Inside-out Submitochondrial Particles Research Articles

Different effects of guanine nucleotides (GDP and GTP) on protein-mediated mitochondrial proton leak.

In this study, we compared the influence of GDP and GTP on isolated mitochondria respiring under conditions favoring oxidative phosphorylation (OXPHOS) and under conditions excluding this process, i.e., in the presence of carboxyatractyloside, an adenine nucleotide translocase inhibitor, and/or oligomycin, an FOF1-ATP synthase inhibitor. Using mitochondria isolated from rat kidney and human endothelial cells, we found that the action of GDP and GTP can differ diametrically depending on the conditions. Namely, under conditions favoring OXPHOS, both in the absence and presence of linoleic acid, an activator of uncoupling proteins (UCPs), the addition of 1 mM GDP resulted in the state 4 (non-phosphorylating respiration)-state 3 (phosphorylating respiration) transition, which is characteristic of ADP oxidative phosphorylation. In contrast, the addition of 1 mM GTP resulted in a decrease in the respiratory rate and an increase in the membrane potential, which is characteristic of UCP inhibition. The stimulatory effect of GDP, but not GTP, was also observed in inside-out submitochondrial particles prepared from rat kidney mitochondria. However, the effects of GDP and GTP were more similar in the presence of OXPHOS inhibitors. The importance of these observations in connection with the action of UCPs, adenine nucleotide translocase (or other carboxyatractyloside-sensitive carriers), carboxyatractyloside- and purine nucleotide-insensitive carriers, as well as nucleoside-diphosphate kinase (NDPK) are considered. Because the measurements favoring oxidative phosphorylation better reflect in vivo conditions, our study strongly supports the idea that GDP cannot be considered a significant physiological inhibitor of UCP. Moreover, it appears that, under native conditions, GTP functions as a more efficient UCP inhibitor than GDP and ATP.

Mussel and mammalian ATP synthase share the same bioenergetic cost of ATP

The molecular mechanism by which the membrane-embedded FO sector of the mitochondrial ATP synthase translocates protons, thus dissipating the transmembrane protonmotive force and leading to ATP synthesis, involves the neutralization of the carboxylate residues of the c-ring. Carboxylates are thought to constitute the binding sites for ion translocation. In order to cast light on this mechanism, we exploited N,N'-dicyclohexylcarbodiimide, which covalently binds to FO c-ring carboxylates, and ionophores which selectively modulate the transmembrane electric (Δφ) and chemical (ΔpH) gradients such as valinomycin, nigericin and dinitrophenol. ATP hydrolysis was evaluated in mitochondrial preparations and/or inside-out submitochondrial particles from mussel and mammalian tissues under different experimental conditions. The experiments pointed out striking similarities between mussel and mammalian mitochondrial ATP synthase. Our results support the hypothesis that the ATP synthase of Mytilus galloprovincialis induces intersubunit torque generation and translocates H(+) by coordinating the hydronium ion (H3O(+)) in the ion binding site of FO. Our results are consistent with the hypothesis that in mussel mitochondria the main component of the electrochemical gradient driving proton flux and ATP synthesis is Δφ. Therefore, mussel FO probably contains a small c-ring, which implies a low bioenergetic cost of making ATP as in mammals. These features which make mussel mitochondria as efficient in ATP production as mammalian ones may be especially advantageous in facultative aerobic species which intermittently exploit mitochondrial respiration to generate ATP.

Partitioning of superoxide and hydrogen peroxide production by mitochondrial respiratory complex I

Membrane-bound respiratory complex I in inside-out submitochondrial particles (SMP) catalyzes both superoxide and hydrogen peroxide formation in NADH- and/or succinate-supported reactions. At optimal NADH concentration (50μM), the complex I-mediated process results in a formation of two superoxide anions and H2O2 as the reaction products in approximately 0.7 ratio. Almost the same ratio is found for purified complex I (0.6) and for the aerobic succinate-supported reverse electron transfer reaction. Superoxide production is depressed at high, more physiologically relevant NADH concentrations, whereas hydrogen peroxide formation is insensitive to the elevated level of NADH. The rates of H2O2 formation at variable NAD+/NADH ratios satisfactorily fit the Nernst equation for a single reactive two-electron donor component equilibrated with ambient midpoint redox potential of −347mV (0.13 NAD+/NADH ratio, pH 8.0). Half-maximal superoxide production rate proceeds at significantly higher NAD+/NADH ratio (0.33). Guanidine strongly stimulates NADH-supported hydrogen peroxide and superoxide production at any NADH concentration and activates NADH:ferricyanide and inhibits NADH:hexaammineruthenium (III) reductase activities while showing no effects on NADH oxidase of SMP. In the low range of NADH concentration, superoxide production rate shows a simple hyperbolic dependence on NADH with apparent KmNADH of 0.5μM, whereas sigmoidal dependence of hydrogen peroxide production is seen with half-maximal rate at 25μM NADH. We interpret the data as to suggest that at least two sites participate in complex I-mediated ROS generation: FMNH– that produces hydrogen peroxide, and an iron–sulfur center (likely N-2) that produces superoxide anion.

Tri-n-butyltin binding to a low-affinity site decreases the F1FO-ATPase sensitivity to oligomycin in mussel mitochondria

In mussel digestive gland mitochondria the environmental pollutant tri-n-butyltin (TBT), other than strongly inhibiting ATPase activity at <1.0 μ m, at ≥1.0 μ m concentration was previously found to desensitize F1FO-ATPase to the antibiotic oligomycin. While F1FO-ATPase inhibition is widely known as one of the main mitochondrial damages caused by TBT, the enzyme's desensitization to oligomycin was quite unexpected. The possible mechanisms involved are here stepwise approached, aiming at enlightening the molecular mechanism(s) of TBT toxicity and the still poorly investigated oligomycin interaction with FO. The findings strongly suggest that the oligomycin desensitization directly stems from the covalent binding of TBT to monothiols of the F1FO-ATPase. This binding implies sulfur oxidation, irrespective of the possible formation of radical species in mitochondria, a mechanism which does not seem to be involved here. It is hypothesized that TBT interacts with the enzyme complex in at least two sites distinguished by different affinities: TBT binding to the high-affinity site would lead to ATPase inhibition, while TBT binding to monothiols in the low-affinity site could mirror the decrease in F1FO-ATPase oligomycin sensitivity at ≥1.0 μ m TBT. Experiments carried out on inside-out submitochondrial particles hint that TBT binding destabilizes the oligomycin-blocked FO conformation, allowing proton flux recovery within FO, without uncoupling the catalytic function from proton channeling. Copyright © 2012 John Wiley & Sons, Ltd.

Submitochondrial fragments of brain mitochondria: General characteristics and catalytic properties of NADH:ubiquinone oxidoreductase (Complex I)

A number of genetic or drug-induced pathophysiological disorders, particularly neurodegenerative diseases, have been reported to correlate with catalytic impairments of NADH:ubiquinone oxidoreductase (mitochondrial complex I). The vast majority of the data on catalytic properties of this energy-transducing enzyme have been accumulated from studies on bovine heart complex I preparations of different degrees of resolution, whereas almost nothing is known about the functional activities of the enzyme in neuronal tissues. Here a procedure for preparation of coupled inside-out submitochondrial particles from brain is described and their NADH oxidase activity is characterized. The basic characteristics of brain complex I, particularly the parameters of A/D-transition are found to be essentially the same as those previously reported for heart enzyme. The results show that coupled submitochondrial particles prepared from either heart or brain can equally be used as a model system for in vitro studies aimed to delineate neurodegenerative-associated defects of complex I.

Effect of the ATPase inhibitor protein IF 1 on H + translocation in the mitochondrial ATP synthase complex

The H + F oF 1-ATP synthase complex of coupling membranes converts the proton-motive force into rotatory mechanical energy to drive ATP synthesis. The F 1 moiety of the complex protrudes at the inner side of the membrane, the F o sector spans the membrane reaching the outer side. The IF 1 component of the mitochondrial complex is a basic 10 kDa protein, which inhibits the F oF 1-ATP hydrolase activity. The mitochondrial matrix pH is the critical factor for the inhibitory binding of the central segment of IF 1 (residue 42–58) to the F 1-α/β subunits. We have analyzed the effect of native purified IF 1 the IF 1-(42–58) synthetic peptide and its mutants on proton conduction, driven by ATP hydrolysis or by [K +] gradients, in bovine heart inside-out submitochondrial particles and in liposome-reconstituted F oF 1 complex. The results show that IF 1, and in particular its central 42–58 segment, displays different inhibitory affinity for proton conduction from the F 1 to the F o side and in the opposite direction. Cross-linking of IF 1 to F 1-α/β subunits inhibits the ATP-driven H + translocation but enhances H + conduction in the reverse direction. These observation are discussed in terms of the rotary mechanism of the F oF 1 complex.

Methylmalonate inhibits succinate‐supported oxygen consumption by interfering with mitochondrial succinate uptake

The effect of methylmalonate (MMA) on mitochondrial succinate oxidation has received great attention since it could present an important role in energy metabolism impairment in methylmalonic acidaemia. In the present work, we show that while millimolar concentrations of MMA inhibit succinate-supported oxygen consumption by isolated rat brain or muscle mitochondria, there is no effect when either a pool of NADH-linked substrates or N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD)/ascorbate were used as electron donors. Interestingly, the inhibitory effect of MMA, but not of malonate, on succinate-supported brain mitochondrial oxygen consumption was minimized when nonselective permeabilization of mitochondrial membranes was induced by alamethicin. In addition, only a slight inhibitory effect of MMA was observed on succinate-supported oxygen consumption by inside-out submitochondrial particles. In agreement with these observations, brain mitochondrial swelling experiments indicate that MMA is an important inhibitor of succinate transport by the dicarboxylate carrier. Under our experimental conditions, there was no evidence of malonate production in MMA-treated mitochondria. We conclude that MMA inhibits succinate-supported mitochondrial oxygen consumption by interfering with the uptake of this substrate. Although succinate generated outside the mitochondria is probably not a sig-nificant contributor to mitochondrial energy generation, the physiopathological implications of MMA-induced inhibition of substrate transport by the mitochondrial dicarboxylate carrier are discussed.

Topography and chemical reactivity of the active–inactive transition-sensitive SH-group in the mitochondrial NADH:ubiquinone oxidoreductase (Complex I)

The spatial arrangement and chemical reactivity of the activation-dependent thiol in the mitochondrial Complex I was studied using the membrane penetrating N-ethylmaleimide (NEM) and non-penetrating anionic 5,5′-dithiobis-(2-nitrobenzoate) (DTNB) as the specific inhibitors of the enzyme in mitochondria and inside-out submitochondrial particles (SMP). Both NEM and DTNB rapidly inhibited the de-activated Complex I in SMP. In mitochondria NEM caused rapid inhibition of Complex I, whereas the enzyme activity was insensitive to DTNB. In the presence of the channel-forming antibiotic alamethicin, mitochondrial Complex I became sensitive to DTNB. Neither active nor de-activated Complex I in SMP was inhibited by oxidized glutathione (10 mM, pH 8.0, 75 min). The data suggest that the active/de-active transition sulfhydryl group of Complex I which is sensitive to inhibition by NEM is located at the inner membrane–matrix interface. These data include the sidedness dependency of inhibition, effect of pH, ionic strength, and membrane bilayer modification on enzyme reactivity towards DTNB and its neutral analogue.

Oxidation and reduction of pyridine nucleotides in alamethicin-permeabilized plant mitochondria.

The inner mitochondrial membrane is selectively permeable, which limits the transport of solutes and metabolites across the membrane. This constitutes a problem when intramitochondrial enzymes are studied. The channel-forming antibiotic AlaM (alamethicin) was used as a potentially less invasive method to permeabilize mitochondria and study the highly branched electron-transport chain in potato tuber (Solanum tuberosum) and pea leaf (Pisum sativum) mitochondria. We show that AlaM permeabilized the inner membrane of plant mitochondria to NAD(P)H, allowing the quantification of internal NAD(P)H dehydrogenases as well as matrix enzymes in situ. AlaM was found to inhibit the electron-transport chain at the external Ca2+-dependent rotenone-insensitive NADH dehydrogenase and around complexes III and IV. Nevertheless, under optimal conditions, especially complex I-mediated NADH oxidation in AlaM-treated mitochondria was much higher than what has been previously measured by other techniques. Our results also show a difference in substrate specificities for complex I in mitochondria as compared with inside-out submitochondrial particles. AlaM facilitated the passage of cofactors to and from the mitochondrial matrix and allowed the determination of NAD+ requirements of malate oxidation in situ. In summary, we conclude that AlaM provides the best method for quantifying NADH dehydrogenase activities and that AlaM will prove to be an important method to study enzymes under conditions that resemble their native environment not only in plant mitochondria but also in other membrane-enclosed compartments, such as intact cells, chloroplasts and peroxisomes.

In situ assay of the intramitochondrial enzymes: use of alamethicin for permeabilization of mitochondria

The channel-forming antibiotic alamethicin was used to permeabilize mitochondrial membranes for the low molecular mass hydrophilic substrates NADH and ATP. Alamethicin-treated mitochondria show high rotenone-sensitive NADH oxidase, NADH-quinone reductase, and oligomycin-sensitive and carboxyatractylate-insensitive ATPase activities. Alamethicin does not affect Complex I and ATPase activities in inside-out submitochondrial particles. Permeabilized mitochondria quantitatively retain their aconitase and iso-citrate dehydrogenase activities. Electron microscopy of alamethicin-treated mitochondria reveals no disruption of their outer and inner membranes. From the results obtained it is recommended, that alamethicin be used for the in situ catalytic assay of intramitochondrially located enzymes.

Assessment of Membrane-Bound Mammal Mitochondrial Adenine Nucleotide Translocase Topography by Experimental Antibodies

To gain insight into the immunogenicity of mitochondrial adenine nucleotide translocase (ANT), we raised antibodies against purified bovine heart ANT by induction of ascitic fluid in male Balb/c mice. We identified the antigenic determinants detected by these antibodies by (1) immunodetection of GST-ANT fusion proteins and selected partial constructs of ANT, (2) immunodetection of chemically synthesized overlapping peptides on solid support, and (3) back-titration ELISA. Results revealed a short epitope spreading of the antibodies, resulting in a small number of antigenic determinants. Thus, each antibody detects one or two major epitopes located in the putative hydrophilic loops M2 and M3. No evidence for the antigenicity of the first 133 amino acids of ANT was obtained. These well-characterized antibodies were used to study the topography of the membrane-bound ANT by back-titration ELISA with mitochondrial membranes. We demonstrated that amino acids 145-150 and 230-237 are fully accessible to the antibodies in native ANT, whereas regions 133-140 and 244-251 are not. Furthermore, we used mitochondria devoid of the outer membrane (mitoplasts) and inside-out submitochondrial particles (SMP) to establish the matrix or cytosolic orientation of loops M2 and M3. The results clearly show that these loops have a matrix orientation and thus support the six transmembrane segment model of ANT topography in the inner mitochondrial membrane.

Catalytic Activity of NADH-ubiquinone Oxidoreductase (Complex I) in Intact Mitochondria: EVIDENCE FOR THE SLOW ACTIVE/INACTIVE TRANSITION

The mammalian purified dispersed NADH-ubiquinone oxidoreductase (Complex I) and the enzyme in inside-out submitochondrial particles are known to be the slowly equilibrating mixture of the active and de-activated forms (Vinogradov, A. D. (1998) Biochim. Biophys. Acta 1364, 169-185). We report here the phenomenon of slow active/de-active transition in intact mitochondria where the enzyme is located within its natural environment being exposed to numerous mitochondrial matrix proteins. A simple procedure for permeabilization of intact mitochondria by channel-forming antibiotic alamethicin was worked out for the "in situ" assay of Complex I activity. Alamethicin-treated mitochondria catalyzed the rotenone-sensitive NADH-quinone reductase reaction with exogenousely added NADH and quinone-acceptor at the rates expected if the enzyme active sites would be freely accessible for the substrates. The matrix proteins were retained in alamethicin-treated mitochondria as judged by their high rotenone-sensitive malate-cytochrome c reductase activity in the presence of added NAD(+). The sensitivity of Complex I to N-ethylmaleimide and to the presence of Mg(2+) was used as the diagnostic tools to detect the presence of the de-activated enzyme. The NADH-quinone reductase activity of alamethicin-treated mitochondria was sensitive to neither N-ethylmaleimide nor Mg(2+). After exposure to elevated temperature (37 degrees C, the conditions known to induce de-activation of Complex I) the enzyme activity became sensitive to the sulfhydryl reagent and/or Mg(2+). The sensitivity to both inhibitors disappeared after brief exposure of the thermally de-activated mitochondria with malate/glutamate, NAD(+), and cytochrome c (the conditions known for the turnover-induced reactivation of the enzyme). We conclude that the slow active/de-active Complex I transition is a characteristic feature of the enzyme in intact mitochondria and discuss its possible physiological significance.

H+/2e- stoichiometry of the nadh:ubiquinone reductase reaction catalyzed by submitochondrial particles.

Mitochondrial NADH:ubiquinone-reductase (Complex I) catalyzes proton translocation into inside-out submitochondrial particles. Here we describe a method for determining the stoichiometric ratio H+/2e- (n) for the coupled reaction of NADH oxidation by the quinone acceptors. Comparison of the initial rates of NADH oxidation and alkalinization of the surrounding medium after addition of small amounts of NADH to coupled particles in the presence of Q1 gives the value of n = 4. Thermally induced deactivation of Complex I [1, 2] results in complete inhibition of the NADH oxidase reaction but only partial inhibition of the NADH:Q1-reductase reaction. N-Ethylmaleimide (NEM) prevents reactivation and thus completely blocks the thermally deactivated enzyme. The residual NADH:Q1-reductase activity of the deactivated, NEM-treated enzyme is shown to be coupled with the transmembraneous proton translocation (n = 4). Thus, thermally induced deactivation of Complex I as well as specific inhibitors of the endogenous ubiquinone reduction (rotenone, piericidin A) do not inhibit the proton translocating activity of the enzyme.

Applications of aqueous two-phase partition to isolation of membranes from plants: A periodic NADH oxidase activity as a marker for right side-out plasma membrane vesicles

Phase separations using standardized mixtures of polyethylene glycol, dextran and potassium phosphate are used widely to prepare highly purified plasma membranes from plants and in the preparation of chloroplast subfractions. Other uses include the removal of right side-out plasma membrane vesicles as contaminants from Golgi apparatus, endoplasmic reticulum and tonoplast (vacuole membrane) fractions and separation of right side-out and inside-out plasma membrane vesicles. The higher degree of separation between plasma membranes into the upper phase and internal membranes into the lower phase is in large measure due to the fact that only plasma membranes are oriented cytoplasmic side in. Most other membranes are oriented cytoplasmic side-out. This property extends to separations of right side-out and inside-out plasma membrane vesicles and to the separation of right side-out and inside-out sub-mitochondrial particles. The inside-out vesicles partition into the lower phase whereas the right side-out vesicles remain in the upper phase. The lack of efficacy of aqueous two-phase partitioning in other types of membrane separations is apparently due to the fact that surface characteristics that may distinguish different internal membranes are not located at the cytosolic membrane surface. At present there are no direct enzymatic markers for right side-out plasma membrane vesicles from plants. Demonstrations of sidedness and estimates of fraction purity are based on measurements of latency of marker enzymes, e.g., ATPases, at the cytosolic surface. This report describes a periodic NADH oxidase as an enzyme marker for right side-out plasma membrane vesicles not requiring detergent disruptions of vesicles for measurement of activity.

Two Separate Transhydrogenase Activities Are Present in Plant Mitochondria

Inside-out submitochondrial particles from both potato tubers and pea leaves catalyze the transfer of hydride equivalents from NADPH to NAD+ as monitored with a substrate-regenerating system. The NAD+ analogue acetylpyridine adenine dinucleotide is also reduced by NADPH and incomplete inhibition by the complex I inhibitor diphenyleneiodonium (DPI) indicates that two enzymes are involved in this reaction. Gel-filtration chromatography of solubilized mitochondrial membrane complexes confirms that the DPI-sensitive TH activity is due to NADH–ubiquinone oxidoreductase (EC 1.6.5.3, complex I), whereas the DPI-insensitive activity is due to a separate enzyme eluting around 220 kDa. The DPI-insensitive TH activity is specific for the 4B proton on NADH, whereas there is no indication of a 4A-specific activity characteristic of a mammalian-type energy-linked TH. The DPI-insensitive TH may be similar to the soluble type of transhydrogenase found in, e.g., Pseudomonas. The presence of non-energy-linked TH activities directly coupling the matrix NAD(H) and NADP(H) pools will have important consequences for the regulation of NADP-linked processes in plant mitochondria.

Phosphoproteins and Protein Kinase Activities Intrinsic to Inner Membranes of Potato Tuber Mitochondria

Inside-out submitochondrial particles (IO-SMP) were isolated and purified from potato (Solanum tuberosum L. cv.) tubers. When these IO-SMP were incubated with [γ32P]ATP more then 20 proteins became labelled as a result of phosphorylation. The 32P incorporation was stimulated by the oxidizing reagent ferricyanide. Except for a 17 kDa protein which was phosphorylated only in the absence of divalent cations, the protein phosphorylation required Mg2+. The time for half-maximum 32P incorporation was 4 min for the 22 kDa phospho-F1 δ-subunit and 2 min for the 28 kDa phospho-F0 b-subunit of the proton-ATPase. The K(m) for ATP for the detected phosphoproteins was between 65 μM and 110 μM. The pH optimum for protein phosphorylation in inner membranes was between pH 6 and 8, and for the F1 δ-subunit and the F0 b-subunit the pH optima were 6.5-8 and pH 8, respectively. A 37 kDa phosphoprotein was phosphorylated on a histidine residue while the remainder of the inner membrane proteins were phosphorylated on serine or threonine residues. Two autophosphorylated putative kinases were identified: one at 16.5 kDa required divalent cations for autophosphorylation, while another at 30 kDa did not. A 110 kDa protein was labelled only with [α-32P]ATP, suggesting adenylylation. (Less)

Two Subunits of the FoF1-ATPase Are Phosphorylated in the Inner Mitochondrial Membrane

Inside-out submitochondrial particles from potato tuber mitochondria were incubated with [γ-32P]ATP. More than 16 phosphorylated polypeptides were detected by autoradiography on an SDS-gel. Two phosphoproteins, migrating at 22 and 28 kDa, were excised from the SDS-gel, electroeluted, and purified further by anion chromatography. The phosphoproteins were N-terminally sequenced. Over the regions sequenced, the 22 and 28 kDa phosphoproteins had 100% sequence identity with potato proteins identified as the δ’-subunit of the F1-ATPase and theb-subunit of the FoATPase, respectively. We suggest that phosphorylation of these proteins may control the interaction between F1and Foand regulate energy coupling in oxidative phosphorylation.

Ubiquinone-1 Induces External Deamino-NADH Oxidation in Potato Tuber Mitochondria.

The addition of ubiquinone-1 (UQ-1) induced Ca2+-independent oxidation of deamino-NADH and NADH by intact potato (Solanum tuberosum L. cv Bintje) tuber mitochondria. The induced oxidation was coupled to the generation of a membrane potential. Measurements of NAD+-malate dehydrogenase activity indicated that the permeability of the inner mitochondrial membrane to NADH and deamino-NADH was not altered by the addition of UQ-1. We conclude that UQ-1-induced external deamino-NADH oxidation is due to a change in specificity of the external rotenone-insensitive NADH dehydrogenase. The addition of UQ-1 also induced rotenone-insensitive oxidation of deamino-NADH by inside-out submitochondrial particles, but whether this was due to a change in the specificity of the internal rotenone-insensitive NAD(P)H dehydrogenase or to a bypass in complex I could not be determined.

Purification and Characterization of a 43-kDa Rotenone-insensitive NADH Dehydrogenase from Plant Mitochondria

A 43-kDa NAD(P)H dehydrogenase was purified from red beetroot mitochondria. An antibody against this dehydrogenase was used in conjunction with the membrane-impermeable protein cross-linker 3,3'-dithiobis(sulfosuccinimidylpropionate) to localize the dehydrogenase on the matrix side of the inner membrane. Immunoblotting showed that the dehydrogenase was found in mitochondria isolated from several plant species but not from rat livers. Antibodies against the purified dehydrogenase partially inhibited rotenoneinsensitive internal NADH oxidation by inside-out submitochondrial particles. The level of rotenone-insensitive respiration with NAD-linked substrates correlated with the amount of 43-kDa NAD(P)H dehydrogenase present in mitochondria isolated from different soybean tissues. Based on these results, we conclude that the 43-kDa NAD(P)H dehydrogenase is responsible for rotenone-insensitive internal NADH oxidation in plant mitochondria.

Inhibition by Ca 2+ of the hydrolysis and the synthesis of ATP in Ehrlich ascites tumour mitochondria: relation to the Crabtree effect

Phosphorylation of ADP and hydrolysis of ATP by isolated mitochondria from Ehrlich ascites tumuor cells is greatly reduced when the mitochondria have been preloaded with Ca 2+ (50 nmol/mg protein or more). Translocation of ADP is diminished in Ca 2+-loaded mitochondria. However, ATPase in toluene-permeabilized mitochondria and in inside-out submitochondrial particles is also strongly inhibited by micromolar concentrations of Ca 2+, indicating that, independently of adenine nucleotide transport, F 1F 0-ATPase is also affected. ATP hydrolysis by submitochondrial particles depleted of the inhibitory subunit of F 1F 0-ATPase is also inhibitor) is insensitive to Ca 2+; however, this sensitivity is restored when the particles are supplemented with the inhibitory subunit isolated from beef heart mitochondria. In view of the previous observations that glucose elicits in Ehrlich ascites tumour cells an increase of cytoplasmic free Ca 2+ (Teplova, V.V., Bogucka, K., Czy/.z, A., Evtodienko, YuV., Duszyński, J. and Wojtczak, L. (1003) Biochem. Biophys. Res. Commun. 196, 1148–1154) and that this calcium is then taken up by mitochondria, resulting in a strong inhibition of coupled respiration (Evtodienko, Yu.V.,Teplova, V.V., Duszyński, J., Bogucka, K. and Wojtczak, L. (1994) Cell Calcium 15, 439–446), the present results are discussed in terms of the mechanism of the Crabtree effect in tumour cells.