Mitochondrial boundary membrane contact sites in brain: points of hexokinase and creatine kinase location, and control of Ca2+ transport

The location of hexokinase at the surface of brain mitochondria was investigated by electron microscopy using immuno-gold labelling techniques. The enzyme was located where the two mitochondrial limiting membranes were opposed and contact sites were possible. Disruption of the outer membrane by digitonin did not remove bound hexokinase and creatine kinase from brain mitochondria, although the activity of outer membrane markers and adenylate kinase decreased, suggesting a preferential location of both enzymes in the contact sites. In agreement with that, a membrane fraction was isolated from osmotically lysed rat brain mitochondria in which hexokinase and creatine kinase were concentrated. The density of this kinase-rich fraction was specifically increased by immuno-gold labelling of hexokinase, allowing a further purification by density gradient centrifugation. The fraction was composed of inner and outer limiting membrane components as shown by the specific marker enzymes, succinate dehydrogenase and NADH-cytochrome-c-oxidase (rotenone insensitive). As reported earlier for the enriched contact site fraction of liver mitochondria the fraction from brain mitochondria contained a high activity of glutathione transferase and a low cholesterol concentration. Moreover, the contacts showed a higher Ca2+ binding capacity in comparison to outer and inner membrane fractions. This finding may have regulatory implications because glucose phosphorylation via hexokinase activated the active Ca2+ uptake system and inhibited the passive efflux, resulting in an increase of intramitochondrial Ca2+.     

Further characterization of contact sites from mitochondria of different tissues: topology of peripheral kinases

A membrane fraction of intermediate density between inner and outer membrane was isolated by density gradient centrifugation from osmotically disrupted mitochondria of rat liver, brain, and kidney. The fraction was hexokinase rich and could therefore be further purified using specific antibodies against hexokinase and immunogold labelling techniques. In agreement with recent findings the gradient fraction which cosedimented with hexokinase contained the boundary membrane contact sites because it was composed of outer and inner membrane components and beside hexokinase, was enriched also by activity of creatine kinase and nucleoside diphosphate kinase. In contrast the activity of adenylate kinase appeared to be concentrated beyond the contact sites in the outer membrane fraction. By employing surface proteolysis analysis and specific blockers of the outer membrane pore we observed that the location of the kinases relative to the membrane components in the contact fraction resembled that of intact mitochondria. This specific organization of some peripheral kinases in the contact sites suggested an important role of the voltage dependence of the outer membrane pore, in that the pore may become limiting in anion exchange because of influence of the inner membrane potential on the closely attached outer membrane. Such control of anion exchange would lead to a dynamic compartmentation at the mitochondrial surface by the formation of contact sites, which may explain the preferential utilization of cytosolic creatine by the mitochondrial creatine kinase, as postulated in the phosphocreatine shuttle.     

Novel mitochondrial creatine transport activity. Implications for intracellular creatine compartments and bioenergetics

Immunoblotting of isolated mitochondria from rat heart, liver, kidney, and brain with antibodies made against N- and C-terminal peptide sequences of the creatine transporter, together with in situ immunofluorescence staining and immunogold electron microscopy of adult rat myocardium, revealed two highly related polypeptides with molecular masses of approximately 70 and approximately 55 kDa in mitochondria. These polypeptides were localized by immunoblotting of inner and outer mitochondrial membrane fractions, as well as by immunogold labeling in the mitochondrial inner membrane. In addition, a novel creatine uptake via a mitochondrial creatine transport activity was demonstrated by [(14)C]creatine uptake studies with isolated mitochondria from rat liver, heart, and kidney showing a saturable low affinity creatine transporter, which was largely inhibited in a concentration-dependent manner by the sulfhydryl-modifying reagent NEM, as well as by the addition of the above anti-creatine transporter antibodies to partially permeabilized mitochondria. Mitochondrial creatine transport was to a significant part dependent on the energetic state of mitochondria and was inhibited by arginine, and to some extent also by lysine, but not by other creatine analogues and related compounds. The existence of an active creatine uptake mechanism in mitochondria indicates that not only creatine kinase isoenzymes, but also creatine transporters and thus a certain proportion of the creatine kinase substrates, might be subcellularly compartmentalized. Our data suggest that mitochondria, shown here to possess creatine transport activity, may harbor such a creatine/phosphocreatine pool.     

Effects of ionic strength and sulfhydryl reagents on the binding of creatine phosphokinase to heart mitochondrial inner membranes

The concept that creatine phosphokinase is bound to the outer surface of the heart mitochondrial inner membrane originated from observations that the enzyme is retained by water-swollen heart mitochondria and by digitonintreated heart mitochondria suspended in isotonic sucrose. The present study establishes that digitonin-treated mitochondria release creatine phosphokinase in isotonic KCl, and other investigators have reported an identical response for the water-swollen organelles. These observations suggest that mitochondrial creatine phosphokinase is not bound to the outer surface of the inner membrane at a site adjacent to the adenine nucleotide translocase under physiologic conditions.     

The cationically selective state of the mitochondrial outer membrane pore: a study with intact mitochondria and reconstituted mitochondrial porin.

The outer mitochondrial membrane pore at a voltage above 20 to 30 mV can adopt a state of low conductance which may restrict free permeability of mitochondrial substrates. In order to obtain insight into the physiological meaning of this property we took advantage of the fact that the low conductance pore state could be induced by a polyanion in lipid bilayer membranes as well as in intact mitochondria. Upon reconstitution in artificial bilayers the pore in this substate became exclusively cation selective when the polarity of the applied voltage was negative on the cis-side. This behaviour of the pore would explain why induction of the low conductance pore state in intact mitochondria led to a complete inhibition of mitochondrial intermembranous kinases, such as creatine kinase and adenylate kinase, but not of peripheral kinases, for example hexokinase, when utilizing external ATP. The possibility that the inner membrane potential might be transduced to the outer membrane in the contact sites, suggests the existence of cation selective pores in these sites. This aspect may be important in the regulation of peripheral kinases like creatine kinase, nucleoside diphosphate kinase and adenylate kinase which are located behind the outer mitochondrial membrane.     

Protein import into mitochondria: ATP-dependent protein translocation activity in a submitochondrial fraction enriched in membrane contact sites and specific proteins

To identify the membrane regions through which yeast mitochondria import proteins from the cytoplasm, we have tagged these regions with two different partly translocated precursor proteins. One of these was bound to the mitochondrial surface of ATP-depleted mitochondria and could subsequently be chased into mitochondria upon addition of ATP. The other intermediate was irreversibly stuck across both mitochondrial membranes at protein import sites. Upon subfraction of the mitochondria, both intermediates cofractionated with membrane vesicles whose buoyant density was between that of inner and outer membranes. When these vesicles were prepared from mitochondria containing the chaseable intermediate, they internalized it upon addition of ATP. A non-hydrolyzable ATP analogue was inactive. This vesicle fraction contained closed, right-side-out inner membrane vesicles attached to leaky outer membrane vesicles. The vesicles contained the mitochondrial binding sites for cytoplasmic ribosomes and contained several mitochondrial proteins that were enriched relative to markers of inner or outer membranes. By immunoelectron microscopy, two of these proteins were concentrated at sites where mitochondrial inner and outer membranes are closely apposed. We conclude that these vesicles contain contact sites between the two mitochondrial membranes, that these sites are the entry point for proteins into mitochondria, and that the isolated vesicles are still translocation competent.     

Mitochondrial protein import: differential recognition of various transport intermediates by antibodies

The precursors of the mitochondrial proteins ADP/ATP carrier (AAC) and F1-ATPase subunit beta (F1 beta) were accumulated at the stages of binding to receptor sites on the mitochondrial outer membrane, or in contact sites between outer and inner membranes. Specific antibodies raised against the mature proteins were added to the isolated mitochondria and efficiently bound to these translocation intermediates. Further movement of the precursors to consecutive steps along their import pathway was thereby inhibited. Controls showed that precursor proteins which were inserted into or translocated across the outer membrane were not recognized by the antibodies unless the mitochondrial membranes were disrupted. We conclude that the trapped translocation intermediates have antigenic sites exposed to the outside of the outer membrane.     

Translocation and insertion of precursor proteins into isolated outer membranes of mitochondria

Nuclear-encoded proteins destined for mitochondria must cross the outer or both outer and inner membranes to reach their final sub- mitochondrial locations. While the inner membrane can translocate preproteins by itself, it is not known whether the outer membrane also contains an endogenous protein translocation activity which can function independently of the inner membrane. To selectively study the protein transport into and across the outer membrane of Neurospora crassa mitochondria, outer membrane vesicles were isolated which were sealed, in a right-side-out orientation, and virtually free of inner membranes. The vesicles were functional in the insertion and assembly of various outer membrane proteins such as porin, MOM19, and MOM22. Like with intact mitochondria, import into isolated outer membranes was dependent on protease-sensitive surface receptors and led to correct folding and membrane integration. The vesicles were also capable of importing a peripheral component of the inner membrane, cytochrome c heme lyase (CCHL), in a receptor-dependent fashion. Thus, the protein translocation machinery of the outer mitochondrial membrane can function as an independent entity which recognizes, inserts, and translocates mitochondrial preproteins of the outer membrane and the intermembrane space. In contrast, proteins which have to be translocated into or across the inner membrane were only specifically bound to the vesicles, but not imported. This suggests that transport of such proteins involves the participation of components of the intermembrane space and/or the inner membrane, and that in these cases the outer membrane translocation machinery has to act in concert with that of the inner membrane.     

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.     

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.     

The vectorial orientation of human monoamine oxidase in the mitochondrial outer membrane.

1. The localization of monoamine oxidase in the mitochondrial outer membrane was studied in preparations of human liver mitochondrial and brain-cortex non-synaptosomal and synaptosomal mitochondria. 2. Immunochemical accessibility in iso-osmotic and hypo-osmotic mitochondrial preparations was used to localize the enzyme. 3. It was shown that the immunochemically accessible tyramine-oxidizing activity was distributed approximately equally on both surfaces of the membrane in human liver and brain-cortex non-synaptosomal mitochondria. However, the immunochemically accessible beta-phenethylamine-oxidizing activity was situated predominantly on the outer surface, and the immunochemically accessible 5-hydroxytryptamine-oxidizing activity was situated predominantly on the inner surface of the mitochondrial outer membrane in liver and brain-cortex non-synaptosomal mitochondrial preparations. 4. Considerable variation in the distribution of the enzyme in preparations of synaptosomal mitochondria was seen. 5. The simplest model consistent with our observations is that, in liver and brain-cortex non-synaptosomal mitochondria, the tyramine-oxidizing activity is distributed on both sides of the mitochondrial outer membrane, the beta-phenethylamine-oxidizing activity is located on the outer surface of the outer membrane and the 5-hydroxytryptamine-oxidizing activity is located on the inner surface of the mitochondria outer membrane.     

Integration of porin synthesized in vitro into outer mitochondrial membranes

Porin, an intrinsic protein of outer mitochondrial membranes of rat liver, was synthesized in vitro in a cell-free in a cell-free translation system with rat liver RNA. The apparent molecular mass of porin synthesized in vitro was the same as that of its mature form (34 kDa). This porin was post-translationally integrated into the outer membrane of rat liver mitochondria when the cell-free translation products were incubated with mitochondria at 30 degrees C even in the presence of a protonophore (carbonyl cyanide m-chlorophenylhydrazone). Therefore, the integration of porin seemed to proceed energy-independently as reported by Freitag et al. [(1982) Eur. J. Biochem. 126, 197-202]. Its integration seemed, however, to require the participation of the inner membrane, since porin was not integrated when isolated outer mitochondrial membranes alone were incubated with the translation products. Porin in the cell-free translation products bound to the outside of the outer mitochondrial membrane when incubated with intact mitochondria at 0 degrees C for 5 min. When the incubation period at 0 degrees C was prolonged to 60 min, this porin was found in the inner membrane fraction, which contained monoamine oxidase, suggesting that porin might bind to a specific site on the outer membrane in contact or fused with the inner membrane (a so-called OM-IM site). This porin bound to the OM-IM site was integrated into the outer membrane when the membrane fraction was incubated at 30 degrees C for 60 min. These observations suggest that porin bound to the outside of the outer mitochondrial membrane is integrated into the outer membrane at the OM-IM site by some temperature-dependent process(es).     

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.     

Heterogeneity of ADP diffusion and regulation of respiration in cardiac cells

Heterogeneity of ADP diffusion and regulation of respiration were studied in permeabilized cardiomyocytes and cardiac fibers in situ and in silico. Regular arrangement of mitochondria in cells was altered by short-time treatment with trypsin and visualized by confocal microscopy. Manipulation of matrix volumes by changing K(+) and sucrose concentrations did not affect the affinity for ADP either in isolated heart mitochondria or in skinned fibers. Pyruvate kinase (PK)-phosphoenolpyruvate (PEP) were used to trap ADP generated in Ca,MgATPase reactions. Inhibition of respiration by PK-PEP increased 2-3 times after disorganization of regular mitochondrial arrangement in cells. ADP produced locally in the mitochondrial creatine kinase reaction was not accessible to PK-PEP in intact permeabilized fibers, but some part of it was released from mitochondria after short proteolysis due to increased permeability of outer mitochondrial membrane. In in silico studies we show by mathematical modeling that these results can be explained by heterogeneity of ADP diffusion due to its restrictions at the outer mitochondrial membrane and in close areas, which is changed after proteolysis. Localized restrictions and heterogeneity of ADP diffusion demonstrate the importance of mitochondrial functional complexes with sarcoplasmic reticulum and myofibrillar structures and creatine kinase in regulation of oxidative phosphorylation.     

Enzyme Distribution in Potato Mitochondria

Enzyme distribution in potato mitochondria was investigatedby selectively disrupting the outer and inner membranes withdigitonin. Antimycin-insensitive NADH-cytochrome c reductase,an outer membrane marker, was released at low digitonin concentrations(0.1 mg mg^–1 mitochondrial protein). Soluble matrix enzymes,fumarase and malate dehydrogenase were released at 0.3–0.4mg digitonin mg^–1 protein, as the inner membrane ruptured.Very little (about 10%) cytochrome oxidase activity was released,even at higher digitonin concentrations, in accord with thisenzyme being an integral inner membrane protein. By this criterionadenylate kinase is also firmly bound to the inner membrane.Evidence indicates that it faces the intermembrane space. Malic enzyme activity was released by the same digitonin concentrationthat released fumarase and malate dehydrogenase, indicatingthat malic enzyme is a soluble matrix enzyme. No activity wasreleased at low digitonin concentrations which selectively breakthe outer membrane, showing that malic enzyme is not presentin the intermembrane space. Considerable catalase activity (20—40 µmol O₂ min^–1mg^–1 protein) was associated with washed mitochondrialpreparations, but 95% of this was lost upon purification ofmitochondria. The remaining activity was firmly bound to themitochondrial membranes.     

Functional Coupling between Nucleoside Diphosphate Kinase of the Outer Mitochondrial Compartment and Oxidative Phosphorylation

In rat liver mitochondria all nucleoside diphosphate kinase of the outer compartment is associated with the outer surface of the outer membrane (Lipskaya, T. Yu., and Plakida, K. N. (2003) Biochemistry (Moscow), 68, 1136-1144). In the present study, three systems operating as ADP donors for oxidative phosphorylation have been investigated. The outer membrane bound nucleoside diphosphate kinase was the first system tested. Two others employed yeast hexokinase and yeast nucleoside diphosphate kinase. The two enzymes exhibited the same activity but could not bind to mitochondrial membranes. In all three systems, muscle creatine phosphokinase was the external agent competing with the oxidative phosphorylation system for ADP. Determination of mitochondrial respiration rate in the presence of increasing quantities of creatine phosphokinase revealed that at large excess of creatine phosphokinase activity over other kinase activities (of the three systems tested) and oxidative phosphorylation the creatine phosphokinase reaction reached a quasi-equilibrium state. Under these conditions equilibrium concentrations of all creatine phosphokinase substrates were determined and K(eq)app of this reaction was calculated for the system with yeast hexokinase. In samples containing active mitochondrial nucleoside diphosphate kinase the concentrations of ATP, creatine, and phosphocreatine were determined and the quasi-equilibrium concentration of ADP was calculated using the K(eq)app value. At balance of quasi-equilibrium concentrations of ADP and ATP/ADP ratio the mitochondrial respiration rate in the system containing nucleoside diphosphate kinase was 21% of the respiration rate assayed in the absence of creatine phosphokinase; in the system containing yeast hexokinase this parameter was only 7% of the respiration rate assayed in the absence of creatine phosphokinase. Substitution of mitochondrial nucleoside diphosphate kinase with yeast nucleoside diphosphate kinase abolished this difference. It is concluded that oxidative phosphorylation is accompanied by appearance of functional coupling between mitochondrial nucleoside diphosphate kinase and the oxidative phosphorylation system. Possible mechanisms of this coupling are discussed.     

Phosphate-Activated Glutaminase and Mitochondrial Glutamine Transport in the Brain

A review of the properties of purified and tissue bound phosphate activated glutaminase (PAG) in brain and kidney (pig and rat) is presented, based on kinetic, electron microscopic and immunocytochemical studies. PAG is a mitochondrial enzyme and two pools can be separated, a soluble and membrane associated one. Intact mitochondria appear to express PAG accessible only to the outer phase of the inner mitochondrial membrane. This PAG has properties similar to that of the membrane fraction and polymeric form of purified enzyme. PAG in the soluble fraction has properties similar to that of the monomeric form of purified enzyme and is assumed to be dormant due to the high matrix concentration of the inhibitor glutamate. A hypothetical model for the localization of PAG in the mitochondria is presented. The activity of PAG in vivo is assumed to be regulated by cytosolic glutamate and other compounds, that affect the activation by phosphate. Glutamine is transported into brain and kidney mitochondria by a protein catalyzed energy requiring process, which may be mediated by more than one protein. There is no correlation between glutamine hydrolysis and transport.     

Improved methods to isolate and subfractionate rat liver mitochondria. Lipid composition of the inner and outer membrane

Rat liver mitochondria were isolated by a combination of differential and Percoll gradient centrifugation, resulting in a highly pure and intact preparation, as assessed by marker enzyme analysis, latency of cytochrome-c oxidase, respiratory control index and electron microscopy. Two different methods were compared for the separation of inner and outer membranes. In the swell-shrink-sonicate procedure glycerol was included resulting in the isolation of one outer membrane and two inner membrane fractions of high purity. Using digitonin a highly selective and gradual solubilization of the outer membrane could be accomplished. Analysis of the phospholipid composition of the intact mitochondria and all subfractions showed that the inner membrane was virtually devoid of phosphatidylinositol and -serine, while the outer membrane contained 23% of the total mitochondrial cardiolipin, which did not originate from inner membrane contamination and therefore is a true component of the outer membrane.     

Influence of mitochondrial creatine kinase on the mitochondrial/extramitochondrial distribution of high energy phosphates in muscle tissue: evidence for a leak in the creatine shuttle

The influence of mitochondrial creatine kinase on subcellular high energy systems has been investigated using isolated rat heart mitochondria, mitoplasts and intact heart and skeletal muscle tissue.In isolated mitochondria, the creatine kinase is functionally coupled to oxidative phosphorylation at active respiratory chain, so that it catalyses the formation of creatine phosphate against its thermodynamic equilibrium. Therefore the mass action ratio is shifted from the equilibrium ratio to lower values. At inhibited respiration, it is close to the equilibrium value, irrespective of the mechanism of the inhibition. The same results were obtained for mitoplasts under conditions where the mitochondrial creatine kinase is still associated with the inner membrane.In intact tissue increasing amounts of creatine phosphate are found in the mitochondrial compartment when respiration and/or muscle work are increased. It is suggested that at high rates of oxidative phosphorylation creatine phosphate is accumulated in the intermembrane space due to the high activity of mitochondrial creatine kinase and the restricted permeability of reactants into the extramitochondrial space. A certain amount of this creatine phosphate leaks into the mitochondrial matrix.This leak is confirmed in isolated rat heart mitochondria where creatine phosphate is taken up when it is generated by the mitochondrial creatine kinase reaction. At inhibited creatine kinase, external creatine phosphate is not taken up. Likewise, mitoplasts only take up creatine phosphate when creatine kinase is still associated with the inner membrane. Both findings indicate that uptake is dependent on the functional active creatine kinase coupled to oxidative phosphorylation.Creatine phosphate uptake into mitochondria is inhibited with carboxyatractyloside. This suggests a possible role of the mitochondrial adenine nucleotide translocase in creatine phosphate uptake.Taken together, our findings are in agreement with the proposal that creatine kinase operates in the intermembrane space as a functional unit with the adenine nucleotide translocase in the inner membrane for optimal transfer of energy from the electron transport chain to extramitochondrial ATP-consuming reactions.     

Studies on the nature of adenosine diphosphatase activity from rat liver mitochondria

Adenosine diphosphatase (ADPase) activity was studied in rat liver with [beta-32P]ADP as a substrate. Mitochondria and outer mitochondrial membrane fractions were isolated and assayed for ADPase and various marker enzymes. ADPase activity was strikingly reduced when the outer membranes were removed from the mitochondria whether by digitonin treatment or osmotic shock. Addition of the inter-membrane space subfraction to the purified outer membranes resulted in enhanced ADPase activity. Addition of the inter-mitochondrial membrane enzyme adenylate kinase to outer membranes also produced a large stimulation of activity. The ADPase activity could also be reconstituted in vitro with adenylate kinase and either mitoplast ATPase or ouabain-sensitive (Na+ + K+ + Mg2+)-ATPase. Chloroform-released ATPase, however, was not capable of producing an ADPase activity when combined with adenylate kinase. Gel permeation chromatography of Triton-solubilised outer mitochondrial membranes was unable to resolve ADPase activity from contaminating ATPase. These results suggest that the majority of ADPase activity in rat liver mitochondria consists of the coupled activity of adenylate kinase and ATPase.