not a specific or preferential source of ATP for hexokinase bound to hepatoma mitochondria. The apparent lack of a channeling mechanism for ATP to hexokinase in these mitochondria is discussed.     

Creatine kinase of rat heart mitochondria. The demonstration of functional coupling to oxidative phosphorylation in an inner membrane-matrix preparation

To define more clearly the interactions between mitochondrial creatine kinase and the adenine nucleotide translocase, the outer membrane of rat heart mitochondria was removed by digitonin, producing an inner membrane-matrix (mitoplast) preparation. This mitoplast fracton was well-coupled and contained a high specific activity of mitochondrial creatine kinase. Outer membrane permeabilization was documented by the loss of adenylate kinase, a soluble intermembrane enzyme, and by direct antibody inhibition of mitochondrial creatine kinase activity. With this preparation, we documented four important aspects of functional coupling. Kinetic studies showed that oxidative phosphorylation decreased the value of the ternary enzyme-substrate complex dissociation constant for MgATP from 140 to 16 microM. Two approaches were used to document the adenine nucleotide translocase specificity for ADP generated by mitochondrial creatine kinase. Exogenous pyruvate kinase (20 IU/ml) could not readily phosphorylate ADP produced by creatine kinase, since added pyruvate kinase did not markedly inhibit creatine + ATP-stimulated respiration. Additionally, when ADP was produced by mitochondrial creatine kinase, the inhibition of the translocase required 2 nmol of atractyloside/mg of mitoplast protein, while only 1 nmol/mg was necessary when exogenous ADP was added. Finally, the mass action ratio of the mitochondrial creatine kinase reaction exceeded the apparent equilibrium constant when ATP was supplied to the creatine kinase reaction by oxidative phosphorylation. Overall, these results are consistent with much data from intact rat heart mitochondria, and suggest that the outer membrane plays a minor role in the compartmentation of adenine nucleotides. Furthermore, since the removal of the outer membrane does not alter the unique coupling between oxidative phosphorylation and mitochondrial creatine kinase, we suggest that this cooperation is the result of protein-protein proximity at the inner membrane surface.     

The regulatory effect of ATP and its non-hydrolyzed analogs on heart adenylate cyclase

The effect of ATP on rabbit heart adenylate cyclase was investigated. The activation of adenylate cyclase by isoproterenol, guanyl nucleotides and NaF increases at a rise in ATP concentration. A similar regulatory effect is exerted by nonhydrolyzed analogs of ATP - adenyl-5'-ilimidodiphosphate and adenosine-5'-(alpha, beta-methylene) triphosphate. Consequently the regulatory influence of ATP is not due to chemical modification of the enzyme or to phosphorylation of endogenous GDP. Earlier we demonstrated that regulation of heart adenylate cyclase by ATP is not mediated by the adenosine binding center (Biokhimiya USSR (1982), 47, 455-464). It is assumed that the regulatory effect of ATP is accomplished by a specific binding site for this nucleotide.     

Adenylate kinase is a source of ATP for tumor mitochondrial hexokinase

It has proposed that hexokinase bound to mitochondria occupies a preferred site to wich ATP from oxidative phosphorylation is channeled directly (Bessman, S. (1966) Am. J. Medicine 40, 740–749). We have investigated this problem in isolated Zajdela hepatoma mitochondria. Addition of ADP to well-coupled mitochondria in the presence of an oxidizable substrate initiates the synthesis of glucose 6-phosphate via bound hexokinase. This reaction is only partially inhibited by oligomycin, carboxyatractyloside, carbonyl cyanide m-chlorophenylhydrazone (CCCP) ot any combination of these, suggesting a source of ATP in addition to oxidative phosphorylation. This source appears to be adenylate kinase, since Ado₂P₅, an inhibitor of the enzyme, suppresses hexokinase activity by about 50% when added alone or suppresses activity completely when added together with any of the inhibitors of oxidative phosphorylation. Ado₂P₅ does not uncouple oxidative phosphorylation nor does it inhibit ADP transport (state 3 respiration) or hexokinase. The relative amount of ATP contributed by adenylate kinase is dependent upon the ADP concentration. At low ADP concentraions, glucose phosphorylation is supported by oxidative phosphorylation, but as the adenine nucleotide translocator becomes saturated the ATP contributed by adenylate kinase increases due to the higher apparent K_m of the enzyme. Under conditions of our standard experiment ([ADP] = 0.5 mM), adenylate kinase provides about 50% of the ATP used by hexokinase in well-coupled mitochondria. In spite of this, externally added ATP supported higher rates of hexokinase activity than ADP. Our findings demonstrate that oxidative phosphorylation is not a specific or preferential source of ATP for hexokinase bound to hepatoma mitochondria. The apparent lack of a channeling mechanism for ATP to hexokinase in these mitochondria is discussed.     

The role of adenine nucleotide translocator in the regulation of oxidative phosphorylation in heart mitochondria

The regulatory role of adenine nucleotide translocase in oxidative phosphorylation was determined by titration of respiration of isolated rabbit heart mitochondria with carboxyatractyloside in the creatine phosphokinase ADP-regenerating system, which is not rate-limiting. It was found that the respiration rate is not controlled by adenine nucleotide translocase in states 3 and 4. Within the physiological region of respiration (30-70% of the maximal rate), the control coefficient for ADP/ATP translocase is 0.62-0.75. Thus, translocase plays a key role in the regulation of oxidative phosphorylation.     

Mathematical modeling of intracellular transport processes and the creatine kinase systems: a probability approach

A probability approach was used to describe mitochondrial respiration in the presence of substrates, ATP, ADP, Cr and PCr. Respiring mitochondria were considered as a three-component system, including: 1) oxidative phosphorylation reactions which provide stable ATP and ADP concentrations in the mitochondrial matrix; 2) adenine nucleotide translocase provides exchange transfer of matrix adenine nucleotides for those from outside, supplied from medium and by creatine kinase; 3) creatine kinase, starting these reactions when activated by the substrates from medium. The specific feature of this system is close proximity of creatine kinase and translocase molecules. This results in high probability of direct activations of translocase by creatine kinase-derived ADP or ATP without their leak into the medium. In turn, the activated translocase with the same high probability directly provides creatine kinase with matrix-derived ATP or ADP. The catalytic complexes of creatine kinase formed with ATP from matrix together with those formed from medium ATP provide activation of the forward creatine kinase reaction coupled to translocase activation. Simultaneously the catalytic complexes of creatine kinase formed with ADP from matrix together with those formed from medium ADP provide activation of the reverse creatine kinase reaction coupled to translocase activation. The considered probabilities were arranged into a mathermatical model. The model satisfactorily simulates the available experimental data by several groups of investigators. The results allow to consider the observed kinetic and thermodynamic iriegularities in behavior of structurally bound creatine kinase as a direct consequence of its tight coupling to translocase.     

Effect of ATP analogs of DNA synthesis in isolated nuclei

Optimal synthesis of DNA in Ehrlich ascites cell nuclei is shown to be dependent upon the presence of both ATP and ADP. ATP can be replaced only by dATP. An ATP regenerating system is less effective than ATP alone or ATP in combination with ADP. ATP does not stimulate DNA synthesis primarily by maintenance of deoxyribonucleotide triphosphate levels. When the inhibition of DNA synthesis by high ATP levels is taken into account, the ATP analogs adenosine 5'-(alpha,beta-methylene)triphosphate, adenosine 5'-(beta, gamma-methylene)-triphosphate, and adenosine 5'-(beta, gamma-imino)triphosphate can neither substitute for ATP nor inhibit the ATP stimulation of DNA synthesis. Adenosine 5'-(3-thio)triphosphate, however, is a competitive inhibitor of DNA synthesis.     

Interaction of creatine kinase with phosphorylating rabbit heart mitochondria and mitoplasts

This paper demonstrates that the mitochondrial isoenzyme of creatine kinase (CKm) can be solubilized from rabbit heart mitochondria, the outer membrane of which has been removed or at least broken by a digitonin treatment or a short hypotonic exposure, but which has retained an important part of the capacity to phosphorylate ADP. Phosphate, ADP, or ATP, at concentrations which are used to study oxidative phosphorylation and creatine phosphate synthesis, solubilize CKm; the same is true with MgCl2 and KCl. The effect of adenine nucleotides does not seem to be due to their interaction with the adenine nucleotide translocase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that CKm is the main protein released in the described conditions; however, it does not amount to more than 1% of the total protein content of the mitoplasts. When the apparent Km for ATP of CKm was estimated by measuring creatine phosphate synthesis, the values obtained using water-treated mitochondria (0.21 mM) were slightly higher than those of intact mitochondria (0.12 mM) but the difference was not significant. In the former preparation 77% of CKm was in a soluble state. If we can extrapolate these results to intact mitochondria and suppose that in this case a fraction of CKm is also soluble in the intermembrane space, this does not support the theory of functional association between CKm and the adenine nucleotide translocase.     

Free pyrimidine nucleotide pool of Ehrlich ascites-tumour cells. Characteristics related to quantitative studies of RNA metabolism

The atractyloside-insensitive accumulation of adenine nucleotides by rat liver mitochondria (as opposed to the exchange-diffusion catalysed by the adenine nucleotide translocase) has been measured by using the luciferin/luciferase assay as well as by measuring [14C]ATP uptake. In foetal rat liver mitochondria ATP is accumulated more rapidly than ADP, whereas AMP is not taken up. The uptake of ATP occurs against a concentration gradient, and the rate of ATP uptake is greater in foetal than in adult rat liver mitochondria. The accumulated [14C]ATP is shown to be present within the mitochondrial matrix space and is freely available to the adenine nucleotide translocase for exchange with ATP present in the external medium. The uptake is specific for ATP and ADP and is not inhibited by adenosine 5'-[beta gamma-imido] triphosphate, GTP, CTP, cyclic AMP or Pi, whereas dATP and AMP do inhibit ATP accumulation. The ATP accumulation is also inhibited by carbonyl cyanide m-chlorophenylhydrazone, KCN and mersalyl but is insensitive to atractyloside. The ATP uptake is concentration-dependent and exhibits Michaelis-Menten kinetics. The divalent cations Mg2+ and Ca2+ greatly enhance ATP accumulation, and the presence of hexokinase inhibits the uptake of ATP by foetal rat liver mitochondria. These latter effects provide an explanation for the low adenine nucleotide content of foetal rat liver mitochondria and the rapid increase that occurs in the mitochondrial adenine nucleotide concentration in vivo immediately after birth.     

Hexokinase of rat brain mitochondria: relative importance of adenylate kinase and oxidative phosphorylation as sources of substrate ATP, and interaction with intramitochondrial compartments of ATP and ADP

Interactions between intramitochondrial ATP-generating, ADP-requiring processes and ATP-requiring, ADP-generating phosphorylation of glucose by mitochondrially bound hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) have been investigated using well-coupled mitochondria isolated from rat brain. ADP generated by mitochondrially bound hexokinase was more effective at stimulating respiration than was ADP generated by hexokinase dissociated from the mitochondria, and pyruvate kinase was less effective as a scavenger of ADP generated by the mitochondrially bound hexokinase than was the case with ADP generated by the dissociated enzyme. These results indicate that ADP generated by the mitochondrially bound enzyme is at least partially sequestered and directed toward the mitochondrial oxidative phosphorylation apparatus. Under the conditions of these experiments, the maximum rate of ATP production by oxidative phosphorylation was approximately 10-fold greater than the maximum rate of ATP generation by the adenylate kinase reaction. Moreover, during periods of active oxidative phosphorylation, adenylate kinase made no detectable contribution to ATP production. Thus, adenylate kinase does not represent a major source of ATP for hexokinase bound to actively phosphorylating brain mitochondria. With adenylate kinase as the sole source of ATP, a steady state was attained in which ATP formation was balanced by utilization in the hexokinase reaction. In contrast, when oxidative phosphorylation was the source of ATP, a steady state rate of Glc phosphorylation was attained, but it was equivalent to only about 40-50% of the rate of ATP production and thus there was a continued net increase in ATP concentration in the system. Rates of Glc phosphorylation with ATP generated by oxidative phosphorylation exceeded those seen with equivalent levels of exogenously added ATP. Moreover, at total ATP concentrations greater than approximately 0.2 mM, hexokinase bound to actively phosphorylating mitochondria was unresponsive to continued slow increases in ATP levels; acute increase in ATP (by addition of exogenous nucleotide) did, however, result in increased hexokinase activity. The relative insensitivity of mitochondrially bound hexokinase to extramitochondrial ATP suggested dependence on an intramitochondrial pool (or pools) of ATP during active oxidative phosphorylation. Two intramitochondrial compartments of ATP were identified based on their selective release by inhibitors of electron transport or oxidative phosphorylation. These compartments were distinguished by their sensitivity to inhibitors and the kinetics with which they were filled with ATP generated by oxidative phosphorylation. Exogenous glycerol kinase competed effectively with mitochondrially bound hexokinase for extramitochondrial ATP, with relatively low levels of glycerol kinase completely inhibiting phosphorylation of Glc.(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies on photophosphorylation utilizing methylene diphosphonate analogs of ADP and ATP.

Spinach chloroplasts were able to photophosphorylate the ADP analog alpha,beta-methylene adenosine 5'-diphosphate (AOPCP). Phosphorylation of AOPCP was catalyzed by chloroplasts that were washed or dialyzed to remove free endogenous nucleotides. In the presence of glucose, hexokinase, AOPCP and 32Pi, the 32P label was incorporated into alpha,beta-methylene adenosine 5'-triphosphate (AOPCPOP). In contrast to photophosphorylation of AOPCP, the ATP analog AOPCPOP was a poor substrate for the ATP-Pi exchange reaction and its hydrolysis was neither stimulated by light and dithiothreitol nor inhibited by Dio-9. Photophosphorylation of AOPCP was inhibited by the alpha,beta- and beta,gamma-substituted methylene analogs of ATP, while phosphorylation of ADP was unaffected by them. The ATP-Pi exchange was also unaffected by both ATP analogs, while the weak AOPCPOP-Pi exchange was inhibited by the beta,gamma-methylene analog of ATP. Direct interaction of methylene analogs with the chloroplast coupling factor ATPase was indicated by the enzymatic hydrolysis of AOPCPOP on polyacrylamide gels.     

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.     

Involvement of inositol 1,4,5-trisphosphate and calcium in the action of adenine nucleotides on aortic endothelial cells

ADP and ATP, in the 1-100 microM range of concentrations, increased the formation of inositol phosphates in bovine aortic endothelial cells. The accumulation of inositol trisphosphate in response to adenine nucleotides was rapid (maximum at 15 s) and transient. This material was identified as the biologically active isomer inositol 1,4,5-trisphosphate on the basis of its retention time by high-performance liquid chromatography on an anion-exchange resin. AMP and adenosine have no effect on inositol phosphates. The action of ATP and ADP was mimicked with an equal potency and activity by their phosphorothioate analogs, ATP gamma S and ADP beta S, and with a lower potency by adenosine 5'-(beta,gamma-imido)triphosphate, whereas adenosine 5'-(alpha,beta-methylene)triphosphate, was inactive. In the same range of concentrations, ADP and ATP induced an efflux of 45Ca2+ from prelabeled bovine aortic endothelial cells and increased the fluorescence emission by cells loaded with quin-2. Here, too, AMP and adenosine were completely inactive. The outflow of 45Ca2+ induced by ADP was partially maintained in a calcium-free medium. These data suggest that in aortic endothelial cells, P2-purinergic receptors, of the P2Y subtype, are coupled to the hydrolysis of phosphatidylinositol bisphosphate by a phospholipase C. It is likely that the release of prostacyclin and endothelium-derived relaxing factor in response to ADP and ATP is a consequence of this initial event.     

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.     

Functional evidence for involvement of P2 purinoceptors in the ATP stimulation of phosphatidylcholine secretion in type II alveolar epithelial cells

There is evidence that phosphatidylcholine secretion in type II pneumocytes is stimulated by adenosine and adenine nucleotides and that the effect of adenosine is mediated by the A2 subtype of the P1 purinoceptor. To determine if the effect of ATP is also mediated by the same receptor following its catabolism to adenosine or by the P2 purinoceptor we compared the effects of adenosine and ATP. Adenosine and terbutaline stimulated phosphatidylcholine secretion approx. 2-fold, while ATP stimulated it by more than 3-fold, essentially to the same extent as the protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate. The stimulatory effect of adenosine but not of ATP was abolished by adenosine deaminase. The effect of ATP was markedly diminished by the P2 desensitizing agent alpha,beta-methylene ATP, but only slightly by the P1 antagonist 8-phenyltheophylline. Adenosine increased the cAMP content of type II cells while ATP had little effect. The effects of ATP and terbutaline were additive while those of adenosine and terbutaline were not. These data show that ATP and adenosine stimulate phosphatidylcholine secretion via different mechanisms. Therefore, the effect of ATP is not mediated via catabolism to adenosine. Metabolically resistant analogs of ATP also stimulated secretion in a concentration-dependent manner although none were as potent as ATP. The order of potency was ATP greater than beta,gamma-methylene ATP = 2-methylthio ATP = 2-deoxy ATP greater than or equal to 8-bromo ATP greater than alpha,beta-methylene ATP. The facts that ATP analogs also stimulate secretion and that the effect of ATP was antagonized by alpha,beta-methylene ATP suggest that the stimulatory effect of ATP is mediated by the P2 purinoceptor.     

Decrease in mitochondrial levels of adenine nucleotides and concomitant mitochondrial dysfunction in ischemic rat liver

The process of mitochondrial dysfunction in ischemic rat liver was studied. A close correlation was found between decrease in the mitochondrial adenine nucleotide content and deterioration of oxidative phosphorylation capacity. The level of total adenine nucleotides, which was 15--20 nmol/mg protein in mitochondria isolated from normal liver, fell to 1--2 nmol/mg protein with concomitant loss of oxidative phosphorylation capacity after anoxic incubation in vitro or in vivo for 120 min. However, neither the permeability barrier to adenine nucleotides nor matrix enzymes were affected under these conditions. The loss of adenine nucleotides was ascribed to degradation of AMP to adenosine and then leakage of the latter. Conventional procedures for maintenance of oxidative phosphorylation capacity of isolated mitochondria, preservation in the cold and addition of ATP or a respiratory substrate under aerobic conditions, were very effective in maintaining the intramitochondrial levels of adenine nucleotides. Of the three species of adenine nucleotides, only AMP was ineffective in maintaining mitochondrial function; mitochondria containing more than 5 nmol of ATP plus ADP/mg protein exhibited normal activity of oxidative phosphorylation, but with less than 2 nmol they showed no activity.     

Modeling of oxygen transport and cellular energetics explains observations on in vivo cardiac energy metabolism

Observations on the relationship between cardiac work rate and the levels of energy metabolites adenosine triphosphate (ATP), adenosine diphosphate (ADP), and phosphocreatine (CrP) have not been satisfactorily explained by theoretical models of cardiac energy metabolism. Specifically, the in vivo stability of ATP, ADP, and CrP levels in response to changes in work and respiratory rate has eluded explanation. Here a previously developed model of mitochondrial oxidative phosphorylation, which was developed based on data obtained from isolated cardiac mitochondria, is integrated with a spatially distributed model of oxygen transport in the myocardium to analyze data obtained from several laboratories over the past two decades. The model includes the components of the respiratory chain, the F₀F₁-ATPase, adenine nucleotide translocase, and the mitochondrial phosphate transporter at the mitochondrial level; adenylate kinase, creatine kinase, and ATP consumption in the cytoplasm; and oxygen transport between capillaries, interstitial fluid, and cardiomyocytes. The integrated model is able to reproduce experimental observations on ATP, ADP, CrP, and inorganic phosphate levels in canine hearts over a range of workload and during coronary hypoperfusion and predicts that cytoplasmic inorganic phosphate level is a key regulator of the rate of mitochondrial respiration at workloads for which the rate of cardiac oxygen consumption is less than or equal to approximately 12 μmol per minute per gram of tissue. At work rates corresponding to oxygen consumption higher than 12 μmol min⁻¹ g⁻¹, model predictions deviate from the experimental data, indicating that at high work rates, additional regulatory mechanisms that are not currently incorporated into the model may be important. Nevertheless, the integrated model explains metabolite levels observed at low to moderate workloads and the changes in metabolite levels and tissue oxygenation observed during graded hypoperfusion. These findings suggest that the observed stability of energy metabolites emerges as a property of a properly constructed model of cardiac substrate transport and mitochondrial metabolism. In addition, the validated model provides quantitative predictions of changes in phosphate metabolites during cardiac ischemia.     

Brown adipose tissue mitochondria: recoupling caused by substrate level phosphorylation and extramitochondrial adenosine phosphates.

1. Uncoupled oxidative phosphorylation in isolated guinea pig brown-adipose-tissue mitochondria is reflected by a low phosphorylation state of adenosine phosphates in the mitochondrial matrix and in the extramitochondrial space during oxidation of succinate or glycerol 1-phosphate in the presence of serum albumin and 100 muM ADP. Recoupling of respiration and phosphorylation in the mitochondria is indicatdd by a dramatic increase in the phosphorylation state of adenine nucleotides in both compartments, when substrates inducing substrate level phosphorylation are respired. In this case ATP/ADP ratios in the extramitochondrial compartment are 10-15 times higher than in the mitochondrial matrix. 2. Recoupling mediated by substrate level phosphorylation depends on the presence of extramitochondrial adenosine phosphate and on intact adenine nucleotide translocation. In the presence of substrate level phosphorylation the amount of extramitochondrial ADP required to restore energy coupling can be extremely low (20 muM ADP or 10 nmol ADP/mg mitochondrial protein respectively). If substrate level phosphorylation is prevented by rotenone or in the presence of atractyloside, 20-50 times higher amounts of extramitochondrial adenine nucleotides are necessary to cause coupled oxidative phosphorylation. The recoupling effect of ATP is significantly stronger than that of ADP. 3. GDP (100 muM) causes a rapid increase of the ATP/ADP ratio in both compartments which is independent of substrate level phosphorylation as well as of the extramitochondrial adenosine phosphate concentration and the adenine nucleotide carrier. 4. The amount of extramitochondrial adenosine phosphate in guinea pig brown-adipose-tissue (18 nmol/mg mitochondrial protein or 2.5 mM respectively) would suffice for recoupling of oxidative phosphorylation mediated by substrate level phosphorylation under conditions in vitro; this suggests that substrate level phosphorylation is of essential importance in brown fat in vivo with respect to energy conditions in the tissue during different states of thermogenesis.     

Phosphorylation and the Reduced Nicotinamide Adenine Dinucleotide Oxidase Reaction in Streptococcus agalactiae

Cell-free extracts from aerobically grown Streptococcus agalactiae cells possess a reduced nicotinamide adenine dinucleotide (NADH) oxidase which is linked to oxygen. It is inhibited by cyanide, although cytochromes evidently are not involved. Adenosine triphosphate (ATP) formation occurs during the reaction, but 66 to 75% of the total ATP is formed nonoxidatively. The remaining 25 to 35% of the ATP formation is related to the oxidation of NADH. The formation of ATP in the oxidative reaction can be prevented by excluding oxygen or adding cyanide to prevent NADH oxidation. It can also be prevented by adding methylene blue or pyruvate, which bypasses electron transport to oxygen, but does not interfere with NADH oxidation. Potential sources of ATP, such as glycolysis, the pyruvate oxidase reaction, or the oxidative pentose cycle, are not present, and the high nonoxidative ATP formation is ascribed to the adenylate kinase reaction. The reaction requires adenosine diphosphate (ADP) as a phosphate acceptor. NADH oxidation is independent of ADP. Antimycin A, amytal, and 2,4-dinitrophenol decreased, but did not prevent, oxidative formation of ATP. P:O ratios ranged from 0.15 to 0.25. All of the oxidative activity was in the soluble portion of the cell-free extracts.     

Relationship between oxidative phosphorylation and adenine nucleotide translocase activity of two populations of cardiac mitochondria and mechanical recovery of ischemic hearts following reperfusion

The possible relationship of the atractyloside-sensitive adenine nucleotide translocase activity, oxidative phosphorylation, and the recovery of ventricular contractility following reperfusion of the ischemic isolated rat heart was studied. Five minutes of total global ischemia without reperfusion produced a significant depression in adenine nucleotide translocase in subsarcolemmal mitochondria (SLM), whereas a minimum of 10 min ischemia was required to observe a significant depression in interfibrillar mitochondria (IFM). Increasing durations of ischemia resulted in a progressively larger depression in translocase activity, with a maximum depression of approximately 75% seen in both populations following 20 min ischemia. In contrast, oxidative phosphorylation was totally unaffected in either mitochondrial population following up to 20 min of ischemia. We assessed whether translocase activity or oxidative phosphorylation were related to contractile recovery in hearts reperfused following various durations of ischemia. In SLM, translocase activity was further depressed following reperfusion compared with pre-reperfusion ischemic values, whereas with IFM only reperfusion following 5 min ischemia produced a further depression in translocase values. Oxidative phosphorylation rates of SLM and IFM were significantly depressed following reperfusion of ischemic hearts, although SLM exhibited a generally higher sensitivity in this regard. In reperfused hearts, an overall significant relationship was found between oxidative phosphorylation rate and adenine translocase activity as well as between translocase activity and post-reperfusion contractile recovery. These data show that ischemia can produce a significant depression in translocase activity in the absence of any change in oxidative phosphorylation. The results also suggest that the depression in mitochondrial ADP/ATP translocase and subsequent inhibition of oxidative phosphorylation in the reperfused heart may represent one of the important contributory mechanisms involved in cardiac failure and injury during acute ischemia and reperfusion.