Ewa Lenartowicz

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

A complex effect of arsenite on the formation of α-ketoglutarate in rat liver mitochondria

Abstract This investigation presents disturbances of the mitochondrial metabolism by arsenite, a hydrophilic dithiol reagent known as an inhibitor of mitochondrial α-keto acid dehydrogenases. Arsenite at concentrations of 0.1–1.0 m m was shown to induce a considerable oxidation of intramitochondrial NADPH, NADH, and glutathione without decreasing the mitochondrial membrane potential. The oxidation of NAD(P)H required the presence of phosphate and was sensitive to ruthenium red, but occurred without the addition of calcium salts. Mitochondrial reactions producing α-ketoglutarate from glutamate and isocitrate were modulated by arsenite through various mechanisms: (i) both glutamate transaminations, with oxaloacetate and with pyruvate, were inhibited by accumulating α-ketoglutarate; however, at low concentrations of α-ketoglutarate the aspartate aminotransferase reaction was stimulated due to the increase of NAD + content; (ii) the oxidation of isocitrate was stimulated at its low concentration only, due to the oxidation of NADPH and NADH; this oxidation was prevented by concentrations of citrate or isocitrate greater than 1 m m ; (iii) the conversion of isocitrate to citrate was suppressed, presumably as a result of the decrease of Mg 2+ concentration in mitochondria. Thus the depletion of mitochondrial vicinal thiol groups in hydrophilic domains disturbs the mitochondrial metabolism not only by the inhibition of α-keto acid dehydrogenases but also by the oxidation of NAD(P)H and, possibly, by the change in the ion concentrations.

Ca2+-Sensitive reduction of 5,5′-dithiobis-(2-nitrobenzoic acid) by rat liver mitochondria

Abstract Energized rat liver mitochondria in the presence of EGTA reduced linearly 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) at the rate of 7 nmol SH/min per mg protein within more than 1 hour at 20°C. The K m for DTNB, 1.4 mM, was decreased by Mg 2+ and spermine to 0.5 and 0.7 mM, respectively. The reaction was suppressed under conditions of decreasing mitochondrial content of NADPH, was blocked by 1,3-bis-(2-chloroethyl)-1-nitrosourea, the inhibitor of disulfide reductases, and was sensitive to external free Ca 2+ in the micromolar range. After lysis of mitochondria the reduction of DTNB required the addition of NADPH and EGTA and was inhibited by 1 mM sodium arsenite. These observations suggest that the reduction of DTNB by mitochondria is catalyzed by Ca 2+ -sensitive thioredoxin reductase (EC 1.6.4.5).

Distribution of non-hydrolysable phosphorus compounds in the body of Galleria mellonella L. larvae

The acid-soluble, especially the non-hydrolysable, phosphate compounds have been quantitatively determined in various organs of the wax moth larva, Galleria mellonella. In all the investigated organs phosphorylethanolamine, phosphorylcholine, and phosphoglycerol have been found, but the amount of these compounds varies considerably. It is highest in the haemolymph (PG-14·1, PCh-21·2, and PEA-29·2 μmole/g) and smallest in the fat body. Orthophosphate and the hydrolysable P compounds are most abundant in the intestine. The content of phosphorylcholine and phosphorylethanolamine in the haemolymph of the wax moth exceeds many times the amounts found in other insects.

Phosphorylethanolamine and phosphorylcholine in the haemolymph of larvae of Galleria mellonella L. during starvation

Abstract During fasting of larvae of the wax moth phosphorylethanolamine is utilized to a much higher degree than phosphorylcholine. This results after prolonged starvation in a sharp decrease of the concentration of phosphorylethanolamine in the larval body and a small increase of that of phosphorylcholine; this is especially conspicuous in the haemolymph, which is a depot for these substances. The changes in the amount of phosphorylethanolamine and phosphorylcholine are discussed in relation to the metabolism of phospholipids. It is suggested that the metabolism of phosphorylethanolamine in larvae of the wax moth is partly independent of that of cephalins.

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 effect of low temperature and starvation on carbohydrate metabolism in larvae of Galleria mellonella L.

Abstract The main effect of a low environmental temperature (2°C) on carbohydrate metabolism of wax moth larvae is the accumulation of glucose. It derives most probably from trehalose since there is a great simultaneous decrease in the amount of this sugar. The utilization of glycogen takes place only during the initial few days of exposure to 2°C. During starvation for 2 to 4 weeks at 30°C the larvae utilize trehalose and monosaccharides, whereas glycogen is used up only at a later period of starvation. In ligated larvae a temporary accumulation of glycogen occurs during the first 10 days of starvation. This could be the result of conversion of trehalose into glycogen. The activities of trehalase and phosphorylase remain unchanged in fasting larvae kept at 2°C but decrease considerably in those kept at 30°C.

The relatioship between the reductive amination and carboxylation of 2-oxoglutarate in rat liver mitochondria

• Two NADPH-linked reductive reactions of 2-oxoglutarate, the amination to glutamate and the carboxylation to isocitrate were studied in isolated rat liver mitochondria. The amination was measured as the production of glutamate plus aspartate and the carboxylation as the bicarbonate-dependent production of citrate plus isocitrate. • The rate of the amination was higher and more dependent on 2-oxoglutarate concentration in State 3 than in State 4. The reaction was correlated with the concentration of NH4+ and the intramitochondrial content of ADP. Incontrast with the amination the carboxylation was higher in State 4 than in State 3, indicating that the reaction was primarily dependent on the degree of NADP reductino. The amination, but not the carboxylation, could attain a rate high enough to increases the levle of NADP+ in mitochondria. • Octanoate stimulated both reactions in State 3, and only the amination in State 4. The stimulation was correlated with an increase of NADP reduction. • During the intense synthesis of glutamate at 10 mM NH4+, the formation of isocitrate from 2-oxoglutarate was strongly suppressed. However, at 1 mM NH4+, the concentration approaching that in the liver cell, 2-oxoglutarate was reduced more quickly to isocitrate than to glutamate in State 4 in the absence of octanoate. In the presence of octanoate the amination was stimulated and its rate exceeded that of the carboxylation only at low concentration of 2-oxoglutarate. Transition to State 3 shifted that balance between these reactions in favour of glutamate synthesis. • The physiological implications of both processes are discussed.

Intramitochondrial reductive carboxylation of 2-oxoglutarate in adipose tissue and its contribution to fatty acid synthesis

The reductive carboxylation of 2-oxoglutarate was found to proceed in mitochondria of rat epididymal fat pads and rabbit perirenal adipose tissue at a rate similar to that in liver mitochondria. In rat fat pads the incorporation of 14C from [5-14C]2-oxoglutarate into fatty acids via the carboxylation was suppressed by butylmalonate by 30%. 2-Oxoglutarate and glutamate stimulated the incorporation into fatty acids of 14C from [2-14C]acetate in rat fat pads with the simultaneous reduction of tissue NADP. These effects persisted after inhibition of succinate dehydrogenase by malonate. It is concluded that in adipose tissue 2-oxoglutarate carboxylation proceeds in both the cytoplasm and mitochondria. Therefore, it can supply carbon atoms as well as NADPH for fatty acid synthesis.