Role of the glycerophosphate shunt in regulating oxidation of cytoplasmic NADH and ATP synthesis by liver mitochondria during rat ontogeny

The activity of alpha-glycerophosphate dehydrogenase rate of alpha-glycerophosphate oxidation and ATP synthesis were determined in the liver mitochondria of 1, 3, 12 and 24-month old rats in norm and two days after thyroxin daily administration in a dose of 0.25 mg per 100 g of body mass. The value of the studied parameters decreases with the age, most considerably--from one to three months and then--from 3 to 24-month age. This evidence for the fact that the role of glycerophosphate shuttle in the energy supply of cells falls with ageing; thyroxin administration evoked almost a two-fold increase of the mentioned parameters in one-month rats and more than three-fold in 12 and 24-month animals. A decrease in the alpha-glycerophosphate dehydrogenase activity with ageing is supposed to be connected with the thyroid gland function.     

The oxidation of exogenous NADH by mitochondria of Euglena gracilis

A novel oxidase activity of external NADH was found in mitochondria of a streptomycin-bleached mutant and the wild strain of Euglena gracilis. In contrast to higher plants the oxidation of external NADH in mitochondria of E. gracilis is sensitive to rotenone and yields the same phosphorylation efficiency as the matrix pool of NADH. Simulation of this activity by the classic complex I of the matrix side of the mitochondrial membrane, as a result of preparation-generated artefacts, is excluded. The external NADH-dehydrogenase activity is bound to the inner mitochondrial membrane with its active side facing the cytosol. State-4 enzyme activity is only slightly influenced by pH in the physiological range, whereas state-3 oxidation indicates an optimum in the physiological pH, as expected from a limitation by the ATPase. The external redox potential of NADH does not control enzyme activity. The results are discussed with respect to the metabolic status of the cells at the time of harvesting.     

Caffeine increases exogenous carbohydrate oxidation during exercise.

Both carbohydrate (CHO) and caffeine have been used as ergogenic aids during exercise. It has been suggested that caffeine increases intestinal glucose absorption, but there are also suggestions that it may decrease muscle glucose uptake. The purpose of the study was to investigate the effect of caffeine on exogenous CHO oxidation. In a randomized crossover design, eight male cyclists (age 27 +/- 2 yr, body mass 71.2 +/- 2.3 kg, maximal oxygen uptake 65.7 +/- 2.2 ml x kg(-1) x min(-1)) exercised at 64 +/- 3% of maximal oxygen uptake for 120 min on three occasions. During exercise subjects ingested either a 5.8% glucose solution (Glu; 48 g/h), glucose with caffeine (Glu+Caf, 48 g/h + 5 mg x kg(-1) x h(-1)), or plain water (Wat). The glucose solution contained trace amounts of [U-13C]glucose so that exogenous CHO oxidation could be calculated. CHO and fat oxidation were measured by indirect calorimetry, and 13C appearance in the expired gases was measured by continuous-flow IRMS. Average exogenous CHO oxidation over the 90- to 120-min period was 26% higher (P < 0.05) in Glu+Caf (0.72 +/- 0.04 g/min) compared with Glu (0.57 +/- 0.04 g/min). Total CHO oxidation rates were higher (P < 0.05) in the CHO ingestion trials compared with Wat, but they were highest when Glu+Caf was ingested (1.21 +/- 0.37, 1.84 +/- 0.14, and 2.47 +/- 0.23 g/min for Wat, Glu, and Glu+Caf, respectively; P < 0.05). There was also a trend (P = 0.082) toward an increased endogenous CHO oxidation with Glu+Caf (1.81 +/- 0.22 g/min vs. 1.27 +/- 0.13 g/min for Glu and 1.12 +/- 0.37 g/min for Wat). In conclusion, compared with glucose alone, 5 mg x kg(-1) x h(-1) of caffeine coingested with glucose increases exogenous CHO oxidation, possibly as a result of an enhanced intestinal absorption.     

The effect of Ca2+ on the oxidation of exogenous NADH by rat liver mitochondria.

The respiratory rate of rat liver mitochondria in the presence of NADH as exogenous substrate is enhanced by the addition of CaCl₂ (> 50 μM) when inorganic phosphate is present in the medium. The Ca-induced oxidation of NADH is inhibited by rotenone but is not affected by uncoupling agents. EDTA, which does not reverse the swelling of mitochondria which occurs in the presence of Ca²⁺ and phosphate, is able to inhibit reversibly the Ca-stimulated NADH oxidation. A stimulation of the rate of oxidation of NADH by Ca²⁺ is also observed in mitochondria partially swollen in a hypotonic medium.     

Stimulation of NADH oxidation during NADPH dependent microsomal electron transport reactions.

Initiation of NADPH oxidation by rat liver microsomes results in a marked stimulation of the rate of NADH oxidation. The amount of NADH rapidly oxidized depends on the ratio of $\text{NADPH}\text{NADH}$ and the presence of various substrates for the function of cytochrome P-450. The present paper illustrates a simple direct method of quantitatively assessing these changes in NADH oxidation and the influence of the anaesthetic, halothane, or the drug, ethylmorphine, on the pattern of changes in NADH oxidation during microsomal electron transport reactions.     

A specific role for Ca2+ in the oxidation of exogenous NADH by Jerusalem-artichoke (Helianthus tuberosus) mitochondria.

1. The addition of chelators to a suspension of mitochondria in a low-cation medium containing 9-aminoacridine caused a decrease in 9-aminoacridine fluorescence. The chelators removed bivalent cations from the membranes and allowed more 9-aminoacridine to move into the diffuse layer. The relative effect of EGTA and EDTA on the fluorescence suggested that the mitochondria are isolated with about equal amounts of Ca2+ and Mg2+ on the membranes. 2. The removal of the bivalent ions by chelators resulted in the inhibition of NADH oxidation. The inhibition could not be removed by adding sufficient decamethylenebistrimethylammonium ion (DM2+) to screen the fixed charges on the membranes and restore the fluorescence of 9-aminoacridine. This observation suggests that bivalent metal ions have a specific role in the oxidation of NADH. 3. Ca2+ and not Mg2+ reversed the inhibition of NADH oxidation caused by EGTA, whereas both reversed the inhibition caused by EDTA. This suggests that Ca2+ plays a specific role and that Mg2+ reverses the inhibition caused by EDTA by displacing the bound calcium from the chelator. 4. The results are interpreted as showing that Ca2+ plays a specific role in the oxidation of external NADH in addition to its ability to screen electrostatically or bind to the fixed charges associated with the surface of the membrane.     

Calmodulin is not involved in the regulation of exogenous NADH oxidation by plant mitochondria

The oxidation of exogenous NADH by mitochondria from potato ( Solanum tuberosum L., cv. Bintje) tubers, measured with different electron acceptors, oxygen, cytochrome c , duroquinone and ubiquinone 1, was greatly enhanced under high salt conditions compared to low salt conditions, confirming the stimulatory effect of electrostatic screeening of negative membrane charges by cations. In addition to this nonspecific stimulation, the oxidation of exogenous NADH showed a specific dependence on Ca²⁺. Results presented here suggest that calmodulin was not directly involved in the regulation of exogenous NADH oxidation by potato mitochondria: (1) Calmodulin antagonists were found to inhibit electron flow at several sites in a nonspecific manner. (2) Using a phenothiazine-Affi Gel column, it was not possible to demonstrate the presence of calmodulin in Triton X-100 solubilized mitochondria. (3) Fractions eluted from a calmodulin-Sepharose column with EGTA [ethyleneglycolbis (β-aminoethylether)-N, N, N', N'-tetraacetic acid] did not display any activity related to mitochondrial electron transport, suggesting that NADH dehydrogenase had no specific affinity for calmodulin. The possible indirect involvement of calmodulin in the regulation of exogenous NADH oxidation by Ca²⁺ is discussed.     

NADH dehydrogenase and NADH oxidation inBacillus megaterium

Only one type (membrane-bound form) of NADH dehydrogenase could be detected in the log-phase cells ofBacillus megaterium. By sonification this enzyme could be effectively solubilized, while NADH oxidase remained bound to the membrane. A molecular weight of about 40 Kd was estimated for the dehydrogenase by gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) with an activity stain. Mercuric chloride and 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) were inhibitors for both the NADH dehydrogenase and oxidase inB. megaterium. The inhibition studies of NADH oxidation suggested that NADH dehydrogenase provided the primary electron source for NADH oxidase in this organismin vitro. NADH dehydrogenase was highly specific for NADH, and K_m was estimated to be 28.2 M. The enzyme was subjected to end-product inhibition of a competitive type.     

A novel phenomenon of burst of oxygen uptake during decavanadate-dependent oxidation of NADH

Oxidation of NADH by decavanadate, a polymeric form vanadate with a cage-like structure, in presence of rat liver microsomes followed a biphasic pattern. An initial slow phase involved a small rate of oxygen uptake and reduction of 3 of the 10 vanadium atoms. This was followed by a second rapid phase in which the rates of NADH oxidation and oxygen uptake increased several-fold with a stoichiometry of NADH: O₂ of 11. The burst of NADH oxidation and oxygen uptake which occurs in phosphate, but not in Tris buffer, was prevented by SOD, catalase, histidine, EDTA, MnCl₂ and CuSO₄, but not by the hydroxyl radical quenchers, ethanol, methanol, formate and mannitol. The burst reaction is of a novel type that requires the polymeric structure of decavanadate for reduction of vanadium which, in presence of traces of H₂O₂, provides a reactive intermediate that promotes transfer of electrons from NADH to oxygen.     

Characterization of oxygen free radicals generated during vanadate-stimulated NADH oxidation

The oxidation of NADH and accompanying reduction of oxygen to H₂O₂ stimulated by polyvanadate was markedly inhibited by SOD and cytochrome c. The presence of decavanadate, the polymeric form, is necessary for obtaining the microsomal enzyme-catalyzed activity. The accompanying activity of reduction of cytochrome c was found to be SOD-insensitive and therefore does not represent superoxide formation. The reduction of cytochrome c by vanadyl sulfate was also SOD-insensitive. In the presence of H₂O₂ all the forms of vanadate were able to oxidize reduced cytochrome c, which was sensitive to mannitol, tris and also catalase, indicating H₂0₂-dependent generation of hydroxyl radicals. Using ESR and spin trapping technique only hydroxyl radicals, but not superoxide anion radicals, were detected during polyvanadate-dependent NADH oxidation.     

ATP synthesis by myosin during pH-jump

It has been shown that a fast increase in pH of myosin solution leads to ATP formation de novo from ADP and Pi (about one ATP molecule per one myosin molecule). The obligatory condition for ATP synthesis was that the value of pH 7.9-8.0 should be crossed in the course of pH-jump. The data obtained are explained in terms of the concept of conformational relaxation in enzyme catalysis.     

The interaction of citrate and calcium in regulating the oxidation of exogenous NADH in plant mitochondria

Plant mitochondria oxidize exogenous NADH via an external NADH dehydrogenase; little is known about the regulation of this dehydrogenase. Data presented here show that citrate caused a non-competitive inhibition of NADH oxidation. This inhibition can be reversed by Ca²⁺. It is postulated that citrate causes the inhibition by extracting Ca²⁺ from the NADH-ubiquinone reductase. Citrate caused a more powerful effect if added before rather than after the NADH. This suggests that the Ca²⁺ is less easily removed in the presence of NADH due to a conformational change in the membrane. Experimentals using a series of electron acceptors suggest that citrate prevents electron transfer between the flavoprotein and the ubiquinone. It is suggested that calcium facilitates electron flux between these components by binding the flavoprotein to the membrane.     

Protein synthesis in transformed 3T3 cells permeabilized by exogenous ATP

Exogenous ATP has been shown to cause a rapid and reversible increase in permeability in transformed 3T3 cells (3T6 and SV3T3) but not in untransformed 3T3 cells. The cells remain viable, but lose intracellular acid-soluble pools. Treatment of transformed cells with ATP greatly reduces incorporation of ¹⁴C-leucine into protein, which is restored by the incubation of the cells with Dulbecco's modified Eagle's medium or by the external additions of certain ions and energy sources. tRNA is not required for the restoration of protein synthesis. In the permeabilized cells the energy for protein synthesis can be provided by glycolysis, oxidative phosphorylation, or direct addition of ATP. These studies demonstrate the usefulness of this method for studying the control of metabolism and macromolecular synthesis in monolayer cultures of transformed mammalian cells.