Analysis of functional coupling: mitochondrial creatine kinase and adenine nucleotide translocase

The mechanism of functional coupling between mitochondrial creatine kinase (MiCK) and adenine nucleotide translocase (ANT) in isolated heart mitochondria is analyzed. Two alternative mechanisms are studied: 1), dynamic compartmentation of ATP and ADP, which assumes the differences in concentrations of the substrates between intermembrane space and surrounding solution due to some diffusion restriction and 2), direct transfer of the substrates between MiCK and ANT. The mathematical models based on these possible mechanisms were composed and simulation results were compared with the available experimental data. The first model, based on a dynamic compartmentation mechanism, was not sufficient to reproduce the measured values of apparent dissociation constants of MiCK reaction coupled to oxidative phosphorylation. The second model, which assumes the direct transfer of substrates between MiCK and ANT, is shown to be in good agreement with experiments—i.e., the second model reproduced the measured constants and the estimated ADP flux, entering mitochondria after the MiCK reaction. This model is thermodynamically consistent, utilizing the free energy profiles of reactions. The analysis revealed the minimal changes in the free energy profile of the MiCK-ANT interaction required to reproduce the experimental data. A possible free energy profile of the coupled MiCK-ANT system is presented.     

Once again about the functional coupling between mitochondrial creatine kinase and adenine nucleotide translocase

The synthesis of creatine phosphate (CP) by mitochondrial creatine kinase during oxidative phosphorylation was terminated when the mass action ratio of the creatine kinase reaction = [ADP]·[CP][ATP]·[Cr] became equal to the apparent equilibrium constant (K _eq ^app) of this reaction. Subsequent excess of over the K _eq ^app was due to an increase in the ADP concentration in the medium. A comparable increase in the ADP concentration also occurred in the absence of creatine (Cr) in the incubation medium. Increase in the ADP concentration was shown to be associated with a decrease in the rate of oxidative phosphorylation and with a relative increase in the ATPase activity of mitochondria during the incubation. A low concentration of ADP (<30 M) and relatively high concentrations (1-6 mM) of other components of the creatine kinase reaction prevented the detection of the reverse reaction within 10 min after exceeded the K _eq ^app, but the reverse reaction became evident on more prolonged incubation. The reverse reaction was accompanied by a further increase in . Low ADP concentration in the medium was also responsible for the lack of an immediate conversion of the excess creatine phosphate added although > K _eq ^app. The findings are concluded to be in contradiction with the concept of microcompartment formation between mitochondrial creatine kinase and adenine nucleotide translocase.     

Molecular dynamics simulations of creatine kinase and adenine nucleotide translocase in mitochondrial membrane patch

Interaction between mitochondrial creatine kinase (MtCK) and adenine nucleotide translocase (ANT) can play an important role in determining energy transfer pathways in the cell. Although the functional coupling between MtCK and ANT has been demonstrated, the precise mechanism of the coupling is not clear. To study the details of the coupling, we turned to molecular dynamics simulations. We introduce a new coarse-grained molecular dynamics model of a patch of the mitochondrial inner membrane containing a transmembrane ANT and an MtCK above the membrane. The membrane model consists of three major types of lipids (phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) in a roughly 2:1:1 molar ratio. A thermodynamics-based coarse-grained force field, termed MARTINI, has been used together with the GROMACS molecular dynamics package for all simulated systems in this work. Several physical properties of the system are reproduced by the model and are in agreement with known data. This includes membrane thickness, dimension of the proteins, and diffusion constants. We have studied the binding of MtCK to the membrane and demonstrated the effect of cardiolipin on the stabilization of the binding. In addition, our simulations predict which part of the MtCK protein sequence interacts with the membrane. Taken together, the model has been verified by dynamical and structural data and can be used as the basis for further studies.     

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.     

Differential coupling of smooth and skeletal muscle pyruvate kinase to creatine kinase.

The interaction of pyruvate kinase from skeletal (SKPK) and smooth (SMPK) muscle with MM-creatine kinase (MMCK) and BB-creatine kinase (BBCK) was assessed using temporal absorbance changes, variations in absorbance at different wavelengths, concentration dependence, association in an electric field, and PK kinetic activity. SKPK exhibits a time course of absorbance increase in the presence of MMCK with a time constant of 29.5 min. This increase occurs at all wavelength from 240 to 1000 nm. At 195 nm, the combination of SKPK and MMCK produces a decrease in absorption with electric fields of both 0 and 204 V/cm. The change in SKPK-MMCK is saturable. SKPK activity is significantly increased by the presence of MMCK in solutions of 0-32% ethanol. These results indicate specific SKPK-MMCK interaction. SMPK and BBCK did not exhibit similar coupling when the BBCK concentration dependence of absorbance or SMPK activity in solutions of 0-32% ethanol was determined. Both MMCK and BBCK increased SKPK activity; neither MMCK nor BBCK increased SMPK activity. The ability to form diazymatic complexes with creatine kinase appears to reside in SKPK. This coupling may account for the increased flux through PK without significant substrate changes seen during skeletal muscle activation. This coupling will not occur in smooth muscle.     

Evidence against direct transfer of the adenine nucleotides by the heart mitochondrial creatine kinase-adenine nucleotide translocase complex

Adenine nucleotide translocase inhibitors, atractyloside and carboxyatractyloside, added to respiration inhibited rat heart mitochondria had no significant effects on the apparent Km's for ATP and ADP and Vmax's of the creatine kinase reaction. The results suggest that there is no sequential, mandatory, direct transfer of the nucleotides between the translocase and the creatine kinase active site. The reaction was run in both the forward and reverse directions in media containing either 0.25 M sucrose or 0.13 M KCl. The apparent Km's, however, were found to be 2–3 times higher in the KCl medium than in sucrose, suggesting the use of the more physiological medium for meaningful kinetic studies.     

Functional expression of human adenine nucleotide translocase 4 in Saccharomyces cerevisiae

The adenine nucleotide translocase (ANT) mediates the exchange of ADP and ATP across the inner mitochondrial membrane. The human genome encodes multiple ANT isoforms that are expressed in a tissue-specific manner. Recently a novel germ cell-specific member of the ANT family, ANT4 (SLC25A31) was identified. Although it is known that targeted depletion of ANT4 in mice resulted in male infertility, the functional biochemical differences between ANT4 and other somatic ANT isoforms remain undetermined. To gain insight into ANT4, we expressed human ANT4 (hANT4) in yeast mitochondria. Unlike the somatic ANT proteins, expression of hANT4 failed to complement an AAC-deficient yeast strain for growth on media requiring mitochondrial respiration. Moreover, overexpression of hANT4 from a multi-copy plasmid interfered with optimal yeast growth. However, mutation of specific amino acids of hANT4 improved yeast mitochondrial expression and supported growth of the AAC-deficient yeast on non-fermentable carbon sources. The mutations affected amino acids predicted to interact with phospholipids, suggesting the importance of lipid interactions for function of this protein. Each mutant hANT4 and the somatic hANTs exhibited similar ADP/ATP exchange kinetics. These data define common and distinct biochemical characteristics of ANT4 in comparison to ANT1, 2 and 3 providing a basis for study of its unique adaptation to germ cells.     

Alteration of mitochondrial oxidative phosphorylation in aged skeletal muscle involves modification of adenine nucleotide translocator

The process of skeletal muscle aging is characterized by a progressive loss of muscle mass and functionality. The underlying mechanisms are highly complex and remain unclear. This study was designed to further investigate the consequences of aging on mitochondrial oxidative phosphorylation in rat gastrocnemius muscle, by comparing young (6 months) and aged (21 months) rats. Maximal oxidative phosphorylation capacity was clearly reduced in older rats, while mitochondrial efficiency was unaffected. Inner membrane properties were unaffected in aged rats since proton leak kinetics were identical to young rats. Application of top-down control analysis revealed a dysfunction of the phosphorylation module in older rats, responsible for a dysregulation of oxidative phosphorylation under low activities close to in vivo ATP turnover. This dysregulation is responsible for an impaired mitochondrial response toward changes in cellular ATP demand, leading to a decreased membrane potential which may in turn affect ROS production and ion homeostasis. Based on our data, we propose that modification of ANT properties with aging could partly explain these mitochondrial dysfunctions.     

Gender-dependent differences of mitochondrial function and oxidative stress in rat skeletal muscle at rest and after exercise training

Objective: This study investigated gender-dependent differences of mitochondrial function and sensitivity to in vitro ROS exposure in rat skeletal muscle at rest and after exercise training.

Methods: Wistar rats underwent running training for 6 weeks. In vitro measurements of hydroxyl radical production, oxygen consumption (under basal and maximal respiration conditions) and ATP production were made on permeabilized fibers. Mitochondrial function was examined after exposure and non-exposure to an in vitro generator system of reactive oxygen species (ROS). Antioxidant enzyme activities and malondialdehyde (MDA) content were also determined.

Results: Compared with sedentary males, females showed a greater resistance of mitochondrial function (oxygen consumption and ATP production) to ROS exposure, and lower MDA content and antioxidant enzyme activities. The training protocol had more beneficial effects in males than females with regard to ROS production and oxidative stress. In contrast to male rats, the susceptibility of mitochondrial function to ROS exposure in trained females was unchanged.

Discussion: Exercise training improves mitochondrial function oxidative capacities in both male and female rats, but is more pronounced in males as a result of different mechanisms. The resistance of mitochondrial function to in vitro oxidative stress exposure and the antioxidant responses are gender- and training-dependent, and may be related to the protective effects of estrogen.     

The enzymes of adenine nucleotide metabolism in developing skeletal muscle

1. During late foetal and early post-natal development of rabbit skeletal muscle the total protein increased more rapidly than the non-protein nitrogen content per g. wet wt. 2. AMP-deaminase activity of rabbit leg muscles increased rapidly over the period 5-15 days after birth. In diaphragm muscle from the same animal the rapid increase to the adult enzymic activity took place at about the time of birth. 3. The rapid increase in AMP-deaminase activity of leg muscle occurred earlier in animals born relatively mature, such as the chick and guinea pig, than in animals less well developed at birth, such as the rabbit and rat. 4. The pattern of enzymic activity shown by AMP deaminase during development in diaphragm, leg and cardiac muscles in a given species was closely paralleled by those of adenylate kinase and creatine phosphokinase. 5. When young rabbits were encouraged to become active at an earlier stage than is normal, the rise in creatine-phosphokinase activity occurred at an earlier age than in the control animals. 6. The results suggest that the activity pattern of the muscle is an important factor in determining the time at which the activities of the enzymes of special significance for muscle rise sharply to the adult values. 7. Development in rabbit leg muscle also involved an increase in aldolase activity. The pattern of change was similar to that obtained with other enzymes studied.     

Mitochondrial creatine kinase in contact sites: interaction with porin and adenine nucleotide translocase, role in permeability transition and sensitivity to oxidative damage

The creatine/phosphocreatine circuit provides an efficient energy buffering and transport system in a variety of cells with high and fluctuating energy requirements. It connects sites of energy production (mitochondria, glycolysis) with sites of energy consumption (various cellular ATPases). The cellular creatine/phosphocreatine pool is linked to the ATP/ADP pool by the action of different isoforms of creatine kinase located at distinct subcellular compartments. Octameric mitochondrial creatine kinase (MtCK), together with porin and adenine nucleotide translocase, forms a microcompartment at contact sites between inner and outer mitochondrial membranes and facilitates the production and export into the cytosol of phosphocreatine. MtCK is probably in direct protein-protein contact with outer membrane porin, whereas interaction with inner membrane adenine nucleotide translocase is rather mediated by acidic phopholipids (like cardiolipin) present in significant amounts in the inner membrane. Octamer-dimer transitions of MtCK as well as different creatine kinase substrates have a profound influence on controlling mitochondrial permeability transition (MPT). Inactivation by reactive oxygen species of MtCK and destabilization of its octameric structure are factors that contribute to impairment of energy homeostasis and facilitated opening of the MPT pore, which eventually lead to tissue damage during periods of ischemia/reperfusion.     

Dependence of creatine kinase and glycogen synthetase activities of skeletal muscles on state of adenine nucleotide phosphorylation and cAMP metabolism

Changes in the contents of adenine nucleotides, creatine phosphate, inorganic phosphate, creatine, glucose-6-phosphate and glycogen and the activity of adenylate cyclase, creatine kinase, glycogen phosphorylase 31:51-AMP-phosphodiesterase and glycogen synthetase in muscles and of blood catecholamines were studied in adult rats before loading, immediately after the cessation of the muscular activity, and at rest. Adenine nucleotides are established to play a regulatory role in catabolic and anabolic processes nucleotides are established to play a regulatory role in catabolic and anabolic processes related to the muscular activity. It is established that compensation and supercompensation of the working losses of muscular creatine phosphate and glycogen are due to activation of anabolic processes under conditions of higher phosphorylation of the adenylic system.     

Specific inhibition of ATP-ADP translocase in cardiac mitoplasts by antibodies against mitochondrial creatine kinase

Mitochondrial creatine kinase was purified from rat hearts and used to produce antibodies in chicken and rabbits. Antibodies were purified to a high degree of homogeneity by an affinity chromatography method. Chicken antibodies against mitochondrial creatine kinase inhibited this enzyme in rat-heart mitochondrial inner membrane and matrix preparation, and simultaneously blocked oxidative phosphorylation. Under these conditions respiratory chain activities remained unchanged, but adenine nucleotide translocase was inhibited. Removal of mitochondrial creatine kinase from the membrane by pretreatment with 0.15 M KCl and 20 mM ADP completely abolished the effect of antibodies against mitochondrial creatine kinase on oxidative phosphorylation. Noninhibitory antibodies from rabbit with high affinity to rat mitochondrial creatine kinase inhibited neither creatine kinase activity nor oxidative phosphorylation. These data show close and specific spatial arrangement of mitochondrial creatine kinase and adenine nucleotide translocase in mitochondria. It is supposed that there is a fixed orientation of these proteins in the cardiolipin domain in the membrane and that their interaction may occur by a frequent collision due to their lateral movement.     

Modification of liver mitochondrial lipids and of adenine nucleotide translocase and oxidative phosphorylation by cold adaptation

The decrease in respiration rate following thyroidectomy is preceded by changes in the lipid composition of the mitochondrial membrane (Hoch, F.L., Subramanian, C., Dhopeshwarkar, G.A. and Mead, J.F. (1981) Lipids 16, 328–334) and in concert, changes in the kinetic parameters of the adenine nucleotide translocase (Mak, I.T., Shrago, E. and Elson, C.E. (1981) Fed. Proc. 40, 398). To demonstrate that physiological adaptation also involves this sequence of events, rats were housed at 8°C for 3–4 weeks. Cold adaptation resulted in a modest (5%) increase in the unsaturation index for the mitochondrial fatty acids comprised of a significant increase in arachidonic acid and a reciprocal decrease in linoleic acid. Phospholipid analysis indicated that cold adaptation increased the mitochondrial phosphatidylethanolamine and reciprocally decreased the phosphatidylcholine content. Concomitantly, cold adaptation resulted in 25–30% increases in rat liver mitochondrial respiratory activities without changing the respiratory control or ADP/O ratios. The kinetic parameters of the adenine nucleotide translocase were determined by the back-exchange method (Pfaff, E. and Klingenberg, M. (1968) Eur. J. Biochem. 6, 66–79). At 0–4 and 10°C, the V_max and K_m of the cold-adapted rat liver adenine nucleotide translocase were not distinguishable from the control values. The K_i values determined by Dixon plot studies for atractylate and palmitoyl-CoA were also comparable between the two groups. However, at 25 and 37°C, cold-adapted rat liver adenine nucleotide translocase exhibited a 20% increase in V_max and a 20% decrease in K_m for external ADP. The results suggest that one adaption to a cold environment involves hormone-mediated changes in the lipid composition in the mitochondrial membranes which in turn modulate the adenine nucleotide translocase and subsequent respiratory activities.     

Purification and crystallization of creatine kinase from rabbit skeletal muscle

Crystallization is the primary rate-limiting step in protein structure determination. It has been our experience over approximately 10 years that crystals are obtained in about 20% of the proteins attempted and that only about 10% of these crystals are sufficiently well ordered to permit atomic resolution structure analysis. In attempts to overcome this limitation, we have investigated the effect on crystallization of microheterogeneity in a protein regarded as pure by conventional criteria. Creatine kinase was purified from rabbit skeletal muscle and crystallized from methylpentanediol. The protein appeared to be nearly pure judging by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high specific activity. The crystals that were obtained were of poor quality, and an extensive survey of precipitants, crystallization conditions, and additives failed to discover conditions from which usable crystals could be obtained. The enzyme was then subjected to a series of further purification steps. After each purification step, the quality of the crystals obtained under almost identical conditions improved. The final purification step was flat-bed isoelectric focusing. Crystals grown from focused creatine kinase are well ordered and diffract to approximately 3-A resolution.