Inhibition of mitochondrial calcium ion transport by an oxo-bridged dinuclear ruthenium ammine complex.

Ruthenium red is a well-known and effective inhibitor of the mitochondrial Ca2+ uniporter; however, Reed and Bygrave [(1974) FEBS Lett. 46, 109-114] tentatively attributed this inhibition to a colorless impurity present in commercial samples of ruthenium red (RR). This component has now been isolated and a derivative, (mu-O) [(HCO2)(NH3)4Ru]2Cl3, structurally characterized.

Human mitochondrial ATP synthase: cloning cDNA for the nuclear-encoded precursor of coupling factor 6.

Coupling factor 6 (F6) is a component of mitochondrial ATP synthase which is required for the interactions of the catalytic and proton-translocating segments. A human fetal muscle cDNA clone encoding this protein was isolated by screening a lambda gt10 library with oligodeoxyribonucleotide probes. The 497-bp F6 cDNA included a 96-bp segment that delineated a presequence of 32 amino acids (aa) in the precursor protein, and 140 bp of 3'-untranslated sequence.

Amino-terminal amino acid sequence of beef heart mitochondrial coupling factor B.

Bovine heart mitochondrial coupling factor B was isolated and purified to homogeneity in its active form. The amino-terminal amino acid sequence of the alkylated protein was determined. Two chains with exactly the same sequence except for the presence of an additional Phe at the amino-terminus on one of them were obtained.

Evidence that coupling factor B is bound to the matrix side of the inner mitochondrial membrane.

Rotenone-sensitive NADH dehydrogenase activity and Lubrol stimulation of cytochrome oxidase activity were measured to assess the opposite membrane polarity of beef heart mitoplast and inside-out particle preparations. The ATP-Pi exchange activity of mitoplasts was not affected by their incubation at pH 8.9 in the presence of 5 mM EDTA (a treatment known to extract coupling factor B (F beta) from submitochondrial particles), nor was it stimulated by the addition of F beta to intact and alkaline treated mitoplast preparations.

Reconstitution of transmembrane K+ transport with a 53 kilodalton mitochondrial protein.

A 53 kDa protein has been purified from a Triton X-100 extract of liver mitochondrial membranes, by affinity chromatography on immobilized quinine, a K+ transport inhibitor. KCl-containing lipid vesicles reconstituted with this protein lose K+ to a medium low in K+ faster than vesicles lacking protein. With bacteriorhodopsin reconstituted in vesicles containing K+, light induces faster development of a pH gradient if the 53 kDa protein is included during vesicle preparation.

Coupling factor B is a component of the Fo proton channel of mitochondrial H+-ATPase.

Repeated extraction of bovine heart submitochondrial particles with ammonia and EDTA (AE) yields a preparation that is highly deficient in coupling factor B (FB). The activity of the thrice extracted particle (AE-P3) in ATP-driven NAD+ reduction by succinate and the 32Pi-ATP exchange activity were substantially stimulated, 8-fold and 5-fold, respectively, by purified FB. To decrease the basal activity of the particle further, the residual FB in AE-P3 was inactivated by treatment with the -SH reagent, 4-vinylpyridine.

Diamide blocks H+ conductance in mitochondrial H+-ATPase by oxidizing FB dithiol.

Effects of diamide on proton conductance of electron transport particles (ETPH), purified H+-ATPase (F1-F0), F0 of the H+-ATPase from beef heart mitochondria and binding of cadmium (109Cd) to the H+-ATPase have been examined in the present paper. When ETPH and purified H+-ATPase are treated with 1 mM diamide, ATP-dependent generation of membrane potential, monitored by the absorbance change produced by the redistribution of oxonol VI, is consistently inhibited. Diamide also blocks passive H+ conductance driven by a K+ diffusion potential in the membrane sector, F0, of H+-ATPase.

Resolution and reconstitution of H+ -ATPase complex from beef heart mitochondria.

Mitochondrial H+ -ATPase complex, purified by the lysolecithin extraction procedure, has been resolved into a "membrane" (NaBr-F0) and a "soluble" fraction by treatment with 3.5 M sodium bromide. The NaBr-F0 fraction is completely devoid of beta, delta, and epsilon subunits of the F, ATPase and largely devoid of alpha and gamma subunits of F1, where F0 is used to denote the membrane fraction and F1, coupling factor 1.

Monoclonal antibodies to mitochondrial coupling factor B.

Two monoclonal antibodies (MAb I and IV) have been prepared which showed high and specific reactions towards bovine heart mitochondrial coupling factor B (FB). Both have been identified as sub-type IgG1 of mouse immunoglobulins. MAb I reacts with purified and functionally active FB, alkylated or oxidized forms of FB and even with peptides formed on digestion of FB with trypsin.

Environment of the sulfhydryl groups in bovine heart mitochondrial H+-ATPase.

Electron transport particles and purified H+-ATPase (F1-F0) vesicles from beef heart mitochondria have been treated with two classes of thiol reagent, viz. membrane-impermeable organomercurials and a homologous series of N-polymethylene carboxymaleimides (Mal-(CH2)x-COOH or AMx). The effect of such treatment on ATP-driven reactions (ATP-Pi exchange and proton translocation) has been examined and compared to the effects on rates of ATP hydrolysis.

Activation of potassium ion transport in mitochondria by cadmium ion.

Low levels of Cd2+ (1-5 microM) produce rapid swelling of mitochondria, which is respiration-dependent and uncoupler-sensitive. No cation requirement is apparent, since the swelling occurs in a medium containing only sucrose and the respiratory substrate. The swelling is inhibited by ruthenium red, suggesting that this effect of Cd2+ requires its entry into mitochondria.

Immunologically reactive multiple species of mitochondrial coupling factor B.

Earlier work had indicated that mitochondrial coupling factor B (FB) could be obtained with differing molecular weights, a highly active 13,000 form, a 29,200 form with low activity, and a partially purified 46,000 form with activity higher than the 29,200 form. We have analyzed FB preparations of different purity and after different types of treatment on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), followed by silver staining or immunostaining either with rabbit anti-FB serum or monoclonal FB antibody. Highly purified preparations which appear as single bands in SDS-PAGE develop additional higher molecular weight bands (silver staining), including a 48,000 and a 68,000 band, after lyophilization or repeated freezing and thawing or if subjected to SDS-PAGE in the absence of thiol compounds.

On the functional role of coupling factor B in the mitochondrial H+ -ATPase.

Evidence for the presence of a functionally important vicinal dithiol in mitochondrial coupling factor B (FB) has been presented earlier (Sanadi, D. R. (1982) Biochim.

Inhibition by dicyclohexylcarbodiimide of ATP synthesis in isolated rat hepatocytes.

Dicyclohexylcarbodiimide (DCCD) inhibits, by 50%, ATP synthesis in isolated hepatocytes. This inhibition is associated with DCCD-binding to a proteolipid fraction present in submitochondrial particles.

Evidence for the involvement of coupling factor B in the H+ channel of the mitochondrial H+-ATPase.

Membrane energization by ATP has been measured in vesicles containing purified bovine heart mitochondrial H+-ATPase (ATP synthase) with the voltage-sensitive dye oxonol VI. The dithiol chelator, Cd2+, and the thiol oxidant, copper o-phenanthroline, produced discharge of the membrane potential when added at the steady state and inhibited its establishment when added prior to energization by ATP. These effects, which were reversed by dithiothreitol, were not accompanied by an increase in the nonspecific H+ permeability of the membrane.

Effects of Cd2+ on ATP-driven membrane potential in beef heart mitochondrial H+-ATPase: a study using the voltage-sensitive probe oxonol VI.

Beef heart mitochondrial H+-ATPase (F1-F0) vesicles were prepared by lysolecithin extraction of ETPH. ATP-driven membrane potential was monitored indirectly by following absorbance changes of the potential-sensitive dye oxonol VI. The steady-state potential was discharged by oligomycin and/or Cd2+ (a dithiol reagent).

Isolation of a highly active H+-ATPase from beef heart mitochondria.

The lysolecithin extraction procedure originally described by Sadler et al. (1974) has been modified to yield a H+-ATPase with high levels of Pi-ATP exchange activity (400-600 nmol x min-1 x mg-1). This activity is further enhanced (1400-1600 nmol x min-1 x mg-1) following sucrose density gradient centrifugation in the presence of asolectin.

On the role of factor B and oligomycin on generation and discharge of the proton gradient.

Bovine mitochondrial coupling factor B (FB) stimulates oxidative phosphorylation as well as other energy-linked reactions which are supported by ATP hydrolysis. Extraction of FB from submitochondrial particles results in a decrease in ATP-dependent proton translocation (delta pH) and binding of the voltage-sensitive dye, oxonol VI. Reconstitution of deficient particles with FB restores ATP-dependent proton translocation and oxonol binding but has little effect on oxonol binding supported by respiratory substrates.

Activation of potassium-dependent H+ efflux from mitochondria by cadmium and phenylarsine oxide.

Addition of Cd2+ or phenylarsine oxide (PhAsO) to respiring rat liver mitochondria results first in acidification of the medium (H+ efflux) followed by disappearance of H+ (discharge of the pH gradient or uncoupling). The first phase of H+ efflux is dependent upon the presence of K+ in the medium, and is not seen in the presence of valinomycin, which is consistent with the conclusion that H+ efflux is linked to membrane potential-dependent uptake of K+. These effects are abolished by low levels of 2,3-dimercaptopropanol but potentiated by excess of 2-mercaptoethanol, showing involvement of a dithiol type of group in the response.

Occurrence of proteins immunoreactive with anti-coupling factor B in phosphorylating membrane preparations.

Coupling factor B has been isolated from beef heart mitochondria, apparently in multiple forms which differ in molecular weight and specific activity. Since it has no known intrinsic catalytic activity, detection and quantitation have been based upon the factor B-dependent stimulation of ATP-linked activities in factor B-deficient sub-mitochondrial particles. This communication reports the development of a reliable and more universally applicable enzyme-linked immunosorbent assay (ELISA) for detection and quantitation of factor B in soluble or membranous preparations.

A simple method for maintaining large, aging populations of Caenorhabditis elegans.

This paper describes a technique capable of establishing and maintaining large, age-synchronous populations of the nematode Caenorhabditis elegans. The technique has three essential components: a rich chemical medium; a method for producing and harvesting mass quantities of eggs; and 5-fluorodeoxyuridine (FUdR), an inhibitor of DNA synthesis. A culture of worms is filtered through glass wool or a wire screen to isolate young larvae.