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.     

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.     

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.     

Carboxyatractyloside-insensitive influx and efflux of adenine nucleotides in rat liver mitochondria

Unidirectional transport (influx and efflux) of adenine nucleotides in rat liver mitochondria was examined using carboxyatractyloside to inhibit rapid exchange of matrix and external adenine nucleotides via the adenine nucleotide translocase. Influx of adenine nucleotides was concentration-dependent. ATP was the preferred substrate with a Km of 2.67 mM and V of the preferred substrate with a Km of 2.67 mM and V of 8.33 nmol/min/mg of protein. For ADP, the Km was 14.7 mM and V was 10.8 nmol/min/mg of protein. Efflux of adenine nucleotides was also concentration-dependent, varying directly as a function of the matrix adenine nucleotide pool size. Any increase in the influx of adenine nucleotides was coupled to an increase in efflux. However, as the external ATP concentration was increased, influx was stimulated to a much greater extent than was efflux. This imbalance suggested that under certain conditions adenine nucleotide movement might be coupled to the movement of an alternate anion such as phosphate. Adenine nucleotide efflux increased as the external phosphate concentration was varied from 0.5 to 4 mM. Also, increasing the external phosphate concentration caused adenine nucleotide influx to decrease, suggesting competition. In the absence of external adenines and phosphate, no efflux occurred. Both adenine nucleotide influx and efflux were depressed if Mg2+ was omitted. Adenine nucleotide efflux in the presence of external phosphate was inhibited much less by lack of Mg2+ than was efflux in the presence of external ATP. This evidence supports a model in which either adenine nucleotides (probably with Mg2+) or phosphate can move across the mitochondrial membrane on a single carrier. Net adenine nucleotide movements can occur when adenine nucleotide movement is coupled to the movement of phosphate in the opposite direction.     

The effect of agaric acid on citrate transport in rat liver mitochondria

The effect of agaric acid (αcetyl citric acid) , a competitive inhibitor of the adenine nucleotide translocase, was studied on the citrate uptake in rat liver mitochondria. The experiments carried out reveal that citrate uptake is progressively inhibited by increasing concentrations of agaric acid, showing a typical competitive type of inhibition. The apparent Ki for agaric acid is 5.2 μM, a concentration lower than that which inhibits the adenine nucleotide translocase. The results also show that mersalyl diminishes the Ki to 3.4 μM; 10 mM KCl reverses the inhibitory action of agaric acid on the ADP and ATP exchange but does not affect the agaric acid induced inhibition of citrate uptake.     

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.     

Heart mitochondria in physiological salt solution: not ionic strength but salt composition is important for association of creatine kinase with the inner membrane surface

In physiological salt solution (PSS) which mimicks the cardiac cells cytoplasm and contains 120 mM K-MES, 10 mM NaCl, 20 mM imidazole, pH 7.2, 20 mM taurine, 15 mM creatine, 15 mM Na2phosphocreatine, 5 mM Na2ATP, 8 mM MgCl2, 5 mM K2HPO4, 3 mM glutamate, 3 mM malate, 0.5 mM dithiothreitol and 10 mg/ml of bovine serum albumine both isolated mitochondria and intracellular structures in skinned fibers stay intact. In PSS mitochondrial creatine kinase remains firmly attached to the inner membrane surface. CKmi-mi is extracted from cardiac mitoplasts in 0.125 M KCl solution, but addition of 10 mM sodium borate to this KCl solution completely inhibits dissociation of CKmi-mi. Therefore, not ionic strength but ion composition is important for association of CKmi-mi with mitochondrial membrane. Functional and structural studies using antibodies against CKmi-mi showed that in PSS CKmi-mi is bound to the inner mitochondrial membrane in spatially close relationship to adenine nucleotide translocase (ANT). Thus, under physiological conditions CKmi-mi is structurally and functionally coupled to ANT in cardiac mitochondria and functions to catalyze almost complete utilization of mitochondrial ATP for aerobic phosphocreatine synthesis.     

Relation between extra- and intramitochondrial ATP/ADP ratios in rat liver mitochondria

Changes of the extra- and intramitochondrial ATP/ADP ratios as a function of the respiratory state were measured in incubations with rat liver mitochondria. ATPase or creatine/creatine kinase was used to change the extramitochondrial ATP/ADP ratio; the separation of the mitochondrial pellet was performed by a Millipore filtration technique. Under all conditions tested, the intramitochondrial ratio changed in the same direction as the extramitochondrial one, except in the presence of atractylate where this correlation was not observed. Furthermore, it could be shown that the oxygen uptake and pyruvate carboxylase activity correlated with the intramitochondrial ATP/ADP ratio and not with the extramitochondrial one. These results do not support the proposal that the adenine nucleotide translocase is rate limiting for respiration.     

Studies of energy transport in heart cells. Mitochondrial isoenzyme of creatine phosphokinase: kinetic properties and regulatory action of Mg2+ ions.

1. The kinetic properties of mitochondrial creatine phosphokinase (Km for all substrates and maximal rates of the forward and reverse reaction) have been studied. Since (a) Km value for MgADP- (0.05 mM) and creatine phosphate (0.5 mM) are significantly lower than Km for MgATP2- (0.7 mM) and creatine (5.0 mM) and (b) maximal rate of the reverse reaction (creatine phosphate + ADP leads to ATP + creatine) equal to 3.5 mumol times min-1 times mg-1 is essentially higher than maximal rate of the forward reaction (0.8 mumol times min-1 times mg-1), ATP synthesis from ADP and creatine phosphate is kinetically preferable over the forward reaction. 2. A possible regulatory role of Mg2+ ions in the creatine phosphokinase reaction has been tested. It has been shown that in the presence of all substrates and products of the reaction the ratio of the rates of forward and reverse reactions can be effectively regulated by the concentration of Mg2+ ions. At limited Mg2+ concentrations creatine phosphate is preferably synthesized while at high Mg2+ concentrations (more ATP in the reaction medium) ATP synthesis takes place. 3. The kinetic (mathematical) model of the mitochondrial creatine phosphokinase reaction has been developed. This model accounts for the existence of a variety of molecular forms of adenine nucleotides in solution and the formation of their complexes with magnesium. It is based on the assumption that the mitochondrial creatine phosphokinase reactions mechanism is analogous to that for soluble isoenzymes. 4. The dependence of the overall rate of the creatine phosphokinase reaction on the concentration of total Mg2+ ions calculated from the kinetic model quantitatively correlates with the experimentally determined dependence through a wide range of substrates (ATP, ADP, creatine and creatine phosphate) concentration. The analysis of the kinetic model demonstrates that the observed regulatory effect of Mg2+ on the overall reaction rate can be expained by (a) the sigmoidal variation in the concentration of the MgADP- complex resulting from the competition between ATP AND ADP for Mg2+ and (b) the high affinity of the enzyme to MgADP-. 5. The results predicted by the model for the behavior of mitochondrial creatine phosphokinase under conditions of oxidative phosphorylation point to an intimate functional interaction of mitochondrial creatine phosphokinase and ATP-ADP translocase.     

Energy-independent protection of the oxidative phosphorylation capacity of mitochondria against anoxic damage by ATP and its non-metabolizable analogs

Preservation of the oxidative phosphorylation capacity of mitochondria by addition of ATP under anaerobic conditions was analyzed by use of non-metabolizable adenine nucleotide analogs. The capacity was well preserved in the presence of ATP and did not require the hydrolysis of ATP, since ATP analogs, such as beta, gamma-methylene adenosine triphosphate (AMPPCP), alpha, beta-methylene adenosine triphosphate (AMPCPP), and adenylyl imidodiphosphate (AMPPNP), were as effective as ATP. These analogs were incorporated into mitochondria through ATP/ADP translocase to maintain the original level of total adenine nucleotides in the mitochondria. ADP apparently had the same effect as ATP, but its effect was shown to be due to ATP generated from it by adenylate kinase in mitochondria. An analog of ADP, alpha, beta-methylene adenosine diphosphate (AMPCP), which was found to be a substrate of the translocase but not of adenylate kinase, could not replace ADP or ATP. From these results, it was concluded that the oxidative phosphorylation capacity of mitochondria was maintained by ATP, but not ADP, through a process not requiring energy.     

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.     

Relation between extra- and intramitochondrial ATP/ADP ratios in rat liver mitochondria

Changes of the extra- and intramitochondrial ATP/ADP ratios as a function of the respiratory state were measured in incubations with rat liver mitochondria. ATPase or creatine/creatine kinase was used to change the extramitochondrial ATP/ADP ratio; the separation of the mitochondrial pellet was performed by a Millipore filtration technique. Under all conditions tested, the intramitochondrial ratio changed in the same direction as the extramitochondrial one, except in the presence of atractylate where this correlation was not observed. Furthermore, it could be shown that the oxygen uptake and pyruvate carboxylase activity correlated with the intramitochondrial ATP/ADP ratio and not with the extramitochondrial one. These results do not support the proposal that the adenine nucleotide translocase is rate limiting for respiration.     

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.     

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.     

Compartmented coupling of chicken heart mitochondrial creatine kinase to the nucleotide translocase requires the outer mitochondrial membrane

The kinetic coupling of mitochondrial creatine kinase (MiMi-CK) to ADP/ATP translocase in chicken heart mitochondrial preparations is demonstrated. Measuring the MiMi-CK apparent Km value for MgATP2- (at saturating creatine) gives a value of 36 microM when MiMi-CK is coupled to oxidative phosphorylation. This Km value is threefold lower than the Km for enzyme bound to mitoplasts or free in solution. The nucleotide translocase Km value for ADP decreases from 20 to 10 microM in the presence of 50 mM creatine only with intact mitochondria. Similar experiments with mitoplasts do not give decreased Km values. The observed Km differences can be used to calculate the concentration of ATP and ADP under steady-state conditions showing that the observed differences in the kinetic constants accurately reflect the enzyme activities of MiMi-CK under the different conditions. The behavior of the Km values provides evidence for what we term compartmented coupling. Therefore, like the rabbit heart system (S. Erickson-Viitanen, P. Viitanen, P. J. Geiger, W. C. T. Yang, and S. P. Bessman (1982) J. Biol. Chem. 257, 14395-14404) compartmented coupling requires an intact outer mitochondrial membrane. The apparent Km values for normal or compartmentally coupled systems can be used to calculate steady-state values of ATP and ADP by coupling enzyme theory. Hence, the overall kinetic parameters accurately reflect the behavior of the enzymes whether free in solution or in the intermembrane space.     

Regulation of respiration in brain mitochondria and synaptosomes: restrictions of ADP diffusion in situ, roles of tubulin, and mitochondrial creatine kinase

The role of ubiquitous mitochondrial creatine kinase (uMtCK) reaction in regulation of mitochondrial respiration was studied in purified preparations of rat brain synaptosomes and mitochondria. In permeabilized synaptosomes, apparent Km for exogenous ADP, Km (ADP), in regulation of respiration in situ was rather high (110 +/- 11 microM) in comparison with isolated brain mitochondria (9 +/- 1 microM). This apparent Km for ADP observed in isolated mitochondria in vitro dramatically increased to 169 +/- 52 microM after their incubation with 1 muM of dimeric tubulin showing that in rat brain, particularly in synaptosomes, mitochondrial outer membrane permeability for ADP, and ATP may be restricted by tubulin binding to voltage dependent anion channel (VDAC). On the other hand, in synaptosomes apparent Km (ADP) decreased to 25 +/- 1 microM in the presence of 20 mM creatine. To fully understand this effect of creatine on kinetics of respiration regulation, complete kinetic analysis of uMtCK reaction in isolated brain mitochondria was carried out. This showed that oxidative phosphorylation specifically altered only the dissociation constants for MgATP, by decreasing that from ternary complex MtCK.Cr.MgATP (K (a)) from 0.13 +/- 0.02 to 0.018 +/- 0.007 mM and that from binary complex MtCK.MgATP (K (ia)) from 1.1 +/- 0.29 mM to 0.17 +/- 0.07 mM. Apparent decrease of dissociation constants for MgATP reflects effective cycling of ATP and ADP between uMtCK and adenine nucleotide translocase (ANT). These results emphasize important role and various pathophysiological implications of the phosphocreatine-creatine kinase system in energy transfer in brain cells, including synaptosomes.     

Affinity modification of creatine kinase and ATP-ADP translocase in heart mitochondria: determination of their stoichiometric ratio

The interaction of mitochondrial creatine kinase and ATP-ADP translocase with 2.3-dialdehyde derivatives of ADP and ATP (oADP and oATP) has been studied. It was shown that these compounds are irreversible and specific inhibitors of creatine kinase (KioADP = 0.6mM, KioATP = 1.12 mM) and ATP-ADP translocase (KioADP = 0.065mM, KioATP = 0.14 mM). The substrates protect both enzymes from inactivation by these compounds. The maximal pseudo-first order rate constants for the 2,3-dialdehyde nucleotide derivative interaction with creatine kinase are 0.2 min-1 for oADP (pH 6.5) and 0.11 min-1 for oATP (pH 7.0). A decrease in the creatine kinase activity correlates with the incorporation of the reagent into the protein. The completely inactivated, isolated and purified enzyme contains 1 mol of oADP per mole of active sites. A procedure for simultaneous determination of the creatine kinase and translocase content in mitochondria and mitoplasts has been developed, which is based on the application of [3H]oADP in combination with specific treatment of mitochondria (or mitoplasts) with carboxyatractyloside 2,4-dinitrofluorobenzene and a mixture of creatine kinase substrates (MgADP + phosphocreatine). It has been found that for heart mitochondria from different animals the content of creatine kinase and translocase is 2.1-2.6 and 2.4-2.9 mol per mol of cytochrome c oxidase, respectively. Thus, the stoiochiometric ratio of creatine kinase and ATP-ADP translocase is close to 1.0 for all mitochondrial preparations under study (i.e. rat, dog, rabbit and chicken).     

Cyclic AMP-induced Mg2+ release from rat liver hepatocytes, permeabilized hepatocytes, and isolated mitochondria.

The addition of norepinephrine, epinephrine, or forskolin to collagenase-dispersed rat liver hepatocytes increase cAMP and result in a 15% loss in total cell Mg2+ within 5 min. Conversely, carbachol and vasopressin induce a 10-15% increase of total cell Mg2+. Permeabilized hepatocytes also mobilize a large pool of Mg2+ when stimulated by ADP or cAMP. This stimulation is completely inhibited by atractyloside and bongkrekic acid, two different specific inhibitors of the mitochondrial adenine nucleotide translocase. cAMP directly mobilizes Mg2+ efflux from isolated rat liver mitochondria. 50 nM cAMP or 250 microM ADP induces in 5 min a mitochondrial loss of about 6 nmol of Mg2+/mg of protein and a stimulation of ATP efflux. The effect of cAMP is specific, is not reproduced by other cyclic or noncyclic nucleotides, and is inhibited by inhibitors of the adenine nucleotide translocase. These data indicate that cAMP is a messenger for a major mobilization of Mg2+ in hepatocytes. A major target for the effect of cAMP are mitochondria, which lose up to 20-25% of their total Mg2+ in 5 min, both within the cell and after isolation. Evidence is presented suggesting that the adenine nucleotide translocase is the target of the cAMP-dependent Mg2+ efflux and that cAMP may change the operation of the translocase. This, in turn, could change within the matrix the substrate of choice of the translocase from ATP to ATP.Mg.     

The lack of direct coupling between ATP-ADP translocase and creatine phosphokinase in isolated rabbit heart mitochondria

The formation of creatine phosphate by isolated rabbit heart mitochondria in the presence of creatine, α-ketoglutarate, ATP, and inorganic phosphate was studied. Creatine phosphate formation was inhibited by oligomycin. This was most probably due to increased concentration of ADP favoring the reverse reaction (formation of creatine and ATP from phosphocreatine and ADP). The inhibitory effect of oligomycin disappeared in the presence of phosphoenolpyruvate and pyruvate kinase. The results do not indicate any direct coupling between mitochondrial creatine phosphokinase and ATP-ADP translocase as has been suggested for rat heart mitochondria.     

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.