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D R Sanadi

Publications and source records attributed to D R Sanadi.

At least 19 recordsLinked to original sources
PubMed1991

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. The active species in solution appears to be the symmetrical oxo-bridged ion, [X(NH3)4Ru-O-Ru(NH3)4X]3+, where X = Cl- or OH-. Its absorption spectrum shows a maximum at 360 nm. The dinuclear ruthenium ammine complex inhibits Ca(2+)-stimulated respiration of rat liver mitochondria with an I50 of 3.5 pmol/mg of protein compared to the value of 60 pmol of RR/mg of protein. The inhibition by the dinuclear compound is noncompetitive with Ca2+. Respiration-linked swelling of mitochondria induced by Cd2+ also responds similarly to both the dinuclear complex and RR. A close correlation was observed between binding to mitochondria as monitored with 103Ru-labeled dinuclear complex and inhibition of Ca2+ transport. A Scatchard plot yielded estimates of maximum specific binding and dissociation constant of 7.5 pmol/mg of protein and 1.3 nM, respectively. The inhibitor has the characteristics of a satisfactory affinity ligand for purification of the uniporter.

Animals↗
PubMed1991

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. The remainder of the cDNA sequence coded for a mature human F6 protein of 76 aa. The deduced primary aa sequence showed 81% homology to that of bovine F6, differing in 14 aa. Almost all of these aa substitutions were conservative and comparison of the hydropathy profiles revealed a similar pattern.

Adenosine Triphosphatases↗
PubMed1990

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. The 55 amino acid sequence appears to be largely hydrophilic with several charged amino acid residues. This sequence showed no homology with the E. coli unc operon, oligomycin sensitivity conferring protein, or coupling factor 6 or any protein in the data base.

Adenosine Triphosphatases↗
PubMed1989

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. In contrast, the exchange activity of inside-out particles was decreased 18 fold by the alkaline/EDTA treatment and was almost completely restored by the addition of F beta to F beta-depleted particles. From these results it is concluded that in beef heart mitochondria, the coupling factor F beta is bound to the matrix-side of the inner mitochondrial membrane.

Adenosine Triphosphatases↗
PubMed1988

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. This effect is like that of valinomycin, which catalyzes K+ efflux, dissipating the membrane potential arising from H+ entry. Evidence that vesicles containing the 53 kDa protein are permeable to K+, but exhibit low permeability to H+, indicates that this protein acts as a K+ uniporter.

Animals↗
PubMed1987

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. The resulting particle was depleted of F1 by treatment with 3.5 M NaBr. This particle was incorporated into asolectin liposomes alone and in the presence of added FB. Passive proton conduction in the FB-deficient proteoliposomes was negligible and restored in the presence of FB. The H+ conductance was inhibited extensively by oligomycin and partially by F1-ATPase. The results show absolute dependence on FB for functioning of the FO proton channel.

Adenosine Triphosphatases↗
PubMed1985

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. Furthermore, diamide treatment drastically reduces the binding of 109Cd2+ to H+-ATPase, showing competition for the FB dithiol group.

Adenosine Triphosphatases↗
PubMed1985

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. When used together, MAb I and IV reacted with FB in immunoblots of normal and urea treated samples of mitochondria, submitochondrial particles, ammonia-EDTA extracted particles, and H+-ATPase. Both MAbs inhibited FB-stimulated ATP-dependent reverse electron flow activity when FB was incubated with the antibody either before or after its addition to FB-deficient AE-particles. Reactivity of MAb I towards FB declined upon exposure of FB to guanidine HC1 while reactivity of MAb IV remained unaltered.

Animals↗
PubMed1985

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. This is confirmed by complete loss of ATPase and Pi-ATP exchange activities. The addition of F1 (400 micrograms X mg-1 F0) results in complete restoration of oligomycin sensitivity without any reduction in the F1-ATPase activity. Presumably, this is due to release of ATPase inhibitor protein from the F1-F0 complex consequent to sodium bromide extraction. Restoration of Pi-ATP exchange and H+ -pumping activities require coupling factor B in addition to F1-ATPase. The oligomycin-sensitive ATPase and 32Pi-ATP exchange activities in reconstituted F1-F0 have the same sensitivity to uncouplers and energy transfer inhibitors as in starting submitochondrial particles from the heavy layer of mitochondria and F1-F0 complex. The data suggest that the altered properties of NaBr-F0 observed in other laboratories are probably inherent to their F1-F0 preparations rather than to sodium bromide treatment itself. The H+ -ATPase (F1-F0) complex of all known prokaryotic (3, 8, 9, 10, 21, 32, 34) and eukaryotic (11, 26, 30, 33, 35-37) phosphorylating membranes contain two functionally and structurally distinct entities. The hydrophilic component F1, composed of five unlike subunits, shows ATPase activity that is cold labile as well as uncoupler- and oligomycin-insensitive. The membrane-bound hydrophobic component F0, having no energy-linked catalytic activity of its own, is indirectly assayed by its ability to regain oligomycin sensitive ATPase and Pi-ATP exchange activities on binding to F1-ATPase (33). The purest preparations of bovine heart mitochondrial F0 show seven or eight major components in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate or SDS-PAGE (1, 2, 12, 14), ranging from 6 to 54 ku in molecular weight (12). The precise structure and polypeptide composition of mitochondrial F0 is not known. The F0 preparations from bovine heart reported so far have been derived from H+ -ATPase preparations isolated in the presence of cholate and deoxycholate (11, 33, 36, 37).(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1979

Synchronous growth and aging of Caenorhabditis elegans in the presence of fluorodeoxyuridine.

Fluorodeoxyuridine (FUdR), an inhibitor of DNA synthesis, was examined for its ability to prevent a synchronous population of C. elegans from reproducing without otherwise interfering with the organism's post-maturational development and aging. When a synchronized population was exposed to 400 micrometer FUdR just as the population reached sexual maturity, the FUdR induced complete sterility within five hours by preventing eggs from hatching. Any larvae that hatched from eggs made before the FUdR was added remained small in the presence of FUdR and were easily removed by filtration or sedimentation. FUdR-sterilized adults showed no morphological abnormalities. Age-associated changes seen in controls also occurred in FUdR-treated worms, including atrophy of the gonads, increased pigmentation, sluggishness and increased transparency. Life span was not shortened by FUdR treatment. Our observations suggest that treatment with FUdR under carefully controlled conditions is a reasonable way to maintain synchronously aging populations of C. elegans.

Aging↗
PubMed1977

Photoaffinity labeling of uncoupler binding sites on mitochondrial membrane.

3H 2-azido-4-nitrophenol, a photoactive uncoupler, has been synthesized, and its uncoupling action on oxidative phosphorylation and its binding to the mitochondrial membrane have been studied. The uncoupler bound covalently to the mitochondrial membrane on photoirradiation was 3-4 times that bound reversibly in the absence of light. When irradiation was carried out in the presence of serum albumin, covalent binding was significantly depressed. The pattern of loss of ATP-Pi exchange activity with increasing amounts of the uncoupler suggests that serum albumin prevents the binding of the uncoupler to the functional sites as well. Polyacrylamide gel electrophoresis of photoaffinity labeled submitochondrial particles in the presence of sodium dodecyl sulfate revealed that a 9000 dalton peptide bound high levels of uncoupler. Other proteins in the molecular weight range of 20,000-40,000 and 55,000 were also labeled. Photolysis in the presence of serum albumin or ATP decreased the covalent binding of the uncoupler to all the proteins, but particularly to the 20,000 dalton component. Soluble ATPase and the mitochondrial proteolipid purified from labeled mitochondria showed the presence of label.

Adenosine Triphosphate↗
PubMed1977

Fatty acid oxidation by isolated perfused working hearts of aged rats.

It has been reported that mitochondria isolated from hearts of old rats have lower respiratory activity than mitochondria from young rats. In order to determine the physiological correlates of these changes, the metabolism of hearts from young and old rats has been compared in a perfused working heart preparation. The oxidation of [14C]palmitate to 14CO2, oxygen consumption, and nucleotide levels were measured under different cardiac workloads. The hearts from old animals performed less cardiac work and utilized less oxygen and palmitate in proportion to tissue mass, but the ratio of oxygen consumed to pressure developed was unaltered. There was a small but significant decrease in cardiac efficiency expressed as the ratio between the rate of oxygen consumed and ventricular pressure development. Tissue levels of total carnitine and long-chain acylcarnitine derivatives were greatly reduced in the older heart without significant change in free CoA, acetyl-CoA, or long-chain acyl-CoA. The adenine nucleotide levels were not significantly different in the two groups. The results appear consistent with the in vitro studies on isolated mitochondria.

Acetyl Coenzyme A↗