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PubMed1966

Ultrastructural bases for metabolically linked mechanical activity in mitochondria. I. Reversible ultrastructural changes with change in metabolic steady state in isolated liver mitochondria.

By means of a new "quick-sampling" method, micropellets of mouse liver mitochondria were rapidly prepared for electron microscopy during the recording of steady state metabolism. Reversible ultrastructural changes were found to accompany change in metabolic steady states. The most dramatic reversible ultrastructural change occurs when ADP is added to systems in which only phosphate acceptor is deficient, i.e., during the State IV to State III transition as defined by Chance and Williams. After 15 min in State IV, mitochondria display an "orthodox" ultrastructural appearance as is usually observed after fixation within intact tissue. On transition to State III, a dramatic change in the manner of folding of the inner membrane takes place. In addition, the electron opacity of the matrix increases as the volume of the matrix decreases, but total mitochondrial volume does not appear to change during this transition. This conformation is called "condensed." Isolated mitochondria were found to oscillate between the orthodox and condensed conformations during reversible transitions between State III and State IV. Various significant ultrastructural changes in mitochondria also occur during transitions in other functional states, e.g., when substrate or substrate and acceptor is made limiting. Internal structural flexibility is discussed with respect to structural and functional integrity of isolated mitochondria. Reversible changes in the manner of folding of the inner membrane and in the manner of packing of small granules in the matrix as respiration is activated by ADP represent an ultrastructural basis for metabolically linked mechanical activity in tightly coupled mitochondria.

Adenine Nucleotides↗
PubMed1992

Ubiquinone biosynthesis by mitochondria, sonicated mitochondria, and mitoplasts of rat liver.

Ubiquinone was biosynthesized when rat liver mitochondria were incubated with S-adenosyl-L-methionine, solanesyl diphosphate, and [U-14C]p-hydroxybenzoate. The intermediates of ubiquinone biosynthesis but not ubiquinone were accumulated in mitochondria incubated without S-adenosyl-L-methionine and the accumulated intermediates were converted to ubiquinone by the addition of the methyl group donor and an excess of cold p-hydroxybenzoate. No solaneylated compounds except nonaprenyl p-hydroxybenzoate were found in sonicated mitochondria, while the biosynthesis of ubiquinone was observed in the sonicated preparation of mitochondria in which the intermediates accumulated. The results indicate that the initial decarboxylation reaction is completely abolished and the subsequent reactions of hydroxylation and methylation are not completely inhibited by the sonication treatment and therefore the decarboxylation reaction is the next step after nonaprenylation of p-hydroxybenzoate. Mitoplasts could biosynthesize ubiquinone with activity comparable to that of intact mitochondria, suggesting that components of the outer membrane and the intermembranous space of mitochondria are not involved in ubiquinone biosynthesis.

Animals↗
PubMed1969

The respiratory chain of plant mitochondria. II. Oxidative phosphorylation in skunk cabbage mitochondria.

Mitochondria were prepared from the spadices of skunk cabbage (Symplocarpus foetidus) whose respiratory rate with succinate and malate showed 15% to 30% sensitivity to cyanide inhibition, and which showed respiratory control by added ADP. The observed respiratory control ratios ranged from 1.1 to 1.4. The change in pH of the mitochondrial suspension was recorded simultaneously with oxygen uptake: alkalinization of the medium, expected for phosphorylation of ADP, coincided with the period of acceleration in oxygen uptake caused by addition of an ADP aliquot. The ADP/O ratios obtained were 1.3 for succinate and 1.9 for malate. In the presence of 0.3 mm cyanide, the ADP/O ratio for succinate was zero, while that for malate was 0.7. These results are consistent with the existence of an alternate oxidase which interacts with the flavoprotein and pyridine nucleotide components of the respiratory chain and which, in the presence of cyanide, allows the first phosphorylation site to function with an efficiency of about 70%. In the absence of respiratory inhibitors, the efficiency of each phosphorylation site is also about 70%. This result implies that diversion of reducing equivalents through the alternate oxidase, thereby bypassing the 2 phosphorylation sites associated with the cytochrome components of these mitochondria, occurs to a negligible extent during the oxidative phosphorylation of ADP or State 3. Addition of ADP or uncoupler to skunk cabbage mitochondria respiring in the controlled state or State 4, results in reduction of cytochrome c and the oxidation of the cytochromes b, ubiquinone and pyridine nucleotide. A site of interaction of ADP with the respiratory chain between cytochromes b and cytochrome c is thereby identified by means of the crossover theorem. Flavoprotein measured by fluorescence is also oxidized upon addition of ADP or uncoupler, but flavoprotein measured by optical absorbance changes becomes more reduced under these conditions. Depletion of the mitochondria by pretreatment with ADP and uncoupler prevents reduction of most of the fluorescent flavoprotein by succinate. These results indicate that skunk cabbage mitochondria contain both high and low potential flavo-proteins characterized by different fluorescence/absorbance ratios similar to those demonstrated to be part of the respiratory chain in mitochondria from animal tissues.

Mitochondria↗
PubMed2002

[Decreased ATP-synthesis ability of liver mitochondria in spontaneously hypertensive rats (SHR): role of calcium overload of the mitochondria].

It has been shown previously, that a decrease of ATP amount and changed balance of the other macroergic phosphates are observed in different tissues of spontaneously hypertensive rats (SHR) compared with the normotensive controls (WKY). The aim of the present study was to assess the ability of SHR liver isolated mitochondria to synthesize ATP and to clarify its dependence on extramitochondrial calcium concentration. Macroergic phosphate concentrations were measured by high performance liquid chromatography (HPLC). The present work shows for the first time, that SHR liver mitochondria initially differ from WKY ones by decreased (by 30%) ATP synthesis rate. Respiratory control coefficient and phosphorylation rate in SHR and WKY mitochondria change in different manner when extramitochondrial calcium concentration increases from 0 to 50 microM. When calcium concentration is increased up to 50 microM both parameters in WKY mitochondria are about one half compared with the condition when the medium does not contain calcium. In the conditions, when the medium contains 50 microM of calcium, complete uncoupling of oxidation and phosphorylation in SHR mitochondria is observed. Thus the decrease of ATP synthesis rate in SHR mitochondria in conjunction with reduced tolerance to calcium overload (by respiratory control coefficient and phosphorylation rate) in comparison with WKY may be considered as the main cause of cell energy deficiency, observed in experimental variant of primary arterial hypertension.

Adenosine Triphosphate↗
PubMedDate not supplied

[Myoglobin and mitochondria: kinetics of oxymyoglobin deoxygenation in mitochondria suspension].

The kinetics of whale MbO2 deoxygenation was studied spectrophotometrically in the presence of breathing rat mitochondria under conditions when mitochondria were separated from the protein solution by a semipermeable film capable to transfer only low-molecular-weight compounds and directly in the solution of MbO2 with mitochondria (incubation medium: 15-35 mM succinate, 150 mM sucrose, 100 mM KCl, 0.5 mM EGTA, 5 mM KH2PO4, 10 mM MOPS, pH 7.4). It was shown that the splitting of O2 from MbO2 at physiological pO2 is possible only if it directly contacts mitohondria. The deoxygenation rate does not depend on the protein concentration (zero order on [MbO2] as opposite to the first order reaction in the absence of mitochondria) and completely coincides with the rate of oxygen consumption by mitochondria under the same conditions, as indicated by the polarographic data. The dependence of the MbO2 deoxygenation rate on the concentration of mitochondria and the protein, and on the total charge of the MbO2 molecule was studied using horse MbO2 (pI 7.1), sperm whale MbO2 (pI 8.3), its zinc complex, Zn-MbO2 (pI > 8.3), and the sperm whale MbO2 derivative carboxymethylated at His residues, CM-MbO2 (pI 5.2). The mechanism of MbO2 deoxygenation in the cell obviously actuates its interplay with the mitochondrial membrane. As a result, the affinity of Mb to oxygen decreases several times, which corresponds to a shift of the Mb dissociation curve to higher pO2 values.

Animals↗
PubMed1985

Translocation of proteins into rat liver mitochondria. Existence of two different precursor polypeptides of liver fumarase and import of the precursor into mitochondria.

Two different putative precursor polypeptides of rat liver fumarase were synthesized when RNA prepared from rat liver were translated in vitro using the rabbit reticulocyte lysate system. One of these putative precursor polypeptides (P1) was synthesized as a larger molecular mass than the mature subunit of fumarase (45,000 daltons) by about 5,000 daltons and the other (P2) had the same molecular mass as the mature enzyme. When the 35S-labeled cell-free translation products were incubated with rat liver mitochondria at 30 degrees C, P1 and the 35S-labeled mature size fumarase were associated with the mitochondria. Of these, the 35S-labeled mature size fumarase was resistant to externally added protease, but P1 was not, indicating that the 35S-labeled mature size fumarase was located in the mitochondrial matrix. The following observations strongly suggested that the 35S-labeled mature size fumarase in mitochondria was derived from P1, which was energy-dependently imported and concomitantly processed to the mature size. 1) The amount of the 35S-labeled mature size fumarase recovered from the mitochondria increased proportionally to the duration of incubation, while the amount of P1 recovered from the post-mitochondrial and mitochondrial fractions decreased with the duration of the incubation. 2) Only P1 could bind with the mitochondrial outer membrane at 0 degrees C even in the presence of an uncoupler of the oxidative phosphorylation but P2 did not. 3) P1 bound to the mitochondrial outer membrane was imported into the matrix, when the mitochondria binding only P1 at 0 degrees C was reisolated and incubated at 30 degrees C in the presence of an energy-generating system. The specific receptor was involved in the binding of P1 to mitochondria, since a high concentration of NaCl did not interfere with the binding of P1 to the membrane and did not discharge P1 bound onto the membrane. It was shown that P1 formed an aggregate composed of 6 to 8 molecules and P2 was a dimer in the cell-free translation mixture and that P1 and P2 were enzymatically inactive. These results suggest that the precursor for the mitochondrial enzyme has a larger molecular weight than that of the mature enzyme, whereas the precursor for the cytosolic enzyme has the same molecular weight as the mature enzyme.

Animals↗
PubMed1965

PROTEIN SYNTHESIS IN MITOCHONDRIA. 4. PREPARATION AND PROPERTIES OF MITOCHONDRIA FROM KREBS II MOUSE ASCITES-TUMOUR CELLS.

1. Methods of disrupting Krebs II mouse ascites-tumour cells have been studied. After washing the cells free of ions with sucrose solutions, rapid disruption was obtained in sucrose by use of an Ultra-Turrax disintegrator or a Dounce homogenizer. 2. Disruption of cells after osmotic shock led to the loss of proteins, especially cytochrome c, from the mitochondria. Such losses did not occur when cells were disrupted by shear in 0.3 m-sucrose. 3. The distribution of protein, RNA, DNA, malate dehydrogenase, cytochrome c, cytochrome oxidase and succin-oxidase was measured in the various cell fractions after separation by differential centrifuging. 4. The mitochondrial fraction sedimented at 9500g was further fractionated by equilibrium sedimentation in a sucrose gradient. The distribution of protein and enzyme activity in the gradient indicated that the 9500g pellet contains other material besides mitochondria. 5. Krebs-cell mitochondria contain up to five times as much RNA as do liver mitochondria. 6. After purification by equilibrium centrifugation Krebs-cell mitochondria still contain traces of DNA.

Animals↗
PubMed1981

Mobilization of iron from ferritin by isolated mitochondria. Effects of species compatibility between ferritin and mitochondria and iron content of ferritin.

Mitochondria mobilize iron from ferritin by a mechanism that depends on external FMN. With rat liver mitochondria, the rate of mobilization of iron is higher from rat liver ferritin than from horse spleen ferritin. With horse liver mitochondria, the rate of iron mobilization is higher from horse spleen ferritin than from rat liver ferritin. The results are explained by a higher affinity between mitochondria and ferritins of the same species. The mobilization of iron increases with the iron content of the ferritin and then levels off. A maximum is reached with ferritins containing about 1 200 iron atoms per molecule. The results represent further evidence that ferritin may function as a direct iron donor to the mitochondria.

Animals↗
PubMed2001

Dependence of permeability transition pore opening and cytochrome C release from mitochondria on mitochondria energetic status.

The dependence of Ca2+-induced permeability transition pore (PTP) opening and cytochrome c (Cyt. c) release from mitochondria on mitochondria energetic status was investigated. Results of test the PTP opening and Cyt. c release of isolated rat liver mitochondria with different mitochondrial respiratory substrates, electron transport inhibitors and uncoupler by spectrophotometry and western blotting showed that, Cyt. c release from mitochondria by PTP opening. PTP opening and Cyt. c release showed more sensitive and responsive with FADH-linked succinate than with NADH-linked glutamate plus malate as substrate. Partial or full inhibition of electron flow with electron flow inhibitors resulted in partial or full inhibition of PTP opening and Cyt. c release, respectively. Partial recovery of electron flow with electron sponsors resulted in partial recovery of PTP opening and Cyt. c release. PTP opening and Cyt. c release were completely interrupted by uncoupler. These results indicated PTP opening and Cyt. c release are characterized by respiratory substrate selectivity, electron flow dependence and energy coupling reliance. Hence, PTP opening and Cyt. c release tightly depend on mitochondria energetic status. These findings suggested also that it is possible to regulate apoptosis by altering mitochondrial energetic status to alter PTP opening and Cyt. c release.

Animals↗
PubMed1998

Direct effects of diazoxide on mitochondria in pancreatic B-cells and on isolated liver mitochondria.

1. The direct effects of diazoxide on mitochondrial membrane potential, Ca2+ transport, oxygen consumption and ATP generation were investigated in mouse pancreatic B-cells and rat liver mitochondria. 2. Diazoxide, at concentrations commonly used to open adenosine 5'-triphosphate (ATP)-dependent K+-channels (K(ATP) channels) in pancreatic B-cells (100 to 1000 microM), decreased mitochondrial membrane potential in mouse intact perifused B-cells, as evidenced by an increase of rhodamine 123 fluorescence. This reversible decrease of membrane potential occurred at non-stimulating (5 mM) and stimulating (20 mM) glucose concentrations. 3. A decrease of mitochondrial membrane potential in perifused B-cells was also caused by pinacidil, but no effect could be seen with levcromakalim (500 microM each). 4. Measurements by a tetraphenylphosphonium-sensitive electrode of the membrane potential of rat isolated liver mitochondria confirmed that diazoxide decreased mitochondrial membrane potential by a direct action. Pretreatment with glibenclamide (2 microM) did not antagonize the effects of diazoxide. 5. In Fura 2-loaded B-cells perifused with the Ca2+ channel blocker, D 600, a moderate, reversible increase of intracellular Ca2+ concentration could be seen in response to 500 microM diazoxide. This intracellular Ca2+ mobilization may be due to mitochondrial Ca2+ release, since the reduction of membrane potential of isolated liver mitochondria by diazoxide was accompanied by an accelerated release of Ca2+ stored in the mitochondria. 6. In the presence of 500 microM diazoxide, ATP content of pancreatic islets incubated in 20 mM glucose for 30 min was significantly decreased by 29%. However, insulin secretion from mouse perifused islets induced by 40 mM K+ in the presence of 10 mM glucose was not inhibited by 500 microM diazoxide, suggesting that the energy-dependent processes of insulin secretion distal to Ca2+ influx were not affected by diazoxide at this concentration. 7. The effects of diazoxide on oxygen consumption and ATP production of liver mitochondria varied depending on the respiratory substrates (5 mM succinate, 10 mM alpha-ketoisocaproic acid, 2 mM tetramethyl phenylenediamine plus 5 mM ascorbic acid), indicating an inhibition of respiratory chain complex II. Pinacidil, but not levcromakalim, inhibited alpha-ketoisocaproic acid-fuelled ATP production. 8. In conclusion, diazoxide directly affects mitochondrial energy metabolism, which may be of relevance for stimulus-secretion coupling in pancreatic B-cells.

Adenosine Triphosphate↗
PubMed1975

Investigations on the turnover of adrenocortical mitochondria. III. Stereologic and autoradiographic observations on the effect of chronic treatment with dexamethasone on the rat Zona fasciculata mitochondria.

The effects of chronic administration of dexamethasone (for up to 15 consecutive days) on both the morphology and DNA-synthesis of the mitochondria of the rat adrenal zona fasciculata were investigated by stereologic and autoradiographic techniques. Up to the 3rd day of continuous dexamethasone treatment, the average volume of mitochondria did not change, whereas the number of mitochondria per cell was significantly decreased. From the 3rd to the 15th day of hormonal administration both the volume and number of mitochondria were found to decrease in proportion to the duration of treatment. Autoradiography showed that after the 3rd day of dexamethasone administration there is virtually no incorporation of 3H-thymidine into the mitochondrial compartment. These findings are discussed in the light of evidence indicating that dexamethasone blocks ACTH-release by inhibiting the hypothalamo-hypophyseal axis. The results confirm the view that ACTH controls the maintenance of growth and proliferation of rat adrenocortical mitochondria.

Adrenal Cortex↗
PubMed2004

Tyrosine phosphatase activity in mitochondria: presence of Shp-2 phosphatase in mitochondria.

Tyrosine phosphorylation by unidentified enzymes has been observed in mitochondria, with recent evidence indicating that non-receptorial tyrosine kinases belonging to the Src family, which represent key players in several transduction pathways, are constitutively present in mitochondria. The extent of protein phosphorylation reflects a coordination balance between the activities of specific kinases and phophatases. The present study demonstrates that purified rat brain mitochondria possess endogenous tyrosine phosphatase activity. Mitochondrial phosphatases were found to be capable of dephosphorylating different exogenous substrates, including paranitrophenylphosphate, (32)P-poly(Glu-Tyr)(4:1) and (32)P-angiotensin. These activities are strongly inhibited by peroxovanadate, a well-known inhibitor of tyrosine phosphatases, but not by inhibitors of alkali or Ser/Thr phosphatases, and mainly take place in the intermembrane space and outer mitochondrial membrane. Using a combination of approaches, we identified the tyrosine phosphatase Shp-2 in mitochondria. Shp-2 plays a crucial role in a number of intracellular signalling cascades and is probably involved in several human diseases. It thus represents the first tyrosine phosphatase shown to be present in mitochondria.

Animals↗
PubMed1995

Characterization of a microsomal subfraction associated with mitochondria of the yeast, Saccharomyces cerevisiae. Involvement in synthesis and import of phospholipids into mitochondria.

In the yeast, Saccharomyces cerevisiae, similar to higher eukaryotes most phospholipids are synthesized in microsomes. Mitochondria contribute to the cellular biosynthesis of phospholipids insofar as they harbor phosphatidylethanolamine decarboxylase, and enzymes of phosphatidylglycerol and cardiolipin synthesis. In this paper we present evidence that certain enzymes of phospholipid biosynthesis, namely phosphatidylserine and phosphatidylinositol synthase, are enriched in a special microsomal fraction associated with mitochondria, which we named MAM. This fraction was isolated and characterized with respect to marker enzymes, protein and phospholipid composition, and enzymes of phospholipid synthesis. According to these analyses MAMs are a specialized subfraction of the endoplasmic reticulum, which is distinct from other microsomal subfractions. Phosphatidylserine synthesized in MAMs can be readily imported into mitochondria and converted to phosphatidylethanolamine. Reassociation of MAMs with purified mitochondria led to reconstitution of the import of phosphatidylserine into mitochondria. Organelle contact is suggested as a possible mechanism of this process.

CDP-Diacylglycerol-Inositol 3-Phosphatidyltransfer↗
PubMed1996

Why are mitochondria involved in apoptosis? Permeability transition pores and apoptosis as selective mechanisms to eliminate superoxide-producing mitochondria and cell.

Petit and co-authors have recently summarized results of their studies on the involvement of mitochondria in apoptosis [Petit et al. (1996) FEBS Lett. 396, 7-13]. The mechanism consists in the release to the cytosol of a protein (presumably a protease) that is normally sequestered in the intermembrane space of mitochondria. This protein, when added to isolated nuclei, caused typical apoptotic changes. Its release from mitochondria was shown to occur as a result of disruption of the outer mitochondrial membrane due to swelling of mitochondria caused by opening of so-called permeability transition pores in their inner membranes. Increase in the level of products of the one-electron reduction of O2 (reactive oxygen species, ROS) is known to induce the mitochondrial pores. The hypothesis described here assumes that pore formation and apoptosis are involved in the organization of a defense system preventing ROS formation. It is proposed that ROS-induced pore opening lowers ROS production due to (a) maximal stimulation of mitochondrial O2 consumption and, hence, intracellular [O2] lowering and (b) complete dissipation of mitochondrial membrane potentials and, as a consequence, maximal oxidation of such respiratory chain carriers as CoQ.- which serve as one-electron O2 reductants. ROS decrease allows pore closure. If, nevertheless, ROS are still accumulating in a mitochondrion, long-lived pores cause degradation of the organelle which cannot import and synthesize proteins due to the absence of the membrane potential. In this way, ROS-producing mitochondria can be eliminated (mitochondrial selection). Another result of the long-lived pores is mitochondrial swelling. This disrupts the outer mitochondrial membrane and releases the apoptosis-inducing protein. Apoptosis eliminates ROS-producing cells (cell selection).

Animals↗
PubMed1986

Partial protection against erucoyl-carnitine inhibition in hamster brown-adipose-tissue mitochondria is due to high CoA levels: a comparison with rat brown-adipose-tissue mitochondria.

Brown-adipose-tissue mitochondria isolated from golden hamsters were found to contain more CoA per mg protein than rat brown-fat mitochondria, and after incubation with erucoyl-carnitine, a higher free CoA level remained, than in rat mitochondria. In accordance with the suggestion (Alexson et al. (1985) Biochim. biophys. Acta 834, 149-158) that the inhibitory effect of erucoyl-carnitine on brown-fat mitochondrial respiration is entirely due to CoA sequestration, hamster mitochondria (with more CoA) were less sensitive to erucoyl inhibition than were rat mitochondria. Thus, increased mitochondrial CoA levels may augment the ability of animals to withstand the detrimental effects of a high erucoyl ester content of the diet.

Adipose Tissue, Brown↗
PubMed2005

Why are some mitochondria more powerful than others: insights from comparisons of muscle mitochondria from three terrestrial vertebrates.

We studied the molecular composition of muscle mitochondria to evaluate whether the contents of cytochromes or adenine nucleotide translocase (ANT) or phospholipid acyl compositions reflect differences in mitochondrial oxidative capacities. We isolated mitochondria from three vertebrates of similar size and preferred temperature, the rat (Rattus norvegicus), the cane toad (Bufo marinus) and the bearded dragon lizard (Pogona vitticeps). Mitochondrial oxidative capacities were higher in rats and cane toads than in bearded dragon, whether rates were expressed relative to protein, cytochromes or ANT. Inter-specific differences were least pronounced when rates were expressed relative to cytochrome A, a component of cytochrome C oxidase (CCO), or ANT. In mitochondria from rat and cane toad, cytochrome A was more abundant than C followed by B and then C(1), while in bearded dragon mitochondria, the cytochromes were present in roughly equal levels. Analysis of correlations between mitochondrial oxidative capacities and macromolecular components revealed that cytochrome A explained at least half of the intra- and inter-specific variability in substrate oxidation rates. ANT levels were an excellent correlate of state 3 rates while phospholipid contents were correlated with state 4 rates. As the % poly-unsaturation and the % 20:4n-6 in mitochondrial phospholipids were equivalent in toads and rats, and exceeded the levels in lizards, they may contribute to the inter-specific differences in oxidative capacities. We suggest that the numbers of CCO and ANT together with the poly-unsaturation of phospholipids explain the higher oxidative capacities in muscle mitochondria from rats and cane toads.

Animals↗
PubMed1970

The intracellular localization of enzymes in white-adipose-tissue fat-cells and permeability properties of fat-cell mitochondria. Transfer of acetyl units and reducing power between mitochondria and cytoplasm.

1. A method is described for extracting separately mitochondrial and extramitochondrial enzymes from fat-cells prepared by collagenase digestion from rat epididymal fat-pads. The following distribution of enzymes has been observed (with the total activities of the enzymes as units/mg of fat-cell DNA at 25 degrees C given in parenthesis). Exclusively mitochondrial enzymes: glutamate dehydrogenase (1.8), NAD-isocitrate dehydrogenase (0.5), citrate synthase (5.2), pyruvate carboxylase (3.0); exclusively extramitochondrial enzymes: glucose 6-phosphate dehydrogenase (5.8), 6-phosphogluconate dehydrogenase (5.2), NADP-malate dehydrogenase (11.0), ATP-citrate lyase (5.1); enzymes present in both mitochondrial and extramitochondrial compartments: NADP-isocitrate dehydrogenase (3.7), NAD-malate dehydrogenase (330), aconitate hydratase (1.1), carnitine acetyltransferase (0.4), acetyl-CoA synthetase (1.0), aspartate aminotransferase (1.7), alanine aminotransferase (6.1). The mean DNA content of eight preparations of fat-cells was 109mug/g dry weight of cells. 2. Mitochondria showing respiratory control ratios of 3-6 with pyruvate, about 3 with succinate and P/O ratios of approaching 3 and 2 respectively have been isolated from fat-cells. From studies of rates of oxygen uptake and of swelling in iso-osmotic solutions of ammonium salts, it is concluded that fat-cell mitochondria are permeable to the monocarboxylic acids, pyruvate and acetate; that in the presence of phosphate they are permeable to malate and succinate and to a lesser extent oxaloacetate but not fumarate; and that in the presence of both malate and phosphate they are permeable to citrate, isocitrate and 2-oxoglutarate. In addition, isolated fat-cell mitochondria have been found to oxidize acetyl l-carnitine and, slowly, l-glycerol 3-phosphate. 3. It is concluded that the major means of transport of acetyl units into the cytoplasm for fatty acid synthesis is as citrate. Extensive transport as glutamate, 2-oxoglutarate and isocitrate, as acetate and as acetyl l-carnitine appears to be ruled out by the low activities of mitochondrial aconitate hydratase, mitochondrial acetyl-CoA hydrolyase and carnitine acetyltransferase respectively. Pathways whereby oxaloacetate generated in the cytoplasm during fatty acid synthesis by ATP-citrate lyase may be returned to mitochondria for further citrate synthesis are discussed. 4. It is also concluded that fat-cells contain pathways that will allow the excess of reducing power formed in the cytoplasm when adipose tissue is incubated in glucose and insulin to be transferred to mitochondria as l-glycerol 3-phosphate or malate. When adipose tissue is incubated in pyruvate alone, reducing power for fatty acid, l-glycerol 3-phosphate and lactate formation may be transferred to the cytoplasm as citrate and malate.

Acetates↗
PubMed1995

Evidence that phosphatidylserine is imported into mitochondria via a mitochondria-associated membrane and that the majority of mitochondrial phosphatidylethanolamine is derived from decarboxylation of phosphatidylserine.

Phosphatidylserine is synthesized in both the endoplasmic reticulum and a unique membrane fraction, the mitochondria-associated membrane (MAM) (Vance, J.E. (1990) J. Biol. Chem. 265, 7248-7256). In Chinese hamster ovary cells labeled with [3H]serine or [3H]ethanolamine, we found that the majority of mitochondrial phosphatidylethanolamine was derived from phosphatidylserine decarboxylation. Essentially no mitochondrial phosphatidylethanolamine, especially that in the inner membrane, was imported from the endoplasmic reticulum. We tested the hypothesis that phosphatidylserine made in the endoplasmic reticulum is delivered via the MAM to mitochondria for decarboxylation to phosphatidylethanolamine. Cells were pulse-labeled with [3H]serine and subsequently incubated either in the presence of hydroxylamine (for inhibition of phosphatidylserine decarboxylation) or under conditions for which cellular ATP had been depleted (for inhibition of phosphatidylserine import into mitochondria). In hydroxylamine-treated cells, within 2 h, the amount of radiolabeled phosphatidylserine in the MAM and mitochondria, but not microsomes, was greater than in untreated cells. Moreover, in ATP-depleted, but not in control, cells the amount of radiolabeled phosphatidylserine in the MAM approximately doubled by 3 h. These observations are consistent with the hypothesis that newly synthesized phosphatidylserine normally traverses the MAM en route to mitochondria.

Animals↗