scholarly journals Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria

1980 ◽  
Vol 191 (2) ◽  
pp. 421-427 ◽  
Author(s):  
J F Turrens ◽  
A Boveris
Keyword(s):  
Cytochrome B ◽  
Nadh Dehydrogenase ◽  
Submitochondrial Particles ◽  
Bovine Heart ◽  
Biphasic Effect ◽  
Carbonyl Cyanide ◽  
Autocatalytic Process ◽  
Ph Range ◽  
Energy Dependent ◽  
O2 Production

Submitochondrial particles from bovine heart in which NADH dehydrogenase is reduced by either addition of NADH and rotenone or by reversed electron transfer generate 0.9 +/- 0.1 nmol of O2-/min per mg of protein at pH 7.4 and at 30 degrees C. When NADH is used as substrate, rotenone, antimycin and cyanide increase O2- production. In NADH- and antimycin-supplemented submitochondrial particles, rotenone has a biphasic effect: it increases O2- production at the NADH dehydrogenase and it inhibits O2- production at the ubiquinone-cytochrome b site. The generation of O2- by the rotenone, the uncoupler carbonyl cyanide rho-trifluoromethoxyphenylhydrazone and oligomycin at concentrations similar to those required to inhibit energy-dependent succinate-NAD reductase. Cyanide did not affect O2- generation at the NADH dehydrogenase, but inhibited O2- production at the ubiquinone-cytochrome b site. Production of O2- at the NADH dehydrogenase is about 50% of the O2- generation but the ubiquinone-cytochrome b area at pH 7.4. Additivity of the two mitochondrial sites of O2- generation was observed over the pH range from 7.0 to 8.8. AN O2–dependent autocatalytic process that requires NADH, submitochondrial particles and adrenaline is described.

scholarly journals The activation of non-phosphorylating electron transport by adenine nucleotides in Jerusalem-artichoke (Helianthus tuberosus) mitochondria

1975 ◽  
Vol 152 (3) ◽  
pp. 637-645 ◽  
Author(s):  
R Sotthibandhu ◽  
J M Palmer
Keyword(s):  
Cytochrome B ◽  
Redox State ◽  
Nadh Dehydrogenase ◽  
Adenine Nucleotides ◽  
Plant Mitochondria ◽  
Jerusalem Artichoke ◽  
Simultaneous Oxidation ◽  
Carbonyl Cyanide ◽  
Bongkrekic Acid ◽  
Complex Manner

In isolated plant mitochondria the oxidation of both succinate and exogenous NADH responded in the expected manner to the addition of ADP or uncoupling agents, and the uncoupled rate of respiration was often in excess of the rate obtained in the presence of ADP. However, the oxidation of NAD+-linked substrates responded in a much more complex manner to the addition of ADP or uncoupling agents such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone to mitochondria oxidizing pyruvate plus malate failed to result in a reliable stimulation; this uncoupled rate could be stimulated by adding AMP or ADP in the presence of oligomycin or bongkrekic acid. Spectrophometric measurements showed that the addition of AMP or ADP resulted in the simultaneous oxidation of endogenous nicotinamide nucleotide and the reduction of cytochrome b. ADP was only effective in bringing about these changes in redox state in the presence of Mg2+ whereas AMP did not require Mg2+. It was concluded that AMP activated the flow of electrons from endogenous nicotinamide nucleotide to cytochrome b, possible at the level of the internal NADH dehydrogenase.

scholarly journals NADH- and NADPH-dependent formation of superoxide anions by bovine heart submitochondrial particles and NADH–ubiquinone reductase preparation

1979 ◽  
Vol 180 (1) ◽  
pp. 129-135 ◽  
Author(s):  
Koichiro Takeshige ◽  
Shigeki Minakami
Keyword(s):  
Respiratory Chain ◽  
Complex I ◽  
Inorganic Salts ◽  
Superoxide Anions ◽  
Submitochondrial Particles ◽  
Bovine Heart ◽  
Sensitive Site ◽  
Effects Of Ph ◽  
High Concentrations ◽  
O2 Production

1. Both NADH and NADPH supported the oxidation of adrenaline to adrenochrome in bovine heart submitochondrial particles. The reaction was completely inhibited in the presence of superoxide dismutase, suggesting that superoxide anions (O2−) are responsible for the oxidation. The optimal pH of the reaction with NADPH was at pH7.5, whereas that with NADH was at pH9.0. The reaction was inhibited by treatment of the preparation with p-hydroxymercuribenzoate and stimulated by treatment with rotenone. Antimycin A and cyanide stimulated the reaction to the same extent as rotenone. The NADPH-dependent reaction was inhibited by inorganic salts at high concentrations, whereas the NADH-dependent reaction was stimulated. 2. Production of O2− by NADH–ubiquinone reductase preparation (Complex I) with NADH or NADPH as an electron donor was assayed by measuring the formation of adrenochrome or the reduction of acetylated cytochrome c which does not react with the respiratory-chain components. p-Hydroxymercuribenzoate inhibited the reaction and rotenone stimulated the reaction. The effects of pH and inorganic salts at high concentrations on the NADH- and NADPH-dependent reactions of Complex I were essentially similar to those on the reactions of submitochondrial particles. 3. These findings suggest that a region between a mercurialsensitive site and the rotenone-sensitive site of the respiratory-chain NADH dehydrogenase is largely responsible for the NADH- and NADPH-dependent O2− production by the mitochondrial inner membranes.

scholarly journals The polypeptide composition of the mitochondrial NADH: ubiquinone reductase complex from several mammalian species

1985 ◽  
Vol 230 (3) ◽  
pp. 739-746 ◽  
Author(s):  
M W Cleeter ◽  
C I Ragan
Keyword(s):  
Nadh Dehydrogenase ◽  
Mammalian Species ◽  
Polypeptide Composition ◽  
Submitochondrial Particles ◽  
Protein Subunits ◽  
Bovine Heart ◽  
Nadh:Ubiquinone Reductase ◽  
Monospecific Antisera

The polypeptide composition of isolated mitochondrial NADH:ubiquinone reductase (NADH dehydrogenase) is very similar to that of material immunoprecipitated from detergent-solubilized bovine heart submitochondrial particles by antisera to the holoenzyme. The specificity of the antisera for dehydrogenase polypeptides was determined by immunoblotting, which showed that antisera reacting with only a few proteins were able to immunoprecipitate all others in parallel. The polypeptide compositions of rat, rabbit and human NADH dehydrogenase were determined by immunoprecipitation of the enzyme from solubilized submitochondrial particles and proved to be very similar to that of the bovine heart enzyme, particularly in the high-Mr region. Further homologies in these and other species were explored by immunoblotting with antisera to the holoenzyme and monospecific antisera raised against iron-sulphur-protein subunits of the enzyme.

scholarly journals Characterization of Na+-dependent phosphate uptake in cultured kidney cells (JTC-12) from monkey

1985 ◽  
Vol 230 (3) ◽  
pp. 715-721 ◽  
Author(s):  
Y Takuwa ◽  
E Ogata
Keyword(s):  
Phosphate Uptake ◽  
Phosphate Transport ◽  
Hill Coefficient ◽  
Epithelial Cell Line ◽  
Carbonyl Cyanide ◽  
Dependent Component ◽  
Km Value ◽  
Ph Range ◽  
Energy Dependent

Phosphate uptake was studied in confluent monolayers of an epithelial-cell line (JTC-12) derived from monkey kidney. Phosphate uptake consisted of a saturable, Na+-dependent, component, which accounted for about 80% of the uptake, and a nonsaturable, Na+-independent, component. The saturable component was specifically dependent on the presence of extracellular Na+ and has an apparent Km value for phosphate of 0.12 mM at 137-mM-Na+, which is close to those reported in the brush-border membranes in mammalian kidneys. The presence of Na+ in the uptake solution decreased the Km for phosphate without affecting the Vmax. Phosphate uptake was inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone and ouabain, suggesting that phosphate transport is an active, energy-dependent, process and is dependent on an Na+ gradient across cell membranes. With respect to the effect of external Na+ concentration, a sigmoid relation was seen between the initial velocity of phosphate uptake and Na+ concentrations, and Hill analysis gave a Hill coefficient of 1.8. In the pH range 6.6-7.4, phosphate uptake declined with increasing pH. Phosphate uptake was stimulated when cells were cultured in the presence of insulin, and was also affected by changes in phosphate concentrations in cultured medium. These results indicate that JTC-12 cells have an Na+-dependent phosphate-transport system with many of the features of phosphate transport in the proximal tubule.

scholarly journals A simple and rapid method for the preparation of adenosine triphosphatase from submitochondrial particles

1975 ◽  
Vol 148 (3) ◽  
pp. 533-537 ◽  
Author(s):  
R B Beechey ◽  
S A Hubbard ◽  
P E Linnett ◽  
A D Mitchell ◽  
E A Munn
Keyword(s):  
Electron Microscopy ◽  
Rapid Method ◽  
Adenosine Triphosphatase ◽  
Pure Form ◽  
Heart Mitochondria ◽  
Polyacrylamide Gels ◽  
Submitochondrial Particles ◽  
Bovine Heart

An almost pure form of the bovine heart mitochondrial adenosine triphosphatase (ATPase) is released from the membrane by shaking submitochondrial particles with chloroform. Analyses on polyacrylamide gels and by electron microscopy, and also sensitivity to inhibitors, show that the chloroform-released enzyme is similar to other ATPase preparations from bovine heart mitochondria.

scholarly journals Studies on the electron-transfer mechanism of the human neutrophil NADPH oxidase

1989 ◽  
Vol 262 (2) ◽  
pp. 575-579 ◽  
Author(s):  
J A Ellis ◽  
A R Cross ◽  
O T G Jones
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Nadph Oxidase ◽  
Cytochrome B ◽  
Human Neutrophil ◽  
Sodium Deoxycholate ◽  
Human Neutrophils ◽  
Electron Transfer Mechanism ◽  
State Reduction ◽  
O2 Production

A superoxide-generating NADPH oxidase was solubilized from phorbol 12-myristate 13-acetate-activated human neutrophils with a mixture of sodium deoxycholate (0.125%, w/v) and Lubrol-PX (0.125%, v/v). The solubilized preparation contained FAD (577 pmol/mg of protein) and cytochrome b-245 (479 pmol/mg of protein) and produced 11.61 mol of O2-./s per mol of cytochrome b (340 nmol of O2-./min per mg of protein). On addition of NADPH, the cytochrome b-245 was reduced by 7.9% and the FAD by 38% in the aerobic steady state; NADH addition caused little steady-state reduction of cytochrome b and FAD. In this preparation, and several others, the measured rate of O2-. production correlated with the turnover of cytochrome b calculated from the extent of cytochrome b-245 reduction under aerobic conditions. Addition of diphenyleneiodonium abolished the reduction of both the FAD and cytochrome b-245 components and inhibited O2-. production. The haem ligand imidazole inhibited O2-. generation and cytochrome b reduction while permitting FAD reduction. These results support the suggestion that the human neutrophil NADPH oxidase has the electron-transport sequence: NADPH-FAD-cytochrome b-245-O2.

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