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.

Decarboxylation of branched-chain alpha-ketoacids in hepatocytes from alloxan-diabetic rats. The effect of insulin.

The flux through branched-chain alpha-ketoacid dehydrogenase and the activity of the branched-chain alpha-ketoacid dehydrogenase complex were measured in hepatocytes isolated from fed, starved and alloxan diabetic rats. The highest rate of branched-chain alpha-ketoacid oxidation was found in hepatocytes isolated from starved rats, slightly lower in those from fed rats, and significantly lower in diabetic hepatocytes. The amount of the active form of branched-chain alpha-ketoacid dehydrogenase was only slightly diminished in diabetic hepatocytes, whereas the flux through the dehydrogenase was inversely correlated with the rate of endogenous ketogenesis. The same was observed in hepatocytes isolated from starved rats when branched-chain alpha-ketoacid oxidation was measured in the presence of added oleate. In both cases the diminished flux through the dehydrogenase, restored by a short preincubation of hepatocytes with insulin, was paralleled by a decrease of fatty acid-derived ketogenesis. The significance of these findings is discussed in relation to the role of insulin in branched-chain alpha-ketoacid oxidation in liver of diabetic rats.

Effect of glucagon and phorbol myristate acetate on oxidative demethylation and lipid peroxidation in isolated hepatocytes.

Oxidative demethylation of aminopyrine and peroxidation of endogenous lipids induced by cumene hydroperoxide were studied in hepatocytes isolated from fed male rats. Glucagon and phorbol-12-myristate-13-acetate (PMA) inhibited both processes in the concentration-dependent manner. Pretreatment of hepatocytes with 1 microM glucagon decreased oxidative demethylation by 75% and had a much smaller effect on lipid peroxidation. Preincubation with 1 microM PMA inhibited both processes by 25-30%. Phosphorylation of three isoforms of cytochrome P-450 was observed in microsomes isolated from hepatocytes incubated in the presence of [32P]orthophosphate. After incubation with PMA the phosphorylation of all these proteins was increased by 60-100%, whereas glucagon increased the phosphorylation of only one isoform. Consequences of the phosphorylation of various isoforms of cytochrome P-450 for metabolic functions of the monooxygenase system are discussed.

The effect of Ca2+ and calmodulin on the inhibition of Ca2(+)+Mg2(+)-ATPase in erythrocyte ghost membranes by nonpolar and polar carbodiimides.

N,N'-dicyclohexylcarbodiimide (DCCD) and 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide (CMCD) inhibited calmodulin-dependent Ca2(+)+Mg2(+)-ATPase activity in erythrocyte ghost membranes. The extent of the inhibition caused by carbodiimides strongly depended on their hydrophobicity. Hydrophobic DCCD was a more potent inhibitor then hydrophilic CMCD. Calmodulin (CaM) protected the enzyme against the former carbodiimide, whereas Ca2+ did the same against the latter. In contrast to previous observations made by Villalobo et al., on the purified enzyme, neither carbodiimide affected the calmodulin-independent ATPase activity in ghost membranes. Inhibition of the calmodulin-dependent ATPase activity was due to a decrease of the maximum activity, whereas the Km value for Ca2+ remained unchanged. Titration of erythrocyte ghost membranes with CaM revealed a biphasic response of ATPase to this activator. Two affinity constants were found for CaM, 0.64 nM and 14 nM. DCCD affected the interaction with CaM at high- and low-affinity binding sites in a competitive manner. CMCD acted as a noncompetitive inhibitor for CaM low-affinity sites, whereas it behaved in a competitive way against CaM interaction with high-affinity sites. In E2 form (stabilized by vanadate and EGTA) ATPase was more sensitive to carbodiimides than in E1 form (induced by La3+).

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%.

On the mechanism of the so-called uncoupling effect of medium- and short-chain fatty acids.

Octanoate applied to rat liver mitochondria respiring with glutamate plus malate or succinate (plus rotenone) under resting-state (State 4) conditions stimulates oxygen uptake and decreases the membrane potential, both effects being sensitive to oligomycin but not to carboxyatractyloside. Octanoate also decreases the rate of pyruvate carboxylation under the same conditions, this effect being correlated with the decrease of intramitochondrial content of ATP and increase of AMP. The decrease of pyruvate carboxylation and the change of mitochondrial adenine nucleotides are both reversed by 2-oxoglutarate. Fatty acids of shorter chain length have similar effects, though at higher concentrations. Addition of octanoate in the presence of fluoride (inhibitor of pyrophosphatase) produces intramitochondrial accumulation of pyrophosphate, even under conditions when oxidation of octanoate is prevented by rotenone. In isolated hepatocytes incubated with lactate plus pyruvate, octanoate also increases oxygen uptake and produces a shift in the profile of adenine nucleotides similar to that observed in isolated mitochondria. It decreases the 'efficiency' of gluconeogenesis, as expressed by the ratio between an increase of glucose production and an increase of oxygen uptake upon addition of gluconeogenic substrates (lactate plus pyruvate), and increases the reduction state of mitochondrial NAD. These effects taken together are not compatible with uncoupling, but point to intramitochondrial hydrolysis of octanoyl-CoA and probably also shorter chain-length acyl-CoAs. This mechanism probably functions as a 'safety valve' preventing a drastic decrease of intramitochondrial free CoA under a large supply of medium- and short-chain fatty acids.

Significance of the alanine aminotransferase reaction in the formation of alpha-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.

Stimulation of mitochondrial functions by glucagon treatment, starvation and by treatment of isolated mitochondria with glycogen-bound enzymes.

Liver mitochondria isolated from rats starved overnight, or fed rats injected with glucagon, exhibited a similar increase of the respiration rate with succinate (by 30-40%) and glutamate plus malate (by 20-30%), as compared to mitochondria from control fed animals. The content of mitochondrial adenine nucleotides was elevated by 30-45% by glucagon treatment or starvation. Mitochondrial respiration and citrulline synthesis were stimulated by 30-40% when mitochondria isolated from fed rats were briefly preincubated with the extract from liver glycogen granules, ATP and MgCl2. This effect was abolished by heating the extract at 100 degrees C.

Transport of 2-oxoisocaproate in isolated hepatocytes and liver mitochondria.

The transport of 2-oxoisocaproate into isolated hepatocytes and liver mitochondria of rat was studied using [U-14C]2-oxoisocaproate and the silicone oil filtration procedure. 2-Oxoisocaproate uptake by hepatocytes was composed of: rapid adsorption, unmediated diffusion and carrier-mediated transport. The carrier-mediated transport was strongly inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid and p-chloromercuribenzoate, was less sensitive to alpha-cyano-4-hydroxycinnamate and insensitive to p-chloromercuriphenylsulphonate. Other 2-oxo acids: pyruvate, 2-oxoisovalerate and 2-oxo-3-methylvalerate, were also inhibitory. The kinetic parameters of the carrier-mediated transport were Km 30.6 mM and Vmax 23.4 nmol/min per mg wet wt, at 37 degrees C. It is concluded that at its low, physiological, concentration, 2-oxoisocaproate penetrates the hepatocyte membrane mainly by unmediated diffusion. The uptake of 2-oxoisocaproate by isolated liver mitochondria was partly inhibited by alpha-cyano-4-hydroxycinnamate, the inhibitor of mitochondrial monocarboxylate carrier. The remaining uptake was linearly dependent on 2-oxoisocaproate concentration and represented unmediated diffusion. The carrier-mediated transport exhibited the following kinetic parameters: Km 0.47 mM, Vmax 1.0 nmol/min per mg protein at 6 degrees C; and Km 0.075 mM and Vmax about 8 nmol/min per mg protein at 37 degrees C.

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.

Interrelations between ureogenesis and gluconeogenesis in isolated hepatocytes. The role of antion transport and the competition for energy.

1. Glucose synthesis from lactate plus pyruvate and from lactate plus alanine was measured in the presence or absence of 1mM-oleate or 2mM-octanoate at low (2mM) or high (8mM) concentrations of NH4Cl. 2. Both fatty acids alone or with 2mM-NH4Cl doubled glucose production from lactate plus pyruvate. Glucose synthesis from lactate plus alanine, in the presence of oleate, was decreased 16% by 2mM-NH4Cl. 3. In the presence of fatty acids, 8mM-NH4Cl decreased gluconeogenesis by 60-65% from both lactate plus pyruvate and lactate plus alanine. This inhibition was correlated with a high accumulation of aspartate and a drastic decrease in 2-oxoglutarate and malate in the cells. 4. In the presence of 2mM- or 8 mM-NH4Cl, oleate and glucogenic precursors, the addition of 2.5mM-ornithine stimulated urea synthesis. 5. This was paralleled by a decrease of 16% in glucose synthesis from lactate plus pyruvate in the presence of 2mM-NH4Cl and had no effect at 8mM-NH4Cl. In the system producing glucose from lactate plus alanine, ornithine completely reversed the inhibition caused by 2mM-NH4Cl and only partly that by 8mM-NH4Cl. 6. Gluconeogenesis from pyruvate was also inhibited by 2mM-NH4Cl in the presence of oleate or ethanol. This way due to the decrease of malate, which is the C4 precursor of glucose in this system. 7. The limitation of gluconeogenesis by 2-oxoglutarate and malate concentrations in the liver cell and the competition for energy between glucose and urea synthesis is discussed.

The inhibition of isocitrate oxidation by palmitoyl-l-carnitine and palmitoyl-C0 A in rat liver mitochondria.

Palmitoyl-L carnitine decreases the oxidation of isocitrate in rat liver mitochondria in state 3 by 25-30%. Palmitoyl-L-carnitine acts as an additional substrate raising the rate of oxidative phosphorylation, NAD reduction and ATP/ADP ratio in mitochondria. Palmitoyl-CoA added to mitochondria oxidizing isocitrate in state 3 causes a strong inhibition of isocitrate oxidation and of oxidative phosphorylation and a considerable elevation of intramitochondrial NADH/NAD and ATP/ADP ratios. The effect of palmitoyl-CoA is dependent on its concentration and is competitive with ADP. Carnitine restores only oxidative phosphorylation, but the oxidation of isocitrate remains inhibited. Evidence is presented that the transport of isocitrate is not affected by palmitoyl-CoA is due to the inhibition of adenine nucleotide translocation. The kinetic studies of NAD-dependent isocitrate dehydrogenase in the soluble fraction of sonicated mitochondria revealed that the enzyme is very sensitive towards the inhibition by NADH and only very slightly affected by ATP (Ki for NADH and ATP are 0.017 and 3.6 mM respectively). On the basis of the kinetic data the relative contribution of NADH and ATP in the inhibition of isocitrate oxidation by fatty acids was calculated. It is concluded that the inhibition of isocitrate oxidation caused by palmitoyl-L-carnitine and palmitoyl-CoA is primarily due to the increased reduction of NAD, whereas the increase of ATP/ADP ratio is much less important.