The proton-translocating nicotinamide–dinucleotide (phosphate) transhydrogenase of rat liver mitochondria

1. The NAD(P) transhydrogenase activity of the soluble fraction of sonicated rat liver mitochondrial preparations was greater than the NAD-linked isocitrate dehydrogenase activity, and the NAD-linked and NADP-linked isocitrate dehydrogenase activities were not additive. The NAD-linked isocitrate dehydrogenase activity was destroyed by an endogenous autolytic system or by added nucleotide pyrophosphatase, and was restored by a catalytic amount of NADP. 2. We concluded that the isocitrate dehydrogenase of rat liver mitochondria was exclusively NADP-specific, and that the oxoglutarate/isocitrate couple could therefore be used unequivocally as redox reactant for NADP in experiments designed to operate only the NAD(P) transhydrogenase (or loop 0) segment of the respiratory chain in intact mitochondria. 3. During oxidation of isocitrate by acetoacetate in intact, anaerobic, mitochondria via the rhein-sensitive, but rotenone- and arsenite-insensitive, NAD(P) transhydrogenase, measurements of the rates of carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive and carbonyl cyanide p-trifluoromethoxyphenylhydrazone-insensitive pH change in the presence of various oxoglutarate/isocitrate concentration ratios gave an -->H(+)/2e(-) quotient of 1.94+/-0.12 for outward proton translocation by the NAD(P) transhydrogenase. 4. Measurements with a K(+)-sensitive electrode confirmed that the electrogenicity of the NAD(P) transhydrogenase reaction corresponded to the translocation of one positive charge per acid equivalent. 5. Sluggish reversal of the NAD(P) transhydrogenase reaction resulted in a significant inward proton translocation. 6. The possibility that isocitrate might normally be oxidized via loop 0 at a redox potential of -450mV, or even more negative, is discussed, and implies that a P/O quotient of 4 for isocitrate oxidation might be expected.     

Ketogenesis in isolated rat liver mitochondria. III. Relationship with the rate of β-oxidation

The synthesis of ketone bodies has been studied as a function of the rate of acetyl-CoA production during the oxidation of different fatty acids and of pyruvate. In the presence of hexokinase plus glucose and a low concentration of malate, the Krebs cycle has a fixed capacity which is independent of the rate of acetyl-CoA supply. When the rate of acetyl-CoA production increases beyond this capacity, acetyl-CoA is converted to the synthesis of acetoacetate. Under all conditions tested the rate of ketogenesis and the ratio of acetyl-CoA over free CoASH were positively correlated.The relevance of our results for the control of ketogenesis in vivo is discussed.     

Ketogenesis in isolated rat liver mitochondria. II. Factors affecting the rate of β-oxidation

1. During fatty acid oxidation by rat liver mitochondria, the rate of β-oxidation is dependent on the relative amounts of substrate and mitochondrial protein, on the energy state of the mitochondria, on the chain length and the number of double bonds of the fatty acid and on the concentration of various compounds in the reaction medium (l-carnitine, CoASH, hexokinase, albumin).2. The rate of β-oxidation of long-chain fatty acids decreases when the ratio of albumin over fatty acid is increased. This effect is most marked in the absence of added carnitine.3. Addition of excess hexokinase decreases the rate of β-oxidation in the presence of added carnitine.4. Maximal rates of β-oxidation are observed with octanoate and decanoate (40–60 nmoles acetyl-CoA/min per mg mitochondrial protein at 25 °C).5. Odd-numbered fatty acids are oxidized at a much lower rate than the even-numbered homologues. In a low-energy state propionyl-CoA accumulates; in a high-energy state in the presence of bicarbonate, Krebs-cycle intermediates accumulate.6. l-Carnitine enhances the rate of β-oxidation of all fatty acids except butyrate. The stimulatory effect is most pronounced with odd-numbered and with long-chain fatty acids.7. In the absence of added carnitine the rate of β-oxidation of long-chain fatty acids decreases with the chain length and increases with the number of double bonds. It is suggested that the solubility of the long-chain fatty acids in the aqueous medium is the rate-limiting factor under these conditions.8. In the presence of carnitine and albumin, palmitate, oleate, linoleate and linolenate are all oxidized at about the same rate (25–30 nmoles/min per mg protein at 25 °C).9. Propionyl-CoA is not formed as an intermediate during oxidation of unsaturated fatty acids.     

α-tocopherol β-oxidation localized to rat liver mitochondria

Approximately 40% of Americans take dietary supplements, including vitamin E (α-tocopherol). Unlike other fat-soluble vitamins, α-tocopherol is not accumulated to toxic levels. Rather tissue levels are tightly regulated, in part via increased hepatic metabolism and excretion that could, theoretically, alter metabolism of drugs, environmental toxins, and other nutrients. To date, in vivo subcellular location(s) of α-tocopherol metabolism have not been identified. The proposed pathway of α-tocopherol metabolism proceeds via ω-hydroxylation to 13′-OH-α-tocopherol, followed by successive rounds of β-oxidation to form α-CEHC. To test the hypothesis that α-tocopherol ω-hydroxylation occurs in microsomes while β-oxidation occurs in peroxisomes, rats received daily injections of vehicle, 10 mg α-tocopherol, or 10 mg trolox/100 g body wt for 3 days, and then microsomes, mitochondria, and peroxisomes were isolated from liver homogenates. Homogenate α-tocopherol levels increased 16-fold in α-tocopherol-injected rats, while remaining unchanged in trolox- or vehicle-injected rats. Total α-tocopherol recovered in the three subcellular fractions represented 93 ± 4% of homogenate α-tocopherol levels. In α-tocopherol-injected rats, microsome α-tocopherol levels increased 28-fold, while mitochondria and peroxisome levels increased 8- and 3-fold, respectively, indicating greater partitioning of α-tocopherol to the microsomes with increasing liver α-tocopherol. In α-tocopherol-injected rats, microsome 13′-OH-α-tocopherol levels increased 24-fold compared to controls, and were 7-fold greater than 13′-OH-α-tocopherol levels in peroxisome and mitochondrial fractions of α-tocopherol-injected rats. An unexpected finding was that α-CEHC, the end product of α-tocopherol metabolism, was found almost exclusively in mitochondria. These data are the first to indicate a mitochondrial role in α-tocopherol metabolism.     

Incorporation of cytosol δ-aminolevulinate synthase of rat liver into the mitochondria in vitro

When ¹²⁵I-labeled cytosol δ-aminolevulinate synthase was incubated in suspensions of rat liver mitochondria, the enzyme was incorporated into the mitochondira at the rate that was linear with time and with the [¹²⁵I]enzyme added. Subfractionation of the mitochondria using a digitonin technique revealed that the [¹²⁵I]enzyme was incorporated into the innermembrane-matrix fraction where endogeneous δ-aminolevulinate synthase is located.     

Pyruvate metabolism in rat liver mitochondria. What is optimized at steady state?

A representative model of mitochondrial pyruvate metabolism was broken down into its extremal independent currents and compared with experimental data obtained from liver mitochondria incubated with pyruvate as a substrate but in the absence of added adenosine diphosphate. Assuming no regulation of enzymatic activities, the free-flow prediction for the output of the model shows large discrepancies with the experimental data. To study the objective of the incubated mitochondria, we calculate the conversion cone of the model, which describes the possible input/output behaviour of the network. We demonstrate the consistency of the experimental data with the model because all measured data are within this cone. Because they are close to the boundary of the cone, we deduce that pyruvate is converted very efficiently (93%) to produce the measured extramitochondrial metabolites. We find that the main function of the incubated mitochondria is the production of malate and citrate, supporting the anaplerotic pathways in the cytosol, notably gluconeogenesis and fatty acid synthesis. Finally, we show that the major flow through the enzymatic steps of the mitochondrial pyruvate metabolism can be reliably predicted based on the stoichiometric model plus the measured extramitochondrial products. A major advantage of this method is that neither kinetic simulations nor radioactive tracers are needed.     

Enzymes of carnitine acylation. Is overt carnitine palmitoyltransferase of liver peroxisomal carnitine octanoyltransferase?

Liver mitochondria prepared by differential centrifugation are contaminated by significant quantities of peroxisomes and microsomal fractions. 'Easily solubilized carnitine palmitoyltransferase' prepared from liver mitochondria is thought to originate from the outer surface of the mitochondrial inner membrane. We have characterized the carnitine palmitoyltransferase activities of freeze-thaw extracts of rat liver mitochondrial preparations. Chromatography on Sephadex G-100 yields two broad peaks of carnitine decanoyltransferase activity: one eluted at the end of the void volume, which can be removed (precipitated) by ultracentrifugation; the second peak represents the soluble activity and is eluted at an Mr near 70,000. The activity in the soluble peak is precipitated by an antibody raised against carnitine octanoyltransferase purified from mouse liver peroxisomes. In contrast, antibody raised against carnitine palmitoyltransferase purified from liver mitochondrial membranes had no effect (P. Brady & L. Brady, personal communication). The carnitine acyltransferase activities of the Mr-70,000 peak in the presence or absence of Tween 20 showed maximum activity with decanoyl-CoA and about one-third of this activity with palmitoyl-CoA, similar to peroxisomal carnitine octanoyltransferase. These data show that 7500 g preparations of liver mitochondria isolated by differential centrifugation are enriched by peroxisomal carnitine octanoyltransferase (approx. 20% of the protein of the fraction is peroxisomal) and indicate that this enzyme may be the one reported as 'overt' or 'easily solubilized' mitochondrial carnitine palmitoyltransferase.     

On the inhibition of α-oxobutyrate utilization by fatty acids in rat liver mitochondria

It has been found that free fatty acids and acylcarnitine inhibit α-oxobutyrate utilization in rat liver mitochondria. It has been recognized that the intramitochondrial accumulation of acetyl-CoA, produced by the β-oxidation of activated fatty acids, is responsible for such inhibition. In fact acetyl-CoA is shown to inhibit α-oxobutyrate dehydrogenase (α-oxoglutarate: lipoate oxidoreductase (acceptor acylating) EC 1.2.4.2)     

Characterization of pore-forming activity in liver mitochondria from Anguilla anguilla. Two porins in mitochondria?

A fast purification procedure for the isolation and purification of eukaryotic porin (De Pinto et al., (1987) Biochim. Biophys. Acta 905, 499-502) was applied to liver mitochondria of the fish Anguilla anguilla. A protein preparation was obtained which formed slightly anionically selective pores in reconstitution experiments with lipid bilayer membranes. The distribution of single-channel conductances had two maxima of 2.4 nS and 4.0 nS in 1 M KCl. Sodium dodecylsulfate electrophoretograms of the protein preparation showed the presence of two bands of very similar electrophoretic mobility (32 and 32.5 kDa). Both bands cross-reacted with antibodies raised against purified bovine heart porin and with antibodies raised against the 19 amino acids N-terminal end of human porin. No cross-reactivity was observed with antibodies against yeast porin. The peptide maps of the two bands showed slight differences. The possibility of the presence of two different porins in liver mitochondria of Anguilla anguilla is discussed. An extensive immunological comparison of different mitochondrial porins is presented.     

Specific inhibition of pyruvate transport in rat liver mitochondria and human erythrocytes by α-cyano-4-hydroxycinnamate (Short Communication)

alpha-Cyano-4-hydroxycinnamate greatly inhibits the transport of pyruvate but not that of acetate or butyrate in liver mitochondria and erythrocytes. In the latter, lactate uptake is also inhibited. It is concluded that a specific carrier is involved in membrane transport of pyruvate and that the plasma-membrane carrier may also be involved in lactate transport.     

Energetic,oxidative and ionic exchange in rat brain and liver mitochondria at experimental audiogenic epilepsy (Krushinsky–Molodkina model)

The role of brain and liver mitochondria at epileptic seizure was studied on Krushinsky-Molodkina (KM) rats which respond to sound with an intensive epileptic seizure (audiogenic epilepsy). We didn't find significant changes in respiration rats of brain and liver mitochondria of KM and control rats; however the efficiency of АТР synthesis in the KM rat mitochondria was 10% lower. In rats with audiogenic epilepsy the concentration of oxidative stress marker malondialdehyde in mitochondria of the brain (but not liver) was 2-fold higher than that in the control rats. The rate of H₂O₂ generation in brain mitochondria of КМ rats was twofold higher than in the control animals when using NAD-dependent substrates. This difference was less pronounced in liver mitochondria. In KM rats, the activity of mitochondrial ATP-dependent potassium channel was lower than in liver mitochondria of control rats. The comparative study of the mitochondria ability to retain calcium ions revealed that in the case of using the complex I and complex II substrates, permeability transition pore is easier to trigger in brain and liver mitochondria of KM and КМs rats than in the control ones. The role of the changes in the energetic, oxidative, and ionic exchange in the mechanism of audiogenic epilepsy generation in rats and the possible correction of the epilepsy seizures are discussed.     

α-Lipoic acid dependent regeneration of ascorbic acid from dehydroascorbic acid in rat liver mitochondria

Rat liver mitochondria were examined for their ability to reduce dehydroascorbic acid to ascorbic acid in an -lipoic acid dependent or independent manner. The a-lipoic acid dependent reduction was stimulated by factors that increased the NADH dependent reduction of -lipoic acid to dihydrolipoic acid in coupled reactions. Optimal conditions for dehydroascorbic acid reduction to ascorbic acid were achieved in the presence of pyruvate, -lipoic acid, and ATP. Electron transport inhibitors, rotenone and antimycin A, further enhanced the dehydroascorbic acid reduction. The reactions were strongly inhibited by 1 mM iodoacetamide or sodium arsenite. Mitoplasts were qualitatively similar to intact mitochondria in dehydroascorbate reduction activity. Pyruvate dehydrogenase and -ketoglutarate dehydrogenase reduced dehydroascorbic acid to ascorbic acid in an -lipoic acid, coenzyme A, and pyruvate or -ketoglutarate dependent fashion. Dehydroascorbic acid was also catalytically reduced to ascorbic acid by purified lipoamide dehydrogenase in an -lipoic acid (K _0.5=1.4±0.8 mM) and lipoamide (K _0.5=0.9±0.3 mM) dependent manner.     

Biochemical effects of the hypoglycaemic compound diphenyleneiodonium. Catalysis of anion–hydroxyl ion exchange across the inner membrane of rat liver mitochondria and effects on oxygen uptake

1. The hypoglycaemic compound diphenyleneiodonium causes rapid and extensive swelling of rat liver mitochondria suspended in 150mm-NH(4)Cl, and in 150mm-KCl in the presence of 2,4-dinitrophenol and valinomycin. This indicates that diphenyleneiodonium catalyses a compulsory exchange of OH(-) for Cl(-) across the mitochondrial inner membrane. Br(-) and SCN(-) were the only other anions found whose exchange for OH(-) is catalysed by diphenyleneiodonium. 2. Diphenyleneiodonium inhibited state 3 respiration of mitochondria and slightly stimulated state 4 respiration with succinate or glutamate as substrate in a standard Cl(-)-containing medium. 3. Diphenyleneiodonium did not inhibit state 3 respiration significantly in two Cl(-)-free media (based on glycerol 2-phosphate or sucrose) but caused some stimulation of state 4. 4. In Cl(-)-containing medium diphenyleneiodonium only slightly inhibited the 2,4-dinitrophenol-stimulated adenosine triphosphatase and it had little effect in the absence of Cl(-). 5. The inhibition of respiration in the presence of Cl(-) is dependent on the Cl(-)-OH(-) exchange. 2,4-Dichlorodiphenyleneiodonium is ten times as active as diphenyleneiodonium both in causing swelling of mitochondria suspended in 150mm-NH(4)Cl and in inhibiting state 3 respiration in Cl(-)-containing medium. Indirect evidence suggests that the Cl(-)-OH(-) exchange impairs the rate of uptake of substrate anions. 6. It is proposed that stimulation of state 4 respiration in the absence of Cl(-) depends, at least in part, on an electrogenic uptake of diphenyleneiodonium cations. 7. Tripropyl-lead acetate, methylmercuric iodide and nine substituted diphenyleneiodonium derivatives also catalyse Cl(-)-OH(-) exchange across the mitochondrial membrane. 8. Diphenyleneiodonium is compared with the trialkyltin compounds, which are also known to mediate Cl(-)-OH(-) exchange and which have in addition strong oligomycin-like effects on respiration. It is concluded that diphenyleneiodonium is specific for catalysing anion-OH(-) exchange and will be a useful reagent for investigating membrane-dependent systems.     

Transport of reduced nicotinamide–adenine dinucleotide into mitochondria of rat white adipose tissue

Mitochondria from rat white adipose tissue were prepared, exhibiting good respiratory control and P/O ratios. They would not oxidize NADH unless NNN'N'-tetramethyl-p-phenylenediamine was added as a carrier of reducing equivalents. These mitochondria were found to oxidize neither l-glycerol 3-phosphate nor l-glutamate plus l-malate at significant rates. The activity of aspartate aminotransferase in these mitochondria was found to be low compared with that found in rat liver mitochondria. As a consequence of this, the adipose-tissue mitochondria exhibited very low rates of cytoplasmic NADH oxidation in a reconstituted Borst (1962) cycle compared with liver mitochondria.     

Partial characterization of placental 3ß-hydroxysteroid dehydrogenase (EC 1.1.1.145), Δ4–5isomerase (EC 5.3.3.1) in human term placental mitochondria

A partial characterization of human term placental 3ß-HSDH in mitochondria is reported. Apparent K_M of pregnenolone: 70 nM. A dose-dependent stimulation of 3ß-HSDH by NAD⁺ or NADP⁺ was observed in the range from 10⁻⁶ to 10⁻³ M (K_M value of NAD⁺: 20 μM). At equimolar concentrations NAD⁺ is more than 10-fold as effective a cofactor of the 3ß-HSDH than NADP⁺. pH optimum: 9.5 (glycine-NaOH buffer). Temperature optimum 40–45°C. A rapid loss of 3ß-HSDH activity was found after preincubation of the enzyme at 37°C after 30 min: less than 50% of initial enzyme activity is present. No inhibition was obtained by Mg²⁺, Ca²⁺ Sr²⁺ and Ba²⁺ (1–100 mM). A strong inhibition was achieved with 1 mM Zn²⁺, Cd²⁺, Cu²⁺ and 10 mM and 100 mM Fe²⁺, Mn²⁺, Co²⁺ and Ni²⁺.     

Stimulation of calcium efflux from rat liver mitochondria by adenosine 3′5′ cyclic monophosphate

Summary The uptake or release of Ca²⁺ from rat liver mitochondria was studied by means of a sensitive Ca-electrode. It was found that using palmitoyl coenzyme A together with carnitine and ATP as substrates that Ca²⁺ was released gradually from mitochondria by adenosine 35 cyclic monophosphate. The effect was obtained with either mitochondria preloaded with Ca²⁺ or with their physiological content of Ca²⁺. No such release was obtained with the usual substrates used to provide energy for Ca²⁺ uptake by mitochondria.     

Comprehensive analysis of lncRNA–miRNA–mRNA during proliferative phase of rat liver regeneration

This study aims to reveal the regulatory mechanism of lncRNAs–miRNAs–mRNAs network during the proliferative phase of liver regeneration (LR). High-throughput sequencing technology was performed, and a total of 1,738 differentially expressed lncRNAs (DE lncRNAs), 167 known differentially expressed miRNAs (DE miRNAs), and 2,727 differentially expressed mRNAs were identified. Then, the target DE lncRNAs and DE mRNAs regulated by the same miRNAs were screened and a ceRNA regulatory network containing 32 miRNAs, 107 lncRNAs, and 270 mRNAs was constructed. Insulin signaling pathway, pyrimidine metabolism, axon guidance, carbohydrate digestion and absorption, and pyruvate metabolism were significantly enriched in the network. Through literature review and the regulatory relationship between lncRNAs and miRNAs, nine core lncRNAs were identified, which might play important roles during the proliferative phase of rat LR. This study analyzed lncRNA–miRNA–mRNA regulatory network for the first time during the proliferative phase of rat LR, providing clues for exploring the mechanism of LR and the treatment of liver diseases.     

Discrete poly(A)-RNA species from rat liver mitochondria are fragments of 16S mitochondrial rRNA carrying its 5′-termini

During electrophoresis in polyacrylamide gels containing 7M urea the major discrete components of preparations of rat liver mitochondrial poly(A)+ and poly(A)- RNA species have similar mobilities. Poly(A)- RNA components hybridize to the 16S rRNA gene of mtDNA. Analysis of 5'-terminal sequences of these components revealed their identity to the 5'-terminal sequence of 16S rRNA. These results show that poly(A)- RNA components are fragmentation products of 16S rRNA. Fragmentation occurs nonrandomly from the 3'-end of the original rRNA molecules and lead to formation of products with electrophoretic mobilities similar to those of poly(A)+ RNA components.     

Comparative effect of N-substituted dehydroamino acids and α-tocopherol on rat liver lipid peroxidation activities

Free radical damage has been associated with a growing number of diseases and conditions, such as autoimmune diseases, neurodegenerative disorders and multiple types of cancer. Some dehydroamino acids and corresponding peptides can function as radical scavengers. In this study the in vitro effects on rat liver lipid peroxidation levels of fourteen N-substituted dehydroamino acid derivatives and α-tocopherol were investigated. α-Tocopherol is a powerful antioxidant that is beneficial in the treatment of many free radical related diseases. The results indicated that all the compounds showed very good inhibitory effect on the lipid peroxidation compound with α-tocopherol at 1 mM concentrations and the inhibition rate was in the range of 70–79 % with the exception of compound 5. At 0.1 mM concentrations compounds 1, 2 and 9 were found more active than α-tocopherol. The results confirmed that molecules such as dehydroamino acids which have reactive double bonds can act as a guard in vitro against oxidants.