Anna B. Wojtczak

Factors controlling the rate of fatty acid β-oxidation in rat liver mitochondria

Abstract 1. 1. The effects of mitochondrial swelling, acylcarnitine/camitine ratio, NADH/ NAD ratio, energy state, and of citric acid cycle intermediates on the β-oxidation of (−)[U-14C]palmitylcarnitine in rat liver mitochondria have been studied. The formation of β-hydroxypalmitylcamitine and of acid soluble products (acetyl groups) have been measured. 2. 2. After loss of NAD and CoA from the mitochondria due to swelling, addition of NAD, CoA, and cytochrome c to the reaction mixture gives only a partial reactivation of palmitylcarnitine oxidation. 3. 3. When the acylcamitine/carnitine ratio is lowered the mitochondrial content of acyl-CoA is lowered, and a lower rate of β-oxidation is obtained. A similar effect is obtained with albumin because it binds palmitylcarnitine extramitochondrially. 4. 4. A high NADH/NAD ratio inhibits the rate of β-oxidation, but the inhibition is strong only with very high ratios. 5. 5. With very high NADH/NAD ratios β-hydroxypalmitylcamitine is formed when the palmitylcarnitine/carnitine ratio is low. It is concluded that the NAD-linked oxidation of the β-hydroxypalmityl-CoA is more easily suppressed than the flavoprotein-linked oxidation of palmityl-CoA. 6. 6. Of the citric acid cycle intermediates only succinate has any appreciable ability to suppress β-oxidation of palmitylcarnitine by competing for the electron transport chain. In low energy states succinate promotes β-hydroxypalmitylcamitine formation showing that it interferes mainly with the oxidation of NADH. In a high energy state it also interferes with the initial acyl-CoA dehydrogenation step, thus sparing palmityl groups. 7. 7. The regulation of fatty acid β-oxidation in the liver is discussed.

Uncoupling of oxidative phosphorylation and inhibition of ATPPi exchange by a substance from insect mitochondria

Human and bovine serum or plasma albumins, and to a smaller degree β-lactoglobulin, stimulate oxidative phosphorylation and the ATPPi exchange reaction mitochondria from larvae of the wax-moth, Galleria mellonella L. (Lepidoptera). This effect has been shown to be due to removal by these proteins of an uncoupling agent that is present in isolated insect mitochondria. It has been demonstrated that this uncoupling agent contains fatty acids, of which palmitic, stearic, oleic, linoleic and linolenic acids have been identified by paper chromatography.

Inhibitory action of oxaloacetate on succinate oxidation in rat-liver mitochondria and the mechanism of its reversal

Abstract 1. 1. Oxidation of succinate in rat-liver mitochondria was studied in the presence of added oxaloacetate, an uncoupler of oxidative phosphorylation and rotenone. 2. 2. At certain concentrations of oxaloacetate, succinate and mitochondria a spontaneous reactivation of succinate oxidation, previously inhibited by oxaloacetate, can be observed. This reactivation is completely abolished by either arsenate, arsenite or lewisite or by the absence of phosphate, and is potentiated by oligomycin. The spontaneous reactivation is accompanied by an increase in the intramitochondrial content of ATP generated by substrate-level phosphorylation. 3. 3. ATP added externally reactivates the oxidation of succinate substantially only in the presence of either carnitine or, to a smaller degree, arsenate. 4. 4. The reactivation produced by ATP plus carnitine is potentiated by the addition of small amounts of palmitate. The addition of pyruvate has a small effect and the addition of pyruvate plus arsenate almost none. 5. 5. Freshly isolated rat-liver mitochondria contain 15–30 mμmoles free fatty acids per mg protein. This amount is increased during incubation in the presence of KCN, but during aerobic incubation with succinate fatty acids are oxidized, even if 2,4-dinitrophenol is present. 6. 6. Upon addition of oxaloacetate the oxidation of intramitochondrial nicotinamide nucleotides is observed. The steady-state redox level depends on the concentration of added oxaloacetate and on the presence of oxidizable NAD-linked substrates. In the presence of oxaloacetate, intramitochondrial NAD(P) + is most effectively reduced by isocitrate; ATP plus carnitine having a small effect only. 7. 7. The best protection of succinate oxidation against the inhibition by oxaloacetate is provided by ATP plus carnitine, by ATP plus carnitine plus palmitate, or by palmitoyl-carnitine. 8. 8. It is concluded that the oxidation of fatty acids is the most effective factor removing oxaloacetate from the site of succinate dehydrogenase in liver mitochondria. Its effect is due to (i) generation of NADH which can reduce oxaloacetate to malate, and (ii) provision of acetyl-CoA which can condense with oxaloacetate to form citrate. A smaller effectiveness of other reactions removing oxaloacetate from mitochondria is discussed.

Effect of fatty acids on pyruvate carboxylation in rat liver mitochondria

Free fatty acids have long been regarded as uncouplers of oxidative phosphorylation [l-5]. On the other hand, Wojtczak et al. [6,7] demonstrated that long chain fatty acids inhibit the translocation of adenine nucleotides through mitochondrial membranes. Recently, Pande and Blanchaer [8], Shug et al. [9, lo] and Vaartjes et al. [l 1, 121 have shown that CoA esters of fatty acids are much stronger inhibitors of the translocation than free acids. Carboxylation of pyruvate to oxaloacetate occurs in mitochondria. It requires ATP as energy donor and is inhibited by ADP [ 131. It has been observed that low concentrations of octanoate [ 141 and oleate [ 151 increase the rate of pyruvate carboxylation under conditions of state 3 respiration of mitochondria. This effect has been interpreted [ 141 as being due to the inhibition of adenine nucleotide translocation by fatty acids and an increased ATP/ADP ratio inside mitochondria. Recently, Stucki et al. [ 151 have shown that oleate and octanoate act by different mechanisms; octanoate diminishes the pool size of exchangeable adenine nucleotides while oleate inhibits adenine nucleotide translocase. A similar explanation of the stimulation of carboxylation by medium chain and long chain fatty acids was presented by ourselves [ 161 in a preliminary form. In the present investigation a large spectrum of fatty acids was examined and it was shown that short

Enzymes of gluconeogenesis and the synthesis of glycogen from glycerol in various organs of the lamprey (Lampetra fluviatilis).

Abstract 1. 1. The content of glycogen and ATP is strongly decreased in lamprey liver but remains unchanged in somatic muscles during the spawning migration. 2. 2. Lamprey somatic muscles and the heart contain highly active glycerol-3-phosphate dehydrogenase and glycerol kinase. 3. 3. [14C]Glycerol is rapidly incorporated into glycogen in lamprey muscles both in vivo and in slices. The incorporation into liver glycogen is very slow in vivo and none in slices. 4. 4. Although pyruvate carboxylase and phosphoenolpyruvate carboxykinase are present in liver, it seems unlikely that gluconeogenesis proceeds in this organ during spawning migration. 5. 5. The results suggest that muscles are the main site of gluconeogenesis in the migrating lamprey and that glycerol is an important precursor.

Mitochondrial oxaloacetate decarboxylase from rat liver

Abstract 1. The occurrence of oxaloacetate decarboxylase (EC 4.1.1.3) in rat liver was confirmed. The decarboxylation was found in both mitochondria and the soluble cytoplasm. 2. The mitochondrial enzyme was partially purified. It appeared not to be activated by divalent cations, nor to be inhibited by chelating agents. It was inhibited up to 50% by 0.5 mM p-chloromercuribenzoate. The Km value was 0.55 mM and pH optimum between 6.5 and 7.5. 3. It was found that the enzyme-substrate complex could be reduced by borohydride to a stable compound, thus showing a Schiff base formation. Parallel to this reduction was a loss of the enzymatic activity. 4. Oxaloacetate decarboxylase was also found in mitochondria of kidney and brain but not in mitochondria from heart and skeletal muscles. 5. It was shown that in intact mitochondria, the substrate for the decarboxylase could be both added oxaloacetate and oxaloacetate formed by the oxidation of malate.

Significance of the alanine aminotransferase reaction in the formation of α-ketoglutarate in rat liver mitochondria

The total production of alpha-ketoglutarate from glutamate and isocitrate was estimated in isolated rat liver mitochondria. Mitochondrial alanine aminotransferase converts glutamate to alpha-ketoglutarate [A.K. Groen et al. (1982) Eur. J. Biochem. 122, 87-93], thus participating in the net formation of the tricarboxylic acid cycle intermediates from glutamate. The present investigation indicates a significant contribution of the alanine aminotransferase reaction to glutamate oxidation by isolated rat liver mitochondria in the presence of bicarbonate. It amounted to 41-74 and 7-31% of the total utilization of glutamate in States 4 and 3, respectively, in various conditions in vitro, at pyruvate concentrations in the range of 0.1-10 mM. The participation of glutamate in the total production of alpha-ketoglutarate at physiological concentrations of glutamate, citrate, and isocitrate varied in the range of 72-82%. It was calculated that alpha-ketoglutarate formation by the reaction of alanine aminotransferase amounted to 30 and 5% of the total mitochondrial alpha-ketoglutarate production in States 4 and 3, respectively, at physiological concentrations of its precursors and in the presence of 0.5 mM malate and 0.1 mM pyruvate. It constituted 77-97% of the net production of the tricarboxylic acid cycle intermediates from glutamate in rat liver mitochondria. The importance of alpha-ketoglutarate production via the alanine aminotransferase reaction under various physiological conditions is discussed.

The elucidation of the effect of ammonium chloride on pyruvate distribution and pyruvate dehydrogenase interconversion in isolated rat hepatocytes

The distribution of pyruvate between cell compartments measured in isolated hepatocytes in the presence of lactate was in agreement with delta pH across plasma and mitochondrial membranes. In isolated liver mitochondria NH4Cl decreased the transmembrane potential (delta psi) by about 14 mV, whereas no change of delta pH was observed. In the presence of lactate or alanine NH4Cl increased the mitochondrial pyruvate concentration presumably due to the inhibition of the flux through pyruvate carboxylase. In the presence of lactate or alanine changes in the amount of the active form of pyruvate dehydrogenase (PDHa) were correlated with the mitochondrial pyruvate concentration, NH4Cl increased the amount of PDHa by lowering the mitochondrial ATP/ADP and NADH/NAD+ ratios.

Is monoamine oxidase activity in the outer mitochondrial membrane influenced by the mitochondrial respiratory state

Monoamine oxidase activity was measured in isolated rat liver mitochondria using the radiochemical assay with [14C]tyramine as substrate. With toluene as the extracting solvent the apparent activity in the resting state (State 4) was much higher than in the active state (State 3) in agreement with Smith and Reid (Smith, G.S. and Reid, R.A. (1978) Biochem. J. 176, 1011-1014). However, with ethyl acetate or diethyl ether as extracting solvents, the activity in both states was almost identical and several times higher than that measured with toluene. p-Hydroxyphenylacetaldehyde, p-hydroxyphenylacetalcohol and p-hydroxyphenylacetic acid were identified as final reaction products, the latter one being hardly extractable with toluene. It is concluded that monoamine oxidase activity is not influenced by the respiratory state of mitochondria and that differences found by Smith and Reid are due to different extractability of secondary reaction products. NADPH-dependent aldehyde reductase was tentatively identified in rat liver mitochondria, its specific activity amounting to about one fourth of that in the cytosol.

Use of the glucose oxidase/peroxidase method for glucose assay leads to overestimation of the inhibition of gluconeogenesis by aminopyrine

4-Aminoantipyrine strongly inhibits glucose determination by the glucose oxidase/peroxidase/dianisidine assay but does not interfere in the assay using glucose-6-phosphate dehydrogenase, hexokinase and ATP. As a result, the inhibition of gluconeogenesis by aminopyrine reported to be 50-90% (Bánhegyi, G., Mandl, J., Antoni, F. and Garzó, T. (1987) Biochim. Biophys. Acta 927, 406-416) is strongly overestimated and amounts to only 10-30%.