Interaction of ubisemiquinone with succinate dehydrogenase and the cytochrome chain of mitochondria

The free radical EPR signals of ubisemiquinone in mitochondria and submitochondrial particles (SMP) were investigated. One of the signals observed under the conditions of the respiratory chain highly oxidized and characterized by an unusually short time of the spin-lattice relaxation has previously been termed as SQ-2. The intensity of SQ-2 in SMP strongly depends on pH, the maximal concentration of QH. is reached at about 8.5. The signal is absent in the succinate dehydrogenase-depleted SMP and is highly sensitive to specific inhibitors of succinate: CoQ-oxidoreductase, such as alpha-thenoyltrifluoroacetone and carboxin. In SMP SQ-2 disappears in the presence of low concentrations of ferricyanide, while in mitochondria this non-penetrating oxidant provokes the appearance of SQ-2. The data obtained suggest that SQ-2 belongs to a stable ubisemiquinone which forms a complex with a FeS center of succinate dehydrogenase, is localized at the M-side of the membrane, and is kinetically isolated from the cytochrome chain. Oxidation of the terminal segment of the respiratory chain of mitochondria and SMP reduced by succinate in the presence of antimycin, is in some cases accompanied by an appearance of a strong free radical EPR signal which is stable at 77K but disappears rapidly in the frozen samples at -30- -40 degrees C. It is suggested that the signal is generated by an antimycin-insensitive oxidation of QH2 to QH. via the branch of the respiratory chain comprised of the Rieske FeS-protein and cytochrome c1. The mechanisms of how the two-electron oxidation-reduction of CoQ is coupled with the one-electron transfer through the cytochromes and FeS centers in the respiratory chain are discussed.     

Effect of a short-term fasting and altitude hypoxia on the cytochrome oxidase activity of rat brain mitochondria

The effect of short-term fasting and thirst, prolonged fasting and hypoxic hypoxia upon the activity of cytochrome oxidase was studied in mitochondrial fractions obtained from the brain and the liver. The investigation was carried out in two groups of rats, 5 and 60 days old. a) The activity of cytochrome oxidase in mitochondria isolated from the brain cortex, subcortical regions and the medulla oblongata rises, while the changes in liver mitochondrial fractions are reverse. b) A significant increase of mitochondrial cytochrome oxidase was found in 5-day-old rats after both types of fasting and hypoxia in all regions of the brain, as well as in the liver. c) The cytochrome oxidase activity in brain and liver mitochondria of 60-day-old rats was not affected appreciably after 24 h nutritional deprivation, with the exception of a significant rise of activity in the medulla oblongata. Prolonged fasting and hypoxia again markedly increased the activity of this enzyme in all regions of the brain and in the liver.     

Effect of ruthenium complexes on the activities of succinate dehydrogenase and cytochrome oxidase

In this article, we report the effects of acute administration of ruthenium complexes, trans-[RuCl(2)(nic)(4)] (nic=3-pyridinecarboxylic acid) 180.7 micromol/kg (complex I), trans-[RuCl(2)(i-nic)(4)] (i-nic=4-pyridinecarboxylic acid) 13.6 micromol/kg (complex II), trans-[RuCl(2)(dinic)(4)] (dinic=3,5-pyridinedicarboxylic acid) 180.7 micromol/kg (complex III) and trans-[RuCl(2)(i-dinic)(4)]Cl (i-dinic=3,4-pyridinedicarboxylic acid) 180.7 micromol/kg (complex IV) on succinate dehydrogenase (SDH) and cytochrome oxidase (COX) activities in brain (hippocampus, striatum and cerebral cortex), heart, skeletal muscle, liver and kidney of rats. Our results showed that complex I inhibited SDH activity in hippocampus, cerebral cortex, heart and liver; and inhibited COX in heart and kidney. Complex II inhibited SDH in heart and hippocampus; COX was inhibited in hippocampus, heart, liver and kidney. SDH activity was inhibited by complex III in heart, muscle, liver and kidney. However, COX activity was increased in hippocampus, striatum, cerebral cortex and kidney. Complex IV inhibited SDH activity in muscle and liver; COX activity was inhibited in kidney and increased in hippocampus, striatum and cerebral cortex. In a general manner, the complexes tested in this work decrease the activities of SDH and COX in heart, skeletal muscle, liver and kidney. In brain, complexes I and II were shown to be inhibitors and complexes III and IV activators of these enzymes. In vitro studies showed that the ruthenium complexes III and IV did not alter COX activity in kidney, but activated the enzyme in hippocampus, striatum and cerebral cortex, suggesting that these complexes present a direct action on COX in brain.     

Effect of phenylpyruvate on pyruvate dehydrogenase activity in rat brain mitochondria

1. The effects of phenylpyruvate, a metabolite produced in phenylketonuria, on the pyruvate dehydrogenase-complex activity were investigated in rat brain mitochondria. 2. Pyruvate dehydrogenase activity was measured by two methods, one measuring the release of (14)CO(2) from [1-(14)C]pyruvate and the other measuring the acetyl-CoA formed by means of the coupling enzyme, pigeon liver arylamine acetyltransferase (EC 2.3.1.5). In neither case was there significant inhibition of the pyruvate dehydrogenase complex by phenylpyruvate at concentrations below 2mm. 3. However, phenylpyruvate acted as a classical competitive inhibitor of the coupling enzyme arylamine acetyltransferase, with a K(i) of 100mum. 4. It was concluded that the inhibition of pyruvate dehydrogenase by phenylpyruvate is unlikely to be a primary enzyme defect in phenylketonuria.     

Activation of succinate oxidase in the inner membrane of rat liver mitochondria

It is shown that the process of activation of succinate oxidase from inner membranes of the rat liver mitochondria by succinate and malonate is specific for the succinate dehydrogenase component of oxidase. These activation constants are comparable with those found by other authors in activation of succinate dehydrogenase and succinate oxidase from oxaloacetate-preincubated submitochondrial fragments of the bull heart. Probably, the 4-fold activation of succinate oxidase from inner membranes of the liver mitochondria reported in this paper depends on separation of endogenous oxaloacetate from the succinate dehydrogenase component of oxidase.     

Inner mitochondrial membranes bound to concanavalin A-sepharose display succinate dehydrogenase, ATPase, and cytochrome oxidase activity

A fraction (15-20% of the total protein) of a preparation of bovine submitochondrial particles (SMPs) binds to concanavalin A-sepharose. The bound membranes displayed succinate dehydrogenase, cytochrome oxidase, and ATPase activity, which, as in SMPs, were inhibited by malonate, cyanide, and oligomycin, respectively. These results indicate that the bound membranes are inner mitochondrial membranes and that they contain a glycoprotein which was recognized by concanavalin A. It was possible to repeatedly perform the three enzyme assays, one after the other, in the same gel with the bound membranes. Long-term stability tests (22 days) showed that cytochrome oxidase was much more stable in the membranes bound to the gel than in SMPs, while the ATPase activity decayed at a similar rate in the two conditions. Thus, inner mitochondrial membranes bound to ConA-Sepharose appear to be a potentially interesting model for the study of immobilized multienzymatic complexes.     

Effect of alloxan-diabetes and subsequent treatment with insulin on kinetic properties of succinate oxidase activity from rat liver mitochondria

We evaluated early and late effects of alloxan-diabetes and subsequent insulin treatment on the kinetic properties of succinate oxidase (SO) in rat liver mitochondria. Diabetic state lowered the SO activity; insulin treatment was effective in restoring the activity only in one-week diabetic rats. The energies of activation in low and high temperature ranges (EH and EL) decreased significantly in diabetic animals; once again insulin treatment was partially effective only in the one-week diabetic group. The total phospholipids (TPL) and cholesterol (CHL) contents did not change in one-week groups. In one-month diabetic animals TPL decreased while CHL increased; insulin treatment induced further changes without restoring normality. The lysophospholipid (Lyso), sphingomyelin (SPM), phosphatidylinositol (PI) and phosphatidylserine (PS) content increased in the diabetic state while phosphatidylcholine (PC) and phosphatidylethanolamine (PE) decreased. Insulin treatment had a partial restorative effect. The changes in EH correlated negatively with SPM. The phase transition temperature, Tt, decreased in diabetic and insulin-treated groups. These changes correlated positively with the ratios of TPL/PI and TPL/PS. The membrane fluidity decreased in the diabetic state; insulin had a restorative effect only in the one-week group.     

Monoamine oxidase activity of rat brain under cold exposure

Cold stress and cold adaptation were studied for their effect on the activity and substrate specificity of the monoamine oxidase A and B and on the Km of serotonin deamination in the rat brain mitochondria and supernatant. Mitochondrial monoamine oxidase Km with serotonin is established to increase more than twice under cold stress and decrease considerably in cold adapted rats. The lowering of the mitochondrial monoamine oxidase A activity is accompanied by the appearance of serotonin and the glucosamine deaminating activity in supernatant. The data suggest that decrease in the monoamine oxidase activity under cold stress may be caused by both release of the enzyme from mitochondrial membrane and changes in its catalytic property alteration.     

Effect in vitro of benthiocarb on rat brain succinate dehydrogenase

Kinetic study of succinate dehydrogenase (SDH) was studied in the brain of albino rat to elucidate the interaction of benthiocarb, an organocarbamate with oxidative metabolism. The significant decrease in maximal velocity (Vmax) without appreciable change in Michaelis-Menten constant (Km) indicates that benthiocarb did not affect or interfere with succinate oriented sites on the enzyme and the inhibition is of a classical non-competitive type.     

Marked and variable inhibition by chemical fixation of cytochrome oxidase and succinate dehydrogenase in single motoneurons

The effect of tissue fixation on succinate dehydrogenase and cytochrome oxidase activity in single motoneurons of the rat was demonstrated using a computer image processing system. Inhibition of enzyme activity by chemical fixation was variable, with some motoneurons being affected more than others. It was concluded that quantification of enzymatic activity in chemically fixed tissue provides an imprecise estimate of enzyme activities found in fresh-frozen tissues.     

Dimethyl sulfoxide elicited increase in cytochrome oxidase activity in rat liver mitochondria in vivo and in vitro

A single intraperitoneal injection of dimethyl sulfoxide (275 mg/100 g body wt.) to rats stimulated cytochrome oxidase activity in liver mitochondria 2-5-fold. The enzyme activity remained at this level for as long as 5 days post-injection. There was however only 10.5% increase in the content of cytochromes a and a3 (as determined spectrophotometrically) in the same period in response to DMSO injection. The addition of either DMSO or dimethyl sulfate (a metabolite of DMSO) to isolated liver mitochondria also caused 2-3-fold increase in cytochrome oxidase activity. The results indicate that enhancement in cytochrome oxidase activity in liver mitochondria after administration of DMSO to rats is on account of activation of cytochrome oxidase caused by structural alterations in mitochondrial membranes rather than de novo synthesis of cytochrome oxidase.     

The influence of respiratory state on monoamine oxidase activity in rat liver mitochondria.

Changes in the respiratory state of rat liver mitochondria caused significant changes (up to 10-fold) in the rates of oxidative deamination of tyramine, indicating interactions between the inner coupling membrane and the monoamine oxidase sites in the outer membrane, and suggesting the possibility that monoamine oxidase is regulated by the thermodynamic state of the mitochondria.