Competing peroxidase and oxidase reactions in scopoletin-dependent H2O2-initiated oxidation of NADH by horseradish peroxidase.

Addition of NADH inhibited the peroxidative loss of scopoletin in presence of horseradish peroxidase and H2O2 and decreased the ratio of scopoletin (consumed):H2O2 (added). Concomitantly NADH was oxidized and oxygen was consumed with a stoichiometry of NADH:O2 of 2:1. On step-wise addition of a small concentration of H2O2 a high rate of NADH oxidation was obtained for a progressively decreasing time period followed by termination of the reaction with NADH:H2O2 ratio decreasing from about 40 to 10. The rate of NADH oxidation increased linearly with increase in scopoletin concentration. Other phenolic compounds including p-coumarate also supported this reaction to a variable degree. A 418-nm absorbing compound accumulated during oxidation of NADH. The effectiveness of a small concentration of H2O2 in supporting NADH oxidation increased in presence of SOD and decreased in presence of cytochrome c, but the reaction terminated even in their presence. The results indicate that the peroxidase is not continuously generating H2O2 during scopoletin-mediated NADH oxidation and that both peroxidase and oxidase reactions occur simultaneously competing for an active form of the enzyme.

Ferrous-iron induces lipid peroxidation with little damage to energy transduction in mitochondria.

Addition of ferrous sulfate, but not ferric chloride, in micromolar concentrations to rat liver mitochondria induced high rates of consumption of oxygen. The oxygen consumed was several times in excess of the reducing capacity of ferrous-iron (O:Fe ratios 5-8). This occurred in the absence of NADPH or any exogenous oxidizable substrate. The reaction terminated on oxidation of ferrous ions. Malondialdehyde (MDA), measured as thiobarbituric acid-reacting material, was produced indicating peroxidation of lipids. The ratio of O2:MDA was about 4:1. Pretreatment of mitochondria with ferrous sulfate decreased the rate of oxidation (state 3) with glutamate (+ malate) as the substrate by about 40% but caused little damage to energy transduction process as represented by ratios of ADP:O and respiratory control, as well as calcium-stimulated oxygen uptake and energy-dependent uptake of [45Ca]-calcium. Addition of succinate or ubiquinone decreased ferrous iron-induced lipid peroxidation in intact mitochondria. In frozen-thawed mitochondria, addition of succinate enhanced lipid peroxidation whereas ubiquinone had little effect. These results suggest that ferrous-iron can cause peroxidation of mitochondrial lipids without affecting the energy transduction systems, and that succinate and ubiquinone can offer protection from damage due to such ferrous-iron released from the stores within the cells.

Functions of cytochrome c in regulation of electron transfer and protein folding.

Cytochrome c, a "mobile electron carrier" of the mitochondrial respiratory chain, also occurs in detectable amounts in the cytosol, and can receive electrons from cytochromes present in endoplasmic reticulum and plasma membranes as well as from superoxide and ascorbate. The pigment was found to dissociate from mitochondrial membranes in liver and kidney when rats were subjected to heat exposure and starvation, respectively. Treating cytochrome c with hydroxylamine gives a partially deaminated product with altered redox properties; decreased stimulation of respiration by deficient mitochondria, increased reduction by superoxide, and complete loss of reducibility by plasma membranes. Mitochondria isolated from brown adipose tissue of cold-exposed rats are found to be sub-saturated with cytochrome c. The ability of cytochrome c to reactivate reduced ribonuclease is now reinterpreted as a molecular chaperone role for the hemoprotein.

Preparation of a soluble 58 kDa-3-hydroxy-3-methylglutaryl CoA reductase from liver microsomes and its inhibition by ethoxysilatrane, a hypocholesterolemic compound.

On repeated thawing at room temperature of frozen preparations of heavy microsomes from rat livers, HMGCoA reductase activity was solubilized due to limited proteolysis. This soluble enzyme was partially purified by fractionation with ammonium sulfate and filtration on Sephacryl S-200 column. The active enzyme was coeluted with a major 92 kDa-protein and was identified as a 58 kDa-protein after separation by SDS-PAGE and immunoblotting. Ethoxysilatrane, a hypocholesterolemic compound, which decreased the liver-microsomal activity of HMGCoA reductase on intra-peritonial treatment of animals, showed little effect on the enzyme activity with isolated microsomes or the 50 kDa-soluble enzyme when added in the assay. But it was able to inhibit the activity of the soluble 58 kDa-enzyme in a concentration-dependent, reversible manner. Cholesterol and an oxycholesterol were without effect whereas chlorophenoxyisobutyrate and ubiquinone showed small inhibition under these conditions. The extra region that links the active site domain (50 kDa protein) to the membrane, present in the 58 kDa-protein appears to be involved in mediating the inhibition by silatrane.

Brown adipose tissue mitochondria are cytochrome c-subsaturated.

The oxidative activity of mitochondria freshly isolated from brown adipose tissue of rats was stimulated two-fold on the addition of small concentrations of exogenous cytochrome c to the reaction medium. Loss of membrane-bound cytochrome c did not occur during isolation of mitochondria. Estimation of the high-affinity binding sites on the organelle membrane indicated that less than a third of these sites remained saturated with cytochrome c. The pigment is thus shown to be a functionally limiting electron transport component in brown adipose tissue.

The nature of inhibition of 3-hydroxy-3-methylglutaryl CoA reductase by garlic-derived diallyl disulfide.

A concentration dependent inhibition of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase was found on preincubation of microsomal preparations with diallyl disulfide, a component of garlic oil. This inhibited state was only partially reversed even with high concentrations of DTT. Glutathione, a naturally occurring reducing thiol agent, was ineffective. The substrate, HMG CoA, but not NADPH, was able to give partial protection for the DTT-dependent, but not glutathione-dependent activity. The garlic-derived diallyl disulfide is the most effective among the sulfides tested for inhibition of HMG CoA reductase. Formation of protein internal disulfides, inaccessible for reduction by thiol agents, but not of protein dimer, is likely to be the cause of this inactivation.

Regulation of respiratory activity of brown adipose tissue mitochondria through changes in cytochromes.

Exposure of cold-acclimatized rats to heat (37 degrees C) for a short period decreased brown adipose tissue (BAT) mitochondrial substrate-dependent oxygen uptake and H2O2 generation. Both the concentration and substrate-dependent rate of cytochrome b reduction decreased as early as 3 h of heat exposure. These results identify cytochrome b as the locus of regulation of electron transport in BAT mitochondria under conditions of heat stress.

Investigations on the mechanism of the hypocholesterolemic action of 1-ethoxysilatrane.

Intraperitoneal administration of the nontoxic silicon compound, 1-ethoxysilatrane, to the rat did not cause proliferation of hepatic mitochondria or of endoplasmic reticulum, nor did it affect mitochondrial oxidative phosphorylation. The activities of cholesterol 7 alpha-hydroxylase in hepatic microsomes and of cholesterol oxidase in mitochondria respectively were unaffected by silatrane treatment. The rate of release of bile, whose composition remained unchanged, also was not increased in silatrane-treated animals. The results indicated that the compound did not affect the pathway of cholesterol degradation. A progressive decrease in the activity of hepatic microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase was observed on administration of the compound over a period of three weeks. Consistent with this, cholesterol biosynthesis in liver as measured by incorporation of radioactive precursors, acetate and water but not mevalonate, was significantly decreased in silatrane-treated animals. However, enzyme-linked immunosorbent assay revealed that the concentration of HMGCoA reductase protein was not decreased by the treatment indicating that inactivated enzyme was also present in such microsomes. Addition of silatrane to microsomes in the assay system did not cause inhibition indicating that the inactivation is by an indirect mechanism. It is concluded that the hypocholesterolemic action of the compound rested entirely on the inhibition of cholesterol biosynthesis in vivo by inactivation of the rate-limiting enzyme HMGCoA reductase.

Decavanadate acts like an alpha-adrenergic agonist in redistributing protein kinase C activity.

Perfusion of rat livers with polyvanadate, but not metavanadate, was found to increase in plasma membrane and decrease in cytosol protein kinase C activity, similar to that obtained with phenylephrine, an alpha-adrenergic agonist. The effect was prevented by phenoxybenzamine, but not by propranolol implicating alpha-adrenergic receptor activation. Comparison of crystal structures of decavanadate and nonadrenaline revealed the occurrence of a structural feature of O-O-O(N) with distances of 5.5 A and 2.9 A.

Decrease of oxidative activities in brown adipose tissue mitochondria of cold acclimated rats on short term exposure to heat stress.

Exposure of rats to the cold (4-5 degrees C) caused large (2-3-fold) increases in the mass of interscapular brown adipose tissue (BAT), its mitochondrial content and the basal metabolic rate of the animals. The rate of substrate oxidation by BAT mitochondria also increased about 3-fold. When cold-acclimated animals were exposed to heat (37 degrees C), the BMR decreased by half in 3 h, the earliest time interval tested. Mitochondrial substrate oxidation, as well as substrate-dependent H2O2 generation, showed a proportionate decrease in rates. In these mitochondria, activities of cytochrome c reductases, but not dehydrogenases with NADH, alpha-glycerophosphate and succinate as substrates, also showed a significant decrease. The concentration of cytochromes aa3 and b, but not cytochrome c, also decreased in BAT mitochondria from 12-h heat-exposed animals, while the change in concentration of cytochrome b alone was found as early as 3 h of heat exposure. These results identify the change in cytochromes as a mechanism of regulation of oxidative activities in BAT mitochondria under conditions of acute heat stress.

Changes in thyroid hormone and insulin status of the brown adipose tissue of cold acclimated rats on short term exposure to heat.

Acclimation of rats to cold caused 45% increase in the concentration of triidothyronine (T3) and 35% increase in the concentration of thyroxine (T4) in serum. Exposure of cold-acclimated rats to heat (12 hr, 37 degrees C) failed to decrease the concentrations of thyroid hormones in circulation. The concentration of T3 in brown adipose tissue (BAT) increased almost 10-fold on cold acclimation. Iodothyronine deiodinase activity also registered 3-fold increase. Exposure of cold-acclimated animals to heat caused decrease in the concentration of T3 in BAT without appreciably affecting T4 concentration. In liver tissue, the changes in hormone concentrations were quite small compared to those in BAT. On thyroidectomy or when fed with propyl thiouracil, rats could not survive exposure to the cold. The concentration of insulin in circulation showed small increase, while that in the tissues showed significant decrease on acclimation of rats to the cold. The concentration of the hormone in BAT registered significant increase on exposure of cold-acclimated animals to heat (12 hr, 37 degrees C). The increase in liver was marginal. The temperature-dependent response of T3 indicates an important role for this hormone in rapid physiological response in BAT.

Increase in hepatic microsomal lipid peroxidation mediated by alpha-adrenergic system under cold stress and noradrenaline treatment.

Lipid peroxidation measured both by the formation of malondialdehyde and by oxygen uptake in presence of NADPH, Fe2+ and ADP in hepatic microsomes increased on cold exposure and decreased on heat exposure of rats. Malondialdehyde content of isolated microsomes also showed similar changes. Treatment of animals with noradrenaline or a alpha-adrenergic agonist, phenylephrine, increased lipid peroxidation which was prevented by simultaneous treatment with cycloheximide, a protein synthesis inhibitor. Cold- and noradrenaline-induced increases were not found in animals pretreated with alpha-adrenergic receptor antagonist, phenoxybenzamine, but not with propranolol, a beta-adrenergic blocking agent. The concentration of the microsomal cytochromes P-450 and b5 remained unaffected under these conditions but the activity of NADPH-cytochrome c reductase showed parallel changes. These observations suggest a role for lipid peroxidation in cellular thermogenesis in liver tissue.

Influence of cerebral ischemia and post-ischemic reperfusion on mitochondrial oxidative phosphorylation.

Unilateral ischemia in the right cerebral hemisphere of the rat was induced by ligation of the right common carotid artery coupled with controlled hemorrhage to produce hypotension (25 +/- 8 mm/Hg). Where indicated after 30 min of ischemia, the withdrawn blood was reinfused to restore arterial pressure to normal. Mitochondria isolated from the ipsilateral hemisphere after 30 min of ischemia showed significantly lower respiratory rates than the organelles isolated from the contralateral side. Oxidation of NAD(+)-linked substrates was more sensitive to inhibition in ischemia (30%) than was of ferrocytochrome c (12%), succinate oxidation being intermediate. The activities of membrane-bound dehydrogenases (both NADH and succinate-linked) were also significantly lowered. Ischemia did not affect the cytochrome content of mitochondria. Respiratory activity (NAD(+)-linked) of mitochondria isolated from the ipsilateral hemisphere was twice as sensitive to inhibition by fatty acid as was of preparations from the contralateral side. Mitochondria isolated from cerebral cortex after 90 min of post-ischemic reperfusion showed no significant improvement in the rate of substrate oxidation. Adenine nucleotide translocase activity and energy-dependent Ca2+ uptake, both of which decreased significantly in mitochondria isolated from the ischemic brain, showed little recovery, on reperfusion. These observations suggested the strong possibility that the deleterious effects of ischemia on mitochondrial respiratory function might be mediated by free fatty acids that are known to accumulate in large amounts in ischemic tissues. The pattern of inhibition of ATPase activity was consistent with this view.

Microsomal redox systems in brown adipose tissue: high lipid peroxidation, low cholesterol biosynthesis and no detectable cytochrome P-450.

The presence of redox systems in microsomes of brown adipose tissue (BAT) in cold exposed rats was investigated and compared with liver. BAT microsomes showed high activity of lipid peroxidation measured both by the formation of malondialdehyde (MDA) and by oxygen uptake. NADH and NADPH dependent cytochrome c reductase activity were present in both BAT and liver microsomes. Aminopyrine demethylase and aniline hydroxylase activities, the characteristic detoxification enzymes in liver microsomes could not be detected in BAT microsomes. BAT minces showed very poor incorporation of [1-14C]acetate and [2-14C]mevalonate in unsaponifiable lipid fraction compared to liver. Biosynthesis of cholesterol and ubiquinone, but not fatty acids, and the activity of 3-hydroxy-3-methyl glutaryl CoA reductase appear to be very low in BAT. Examination of difference spectra showed the presence of only cytochrome b5 in BAT microsomes. In addition to the inability to detect the enzyme activities dependent on cytochrome P-450, a protein with the characteristic spectrum, molecular size in SDS-PAGE and interaction with antibodies in double diffusion test, also could not be detected in BAT microsomes. The high activity of lipid peroxidation in microsomes, being associated with large oxygen uptake and oxidation of NADPH, will also contribute to the energy dissipation as heat in BAT, considered important in thermogenesis.

Redistribution of subcellular calcium in rat liver on administration of vanadate.

Mitochondria isolated from the livers of rats administered with sodium meta-, ortho-, or polyvanadate, but not vanadyl sulphate, exhibited enhanced Ca2+ -stimulated respiration and uptake of calcium. These effects were shown also by mitochondria isolated from livers perfused with polyvanadate. The concentration of acid-soluble calcium decreased significantly in the mitochondrial fraction on vanadate treatment, while that in the cytosol showed a corresponding increase. Phenoxybenzamine, an antagonist to alpha-adrenergic receptors, effectively inhibited vanadate-induced Ca2+ mobilization, but surgical sympathectomy was without effect. This is the first demonstration of vanadate mimicking alpha-adrenergic agonists in vivo.

Increase in alpha-glycerophosphate dehydrogenase and other oxidoreductase activities of hepatic mitochondria on administration of vanadate to the rat.

Liver mitochondria isolated from vanadate-administered rats showed increased (20-25%) rates of oxidation of both NAD(+)-linked substrates and succinate. Respiratory control index and ADP/O were unaffected by the treatment. Dormant and uncoupler-stimulated ATPase activity also was not affected by vanadate administration. Membrane-bound, electron-transport-linked dehydrogenase activities (both NAD(+)- and succinate-dependent) increased by 15-20% on vanadate treatment. Mitochondrial alpha-glycerophosphate dehydrogenase activity increased by 50% on vanadate administration. The above effects of vanadate on oxidoreductase activities could be prevented by the prior administration of antagonists to alpha-adrenergic receptors. Substrate-dependent H2O2 generation by mitochondria also showed an increase on vanadate administration.

Oxidative activities in mitochondria-like particles from Setaria digitata, a filarial parasite.

The oxidative metabolic potential of Setaria digitata, a filarial parasite found in the intraperitoneal cavity of cattle, was investigated. These worms showed active wriggling movements which were not affected by respiratory poisons such as cyanide, rotenone and malonate. They also possessed cyanide-insensitive and glucose-independent oxygen consumption pathways. By differential centrifugation of sucrose homogenates, a fraction containing mitochondria-like particles was obtained in which the activity of the marker enzyme, succinate dehydrogenase, was recovered. This fraction catalysed succinate- and NADH-dependent reduction of both cytochrome c and dyes. Oxygen uptake found with succinate, NADH and ascorbate as substrates was not sensitive to cyanide. Cytochromes could not be detected in either this fraction or homogenates of the worms. H2O2 generation with a number of substrates and lipid peroxidation by measuring malondialdehyde formed as well as by accompanying oxygen uptake were demonstrated in the mitochondria-like particles. A lipid quinone, possibly with a short side chain and related to ubiquinone, was detected in the worms. The results suggested the existence of two cyanide-insensitive oxygen-consuming reactions in Setaria: one respiratory substrate-independent lipid peroxidation, and a second substrate-dependent reaction that requires an auto-oxidizable quinone but not a cytochrome system.