The heterogeneity of rat brain mitochondria isolated on continuous sucrose gradients

Abstract— The distribution of a series of enzymes in the post-nuclear supernatant of rat brain homogenates was investigated following continuous density-gradient centrifugation. The enzymes studied were acetyl coenzyme A synthetase, glutamic dehydrogenase, glutamine synthetase, glutaminase I, succinic dehydrogenase and monoamine oxidase. Each of these enzymes with the exception of glutamine synthetase appears predominantly in the mitochondrial region of the gradient. Although about 20 per cent of this enzyme is present in the crude mitochondrial pellet, on density gradient centrifugation no special association of glutamine synthetase with any of the mitochondrial fractions was observed. Each of the other enzymes studied was found to have a characteristic distribution in the gradient; this suggests that brain mitochondria may be heterogeneous both in buoyant density and in their enzyme content. Three principal fractions are described: (i) dense particles containing high concentrations of acetyl coenzyme A synthetase and glutamic dehydrogenase; (ii) a fraction comprising the bulk of the mitochondria with high levels of monoamine oxidase, succinic dehydrogenase and glutaminase I; and (iii) particles in the synaptic ending region of the gradient characterized by relatively high levels of monoamine oxidase and succinic dehydrogenase and containing only small amounts of the other enzymes studied. If the mitochondrial heterogeneity that is observed on centrifugation reflects the existence within brain cells of mitochondria with specialized function, a partial explanation may be available for multiple pools of tricarboxylic acid cycle intermediates which have been postulated from isotopie labelling experiments.     

High content of mitochondrial glycerol-3-phosphate dehydrogenase in pancreatic islets and its inhibition by diazoxide

Homogenates of isolated pancreatic islets contain 40-70 times as much flavin-linked glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) as homogenates of whole pancreas, liver, heart, or skeletal muscle when the activity is assayed with either iodonitrotetrazolium or with dichloroindophenol as an electron acceptor. Intact mitochondria from islets release 3HOH from [2-3H]glycerol phosphate 7 times faster than do skeletal muscle mitochondria. The activity of the cytosolic, NAD-linked, glycerol phosphate dehydrogenase (EC 1.1.1.8) in pancreatic islets is comparable to that of the mitochondrial dehydrogenase so a glycerol phosphate shuttle is possible in pancreatic islets. Diazoxide, an inhibitor of insulin release in vivo and in vitro, inhibits the islet mitochondrial glycerol phosphate dehydrogenase in all three of the assays mentioned above at concentrations that inhibit insulin release and CO2 formation from glucose by isolated pancreatic islets. Diazoxide does not inhibit the dehydrogenase in mitochondria from skeletal muscle, liver, and heart. A slight inhibition in mitochondria from whole pancreas can be accounted for as inhibition of the islet dehydrogenase because no inhibition is observed in mitochondria from pancreas of rats treated with alloxan, an agent that causes diabetes by destroying pancreatic beta cells. The results of this study are compatible with the hypothesis that the mitochondrial glycerol phosphate dehydrogenase has a key role in stimulus-secretion coupling in the pancreatic beta cell during glucose-induced insulin release.     

Preparation and properties of mitochondria derived from synaptosomes.

A method has been developed whereby a fraction of rat brain mitochondria (synaptic mitochondria) was isolated from synaptosomes. This brain mitochondrial fraction was compared with the fraction of "free" brain mitochondria (non-synaptic) isolated by the method of Clark & Nicklas (1970). (J. Biol. Chem. 245, 4724-4731). Both mitochondrial fractions are shown to be relatively pure, metabolically active and well coupled. 2. The oxidation of a number of substrates by synaptic and non-synaptic mitochondria was studied and compared. Of the substrates studied, pyruvate plus malate was oxidized most rapidly by both mitochondrial populations. However, the non-synaptic mitochondria oxidized glutamate plus malate almost twice as rapidly as the synaptic mitochondria. 3. The activities of certain tricarboxylic acid-cycle and related enzymes in synaptic and non-synaptic mitochondria were determined. Citrate synthase (EC 4.1.3.7), isocitrate dehydrogenase (EC 1.1.1.41) and malate dehydrogenase (EC 1.1.1.37) activities were similar in both fractions, but pyruvate dehydrogenase (EC 1.2.4.1) activity in non-synaptic mitochondria was higher than in synaptic mitochondria and glutamate dehydrogenase (EC 1.4.1.3) activity in non-synaptic mitochondria was lower than that in synaptic mitochondria. 4. Comparison of synaptic and non-synaptic mitochondria by rate-zonal separation confirmed the distinct identity of the two mitochondrial populations. The non-synaptic mitochondria had higher buoyant density and evidence was obtained to suggest that the synaptic mitochondria might be heterogeneous. 5. The results are also discussed in the light of the suggested connection between the heterogeneity of brain mitochondria and metabolic compartmentation.     

Riboflavin uptake and FAD synthesis in Saccharomyces cerevisiae mitochondria: involvement of the Flx1p carrier in FAD export

We have studied the functional steps by which Saccharomyces cerevisiae mitochondria can synthesize FAD from cytosolic riboflavin (Rf). Riboflavin uptake into mitochondria took place via a mechanism that is consistent with the existence of (at least two) carrier systems. FAD was synthesized inside mitochondria by a mitochondrial FAD synthetase (EC 2.7.7.2), and it was exported into the cytosol via an export system that was inhibited by lumiflavin, and which was different from the riboflavin uptake system. To understand the role of the putative mitochondrial FAD carrier, Flx1p, in this pathway, an flx1Delta mutant strain was constructed. Coupled mitochondria isolated from flx1Delta mutant cells were compared with wild-type mitochondria with respect to the capability to take up Rf, to synthesize FAD from it, and to export FAD into the extramitochondrial phase. Mitochondria isolated from flx1Delta mutant cells specifically lost the ability to export FAD, but did not lose the ability to take up Rf, FAD, or FMN and to synthesize FAD from Rf. Hence, Flx1p is proposed to be the mitochondrial FAD export carrier. Moreover, deletion of the FLX1 gene resulted in a specific reduction of the activities of mitochondrial lipoamide dehydrogenase and succinate dehydrogenase, which are FAD-binding enzymes. For the flavoprotein subunit of succinate dehydrogenase we could demonstrate that this was not due to a changed level of mitochondrial FAD or to a change in the degree of flavinylation of the protein. Instead, the amount of the flavoprotein subunit of succinate dehydrogenase was strongly reduced, indicating an additional regulatory role for Flx1p in protein synthesis or degradation.     

The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha-Cyano-4-hydroxycinnamate and related compounds.

1. Effects of alpha-cyano-4-hydroxycinnamate and alpha-cyanocinnamate on a number of enzymes involved in pyruvate metabolism have been investigated. Little or no inhibition was observed of any enzyme at concentrations that inhibit completely mitochondrial pyruvate transport. At much higher concentrations (1 mM) some inhibition of pyruvate carboxylase was apparent. 2. Alpha-Cyano-4-hydroxycinnamate (1-100 muM) specifically inhibited pyruvate oxidation by mitochondria isolated from rat heart, brain, kidney and from blowfly flight muscle; oxidation of other substrates in the presence or absence of ADP was not affected. Similar concentrations of the compound also inhibited the carboxylation of pyruvate by rat liver mitochondria and the activation by pyruvate of pyruvate dehydrogenase in fat-cell mitochondria. These findings imply that pyruvate dehydrogenase, pyruvate dehydrogenase kinase and pyruvate carboxylase are exposed to mitochondrial matrix concentrations of pyruvate rather than to cytoplasmic concentrations. 3. Studies with whole-cell preparations incubated in vitro indicate that alpha-cyano-4-hydroxycinnamate or alpha-cyanocinnamate (at concentrations below 200 muM) can be used to specifically inhibit mitochondrial pyruvate transport within cells and thus alter the metabolic emphasis of the preparation. In epididymal fat-pads, fatty acid synthesis from glucose and fructose, but not from acetate, was markedly inhibited. No changes in tissue ATP concentrations were observed. The effects on fatty acid synthesis were reversible. In kidney-cortex slices, gluconeogenesis from pyruvate and lactate but not from succinate was inhibited. In the rat heart perfused with medium containing glucose and insulin, addition of alpha-cyanocinnamate (200 muM) greatly increased the output and tissue concentrations of lactate plus pyruvate but decreased the lactate/pyruvate ratio. 4. The inhibition by cyanocinnamate derivatives of pyruvate transport across the cell membrane of human erythrocytes requires much higher concentrations of the derivatives than the inhibition of transport across the mitochondrial membrane. Alpha-Cyano-4-hydroxycinnamate appears to enter erythrocytes on the cell-membrane pyruvate carrier. Entry is not observed in the presence of albumin, which may explain the small effects when these compounds are injected into whole animals.     

水生动物和大鼠线粒体的呼吸链比较

用陆生哺乳动物线粒体呼吸链与水生动物线粒体呼吸链相比较的研究方法,探讨了呼吸链的功能与环境相适应的关系。研究了淡水中生活的草鱼肝丝线粒体,观察到琥珀酸脱氢酶的活性非常低,而ＮＡＤＨ脱氢酶和泛醌细胞色素Ｃ还原酶的活性较高。但海洋生物海绵的线粒体ＮＡＤＨ脱氢酶和琥垢酸脱氢酶的活性都非常低。     

Use of toluene-permeabilized mitochondria to study the regulation of adipose tissue pyruvate dehydrogenase in situ. Further evidence that insulin acts through stimulation of pyruvate dehydrogenase phosphate phosphatase.

Rat epididymal-adipose-tissue mitochondria were made selectively permeable to small molecules without the loss of matrix enzymes by treating the mitochondria with toluene under controlled conditions. With this preparation the entire pyruvate dehydrogenase system was shown to be retained within the mitochondrial matrix and to retain its normal catalytic activity. By using dilute suspensions of these permeabilized mitochondria maintained in the cuvette of a spectrophotometer, it was possible to monitor changes of pyruvate dehydrogenase activity continuously while the activities of the interconverting kinase and phosphatase could be independently manipulated. Permeabilized mitochondria were prepared from control and insulin-treated adipose tissue, and the properties of both the pyruvate dehydrogenase kinase and the phosphatase were compared in situ. No difference in kinase activity was detected, but increases in phosphatase activity were observed in permeabilized mitochondria from insulin-treated tissue. Further studies showed that the main effect of insulin treatment was a decrease in the apparent Ka of the phosphatase for Mg2+, in agreement with earlier studies with mitochondria made permeable to Mg2+ by using the ionophore A23187 [Thomas, Diggle & Denton (1986) Biochem. J. 238, 83-91]. No effects of spermine were detected, although spermine diminishes the Ka of purified phosphatase preparations for Mg2+. Since effects of insulin on pyruvate dehydrogenase phosphatase activity are not evident in mitochondrial extracts, it is concluded that insulin may act by altering some high-Mr component which interacts with the pyruvate dehydrogenase system within intact or permeabilized mitochondria, but not when the mitochondrial membranes are disrupted.     

Euglena gracilis rhodoquinone:ubiquinone ratio and mitochondrial proteome differ under aerobic and anaerobic conditions

Euglena gracilis cells grown under aerobic and anaerobic conditions were compared for their whole cell rhodoquinone and ubiquinone content and for major protein spots contained in isolated mitochondria as assayed by two-dimensional gel electrophoresis and mass spectrometry sequencing. Anaerobically grown cells had higher rhodoquinone levels than aerobically grown cells in agreement with earlier findings indicating the need for fumarate reductase activity in anaerobic wax ester fermentation in Euglena. Microsequencing revealed components of complex III and complex IV of the respiratory chain and the E1beta subunit of pyruvate dehydrogenase to be present in mitochondria of aerobically grown cells but lacking in mitochondria from anaerobically grown cells. No proteins were identified as specific to mitochondria from anaerobically grown cells. cDNAs for the E1alpha, E2, and E3 subunits of mitochondrial pyruvate dehydrogenase were cloned and shown to be differentially expressed under aerobic and anaerobic conditions. Their expression patterns differed from that of mitochondrial pyruvate:NADP(+) oxidoreductase, the N-terminal domain of which is pyruvate:ferredoxin oxidoreductase, an enzyme otherwise typical of hydrogenosomes, hydrogen-producing forms of mitochondria found among anaerobic protists. The Euglena mitochondrion is thus a long sought intermediate that unites biochemical properties of aerobic and anaerobic mitochondria and hydrogenosomes because it contains both pyruvate:ferredoxin oxidoreductase and rhodoquinone typical of hydrogenosomes and anaerobic mitochondria as well as pyruvate dehydrogenase and ubiquinone typical of aerobic mitochondria. Our data show that under aerobic conditions Euglena mitochondria are prepared for anaerobic function and furthermore suggest that the ancestor of mitochondria was a facultative anaerobe, segments of whose physiology have been preserved in the Euglena lineage.     

Regulation of mitochondrial dehydrogenases by calcium ions

Studies in Bristol in the 1960s and 1970s, led to the recognition that four mitochondrial dehydrogenases are activated by calcium ions. These are FAD-glycerol phosphate dehydrogenase, pyruvate dehydrogenase, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase. FAD-glycerol phosphate dehydrogenase is located on the outer surface of the inner mitochondrial membrane and is influenced by changes in cytoplasmic calcium ion concentration. The other three enzymes are located within mitochondria and are regulated by changes in mitochondrial matrix calcium ion concentration. These and subsequent studies on purified enzymes, mitochondria and intact cell preparations have led to the widely accepted view that the activation of these enzymes is important in the stimulation of the respiratory chain and hence ATP supply under conditions of increased ATP demand in many stimulated mammalian cells. The effects of calcium ions on FAD-isocitrate dehydrogenase involve binding to an EF-hand binding motif within this enzyme but the binding sites involved in the effects of calcium ions on the three intramitochondrial dehydrogenases remain to be fully established. It is also emphasised in this article that these three dehydrogenases appear only to be regulated by calcium ions in vertebrates and that this raises some interesting and potentially important developmental issues.     

Mitochondria are an essential mediator of nitric oxide/cyclic guanosine 3',5'-monophosphate blocking of glucose depletion induced cytotoxicity in human HepG2 cells

It is well known that glucose is a major energy source in tumors and that mitochondria are specialized organelles required for energy metabolism. Previous studies have revealed that nitric oxide (NO) protects against glucose depletion-induced cytotoxicity in mouse liver cells and in rat hepatocytes, but the detailed mechanism is not well understood. Therefore, we investigated the involvement of mitochondria in the NO protective effect in human hepatoma HepG2 cells. In this study, we showed that glucose depletion resulted in a time-dependent decrease in intracellular NO and in the protein expression of NO synthases. This glucose depletion-induced decrease in NO was blocked by NO donors. Next, we showed that the cytoprotective effect of NO is via a cyclic guanosine 3',5'-monophosphate-dependent pathway. Additionally, SNP blocked a glucose depletion-induced decrease in mitochondrial mass, mitochondrial DNA copies, and ATP level in HepG2 cells. Moreover, glucose depletion decreased the expression of various mitochondrial proteins, including cytochrome c, complex I (NADH dehydrogenase), complex III (cytochrome c reductase), and heat shock protein 60; these glucose depletion-induced effects were blocked by SNP. Furthermore, we found that rotenone and antimycin A (mitochondria complex I and III inhibitors, respectively) blocked SNP cytoprotection against glucose depletion-induced cytotoxicity. Taken together, our results indicated that the mitochondria serve as an important cellular mediator of NO during protection against glucose deprivation-induced damage.     

Photometric and fluorimetric studies of protomitochondria from liver of young and adult rats

Protomitochondria (PRM) — young organelles — were isolated from liver cells of young (1 month) and adult (9 months) rats. PRM were characterized by photometric and fluorimetric methods. It was found that the number of PRM of various diameters in young and adult rats differs. PRM and mitochondria (MCH) from young and adult rats differ significantly. It was detected by fluorescence of 1-anilinonaphtalene-8-sulfonate (ANS), indicating a different number of membrane binding sites for this probe. An increase of the cytochrome content was observed during ripening of PRM to MCH, more pronounced in young animals. In contrast, the amount of cytochromes in MCH decreases during maturation of young rats to adult rats. The obtained data suggest that massive transformation of PRM to MCH in the cells of young animals takes place, similar to the transformation in the cells of adult animals observed earlier.     

Mammalian MTHFD2L encodes a mitochondrial methylenetetrahydrofolate dehydrogenase isozyme expressed in adult tissues

Previous studies in our laboratory showed that isolated, intact adult rat liver mitochondria are able to oxidize the 3-carbon of serine and the N-methyl carbon of sarcosine to formate without the addition of any other cofactors or substrates. Conversion of these 1-carbon units to formate requires several folate-interconverting enzymes in mitochondria. The enzyme(s) responsible for conversion of 5,10-methylene-tetrahydrofolate (CH(2)-THF) to 10-formyl-THF in adult mammalian mitochondria are currently unknown. A new mitochondrial CH(2)-THF dehydrogenase isozyme, encoded by the MTHFD2L gene, has now been identified. The recombinant protein exhibits robust NADP(+)-dependent CH(2)-THF dehydrogenase activity when expressed in yeast. The enzyme is localized to mitochondria when expressed in CHO cells and behaves as a peripheral membrane protein, tightly associated with the matrix side of the mitochondrial inner membrane. The MTHFD2L gene is subject to alternative splicing and is expressed in adult tissues in humans and rodents. This CH(2)-THF dehydrogenase isozyme thus fills the remaining gap in the pathway from CH(2)-THF to formate in adult mammalian mitochondria.     

Calpain inhibition by peptide epoxides.

The protein activator of phosphorylated branched-chain 2-oxo acid dehydrogenase complex was purified greater than 1000-fold from extracts of rat liver mitochondria; the specific activity was greater than 1000 units/mg of protein (1 unit gives half-maximum re-activation of 10 munits of phosphorylated complex). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave two bands (Mr 47700 and 35300) indistinguishable from the alpha- and beta-subunits of the branched-chain dehydrogenase component of the complex. On gel filtration (Sephacryl S-300), apparent Mr was 190000. This and other evidence suggests that activator protein is free branched-chain dehydrogenase; this conclusion is provisional until identical amino acid composition of the subunits has been demonstrated. Activator protein (i.e. free branched-chain dehydrogenase) was inhibited (up to 30%) by NaF, whereas branched-chain complex was not inhibited. There was no convincing evidence for interconvertible active and inactive forms of activator protein in rat liver mitochondria. Activator protein was detected in mitochondria from liver (ox, rabbit and rat) and kidney (ox and rat), but not in rat heart or skeletal-muscle mitochondria. In rat liver mitochondrial extracts, branched-chain complex sedimented with the mitochondrial membranes, whereas activator protein remained in the supernatant. Activator protein re-activated phosphorylated (inactive) particulate complex from rat liver mitochondria, but it did not activate dephosphorylated complex. Liver and kidney, but not muscle, mitochondria apparently contain surplus free branched-chain dehydrogenase, which is bound by the complex with lower affinity than is the branched-chain dehydrogenase intrinsic to the complex. It is suggested that this functions as a buffering mechanism to maintain branched-chain complex activity in liver and kidney mitochondria.     

Oxidation of External NAD(P)H by Mitochondria from Taproots and Tissue Cultures of Sugar Beet (Beta vulgaris)

The present study compares the exogenous NAD(P)H oxidation and the membrane potential ([delta][psi]) generated in mitochondria isolated from different tissues of an important agricultural crop, sugar beet (Beta vulgaris}. We observed that mitochondria from taproots, cold-stored taproots, and in vitro-grown tissue cultures contain a functional NADH dehydrogenase, whereas only those isolated from tissue cultures displayed a functional NAD(P)H dehydrogenase. It is interesting that the NADH-dependent [delta][psi] of mitochondria from cold-stored taproots and from tissue cultures was not affected by free Ca2+ ions, whereas free Ca2+ was required for the mitochondrial NADPH oxidation by in vitro-grown cells and cytosolic NADH oxidation by mitochondria from fresh taproots. A tentative model accounting for the different response to Ca2+ ions of the NADH dehydrogenase in mitochondria from cold-stored taproots and tissue cultures of B. vulgaris is discussed.     

The pathway of glutamine and glutamate oxidation in isolated mitochondria from mammalian cells

1. Pyruvate strongly inhibited aspartate production by mitochondria isolated from Ehrlich ascites-tumour cells, and rat kidney and liver respiring in the presence of glutamine or glutamate; the production of (14)CO(2) from l-[U-(14)C]glutamine was not inhibited though that from l-[U-(14)C]glutamate was inhibited by more than 50%. 2. Inhibition of aspartate production during glutamine oxidation by intact Ehrlich ascites-tumour cells in the presence of glucose was not accompanied by inhibition of CO(2) production. 3. The addition of amino-oxyacetate, which almost completely suppressed aspartate production, did not inhibit the respiration of the mitochondria in the presence of glutamine, though the respiration in the presence of glutamate was inhibited. 4. Glutamate stimulated the respiration of kidney mitochondria in the presence of glutamine, but the production of aspartate was the same as that in the presence of glutamate alone. 5. The results suggest that the oxidation of glutamate produced by the activity of mitochondrial glutaminase can proceed almost completely through the glutamate dehydrogenase pathway if the transamination pathway is inhibited. This indicates that the oxidation of glutamate is not limited by a high [NADPH]/[NADP(+)] ratio. 6. It is suggested that under physiological conditions the transamination pathway is a less favourable route for the oxidation of glutamate (produced by hydrolysis of glutamine) in Ehrlich ascites-tumour cells, and perhaps also kidney, than the glutamate dehydrogenase pathway, as the production of acetyl-CoA strongly inhibits the first mechanism. The predominance of the transamination pathway in the oxidation of glutamate by isolated mitochondria can be explained by a restricted permeability of the inner mitochondrial membrane to glutamate and by a more favourable location of glutamate-oxaloacetate transaminase compared with that of glutamate dehydrogenase.     

Fractionation by differential and zonal centrifugation of spheroplasts prepared from a glucose-repressed fission yeast Schizosaccharomyces pombe 972h-.

A method is described for the preparation of spheroplasts in high yield from Schizosaccharomyces pombe, by treating cells grown in the presence of glucose and deoxyglucose with snail digestive enzymes. Gentle disruption of such spheroplasts yielded homogenates, from which marker enzymes for nuclei (NAD pyrophosphorylase) and mitochondria (cytochrome c oxidase activity and spectroscopically-detectable cytochromes a + a3) could be quantitatively sedimented by low-speed centrifugation. In contrast to previous findings with Saccharomyces carlsbergensis, cytochrome c oxidase and another mitochondrial enzyme, succinate dehydrogenase, were completely sedimentable by zonal centrifugation in sucrose gradients in the presence of either 2 mM-MgCl2 or 0-4 mM-EDTA. Mitochondria were apparently smaller and of lower buoyant density in gradients containing EDTA. The bulk of the total units of malate dehydrogenase and NADH; cytochrome c oxidoreductase sedimented with mitochondria, whereas NADPH: cytochrome c oxidoreductase was located in fractions containing no mitochondria. The distributions of mitochondrial enzymes were heterogeneous in populations of mitochondria separated on the basis of size or density. The possible origins of mitochondrial heterogeneity in extracts of S. pombe are discussed with special reference to changes in the enzyme activities of cells during the cell cycle.     

THE USE OF TETRANITRO-BLUE TETRAZOLIUM FOR THE CYTOCHEMICAL LOCALIZATION OF SUCCINIC DEHYDROGENASE : Cytochemical and Cytological Studies of Sarcoma 37 Ascites Tumor Cells

Succinic dehydrogenase (SDH) was localized in the mitochondria of Sarcoma 37 ascites tumor cells by the use of tetranitro-BT (TNBT) and nitro-BT (NBT) in smear preparations. Results with each tetrazolium salt as electron acceptor were evaluated with respect to: (a) size and shape of the formazan precipitate relative to standard mitochondrial morphology; (b) crystallization phenomena of reduced dye; (c) lipid adsorption of formazan. The association of formazan- or iron hematoxylin-stained mitochondria with lipid droplets within the cells was investigated, as was also the influence of formalin fixation, with and without cold acetone pretreatment, on mitochondrial morphology and enzymatic staining. Data from these studies appear to indicate that TNBT is more suitable than NBT for use as a cytochemical reagent in oxidative and/or dehydrogenase enzyme histochemistry and cytochemistry.     

Role of Ca2+ ions in the regulation of intramitochondrial metabolism in rat epididymal adipose tissue. Evidence against a role for Ca2+ in the activation of pyruvate dehydrogenase by insulin.

The sensitivity of rat epididymal-adipose-tissue pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase to Ca2+ ions was studied both in mitochondrial extracts and within intact coupled mitochondria. It is concluded that all three enzymes may be activated by increases in the intramitochondrial concentration of Ca2+ and that the distribution of Ca2+ across the mitochondrial inner membrane is determined, as in rat heart mitochondria, by the relative activities of a uniporter (which transports Ca2+ into mitochondria and is inhibited by Mg2+ and Ruthenium Red) and an antiporter (which allows Ca2+ to leave mitochondria in exchange for Na+ and is inhibited by diltiazem). Previous studies with incubated fat-cell mitochondria have indicated that the increases in the amount of active non-phosphorylated pyruvate dehydrogenase in rat epididymal tissue exposed to insulin are the result of activation of pyruvate dehydrogenase phosphate phosphatase. In the present studies, no changes in the activity of the phosphatase were found in extracts of mitochondria, and thus it seemed likely that insulin altered the intramitochondrial concentration of some effector of the phosphatase. Incubation of rat epididymal adipose tissue with medium containing a high concentration of CaCl2 (5mM) was found to increase the active form of pyruvate dehydrogenase to much the same extent as insulin. However, the increases caused by high [Ca2+] in the medium were blocked by Ruthenium Red, whereas those caused by insulin were not. Moreover, whereas the increases resulting from both treatments persisted during the preparation of mitochondria and their subsequent incubation in the absence of Na+, only the increases caused by treatment of the tissue with insulin persisted when the mitochondria were incubated in the presence of Na+ under conditions where the mitochondria are largely depleted of Ca2+. It is concluded that insulin does not act by increasing the intramitochondrial concentration of Ca2+. This conclusion was supported by finding no increases in the activities of the other two Ca2+-responsive intramitochondrial enzymes (NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in mitochondria prepared from insulin-treated tissue compared with controls.     

l-Lactate metabolism in HEP G2 cell mitochondria due to the l-lactate dehydrogenase determines the occurrence of the lactate/pyruvate shuttle and the appearance of oxaloacetate, malate and citrate outside mitochondria

As part of an ongoing study of l-lactate metabolism both in normal and in cancer cells, we investigated whether and how l-lactate metabolism occurs in mitochondria of human hepatocellular carcinoma (Hep G2) cells. We found that Hep G2 cell mitochondria (Hep G2-M) possess an l-lactate dehydrogenase (ml-LDH) restricted to the inner mitochondrial compartments as shown by immunological analysis, confocal microscopy and by assaying ml-LDH activity in solubilized mitochondria. Cytosolic and mitochondrial l-LDHs were found to differ from one another in their saturation kinetics. Having shown that l-lactate itself can enter Hep G2 cells, we found that Hep G2-M swell in ammonium l-lactate, but not in ammonium pyruvate solutions, in a manner inhibited by mersalyl, this showing the occurrence of a carrier-mediated l-lactate transport in these mitochondria. Occurrence of the l-lactate/pyruvate shuttle and the appearance outside mitochondria of oxaloacetate, malate and citrate arising from l-lactate uptake and metabolism together with the low oxygen consumption and membrane potential generation are in favor of an anaplerotic role for l-LAC in Hep G2-M.     

The ultrastructural cytochemistry of lactic dehydrogenase, succinic dehydrogenase, dihydro-nicotinamide adenine dinucleotide diaphorase and cytochrome oxidase activities in hair cell mitochondria of the guinea pig cochlea.

The use of cinnamyl nitroblue tetrazolium chloride (DS-NBT) in dehydrogenase experiments (lactic dehydrogenase, succinic dehydrogenase, nicotinamide adenine dinucleotide diaphorase) and 3,3'-diaminobenzidine tetrahydrochloride (DAB) in cytochrome oxidase experiments indicated that mitochondrial oxidoreduction reactions from nicotinamide adenine dinucleotide to cytochrome oxidase are located on the inner mitochondrial membrane in the outer compartment and the intracristate spaces. These reactions behave according to the chemiosmotic hypothesis. The cochlear hair cell mitochondria are cytochemically indistinguishable from free liver mitochondria. The heterogeneous mitochondrial staining pattern is related to the osmolarity of the incubation media, solubility of the enzymes and pH of the medium, but not to the fixation method.