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  • 1
    ISSN: 1573-6881
    Keywords: Mitochondria ; oxidative phosphorylation ; steady-state kinetics ; rate-limiting step ; coupling relationship ; succinoxidase ; specific inhibition
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract The linear sequence of steps involved in the oxidation of extramitochondrial succinate by O2 in bovine heart mitochondria was examined by a steady-state kinetic method to determine whether or not freely diffusible intermediates occur between the various inhibitor-sensitive steps. The kinetic method is based on the facts (1) that if two inhibitor-sensitive steps within a sequence are linked by a freely diffusible intermediate, inhibition of one will make the other less rate limiting in the overall reaction and thus will increase the amount of inhibitor of the other step required for half-maximal inhibition of the overall reaction, and (2) that if the two steps are not linked in this manner, inhibition of one will make the other more rate limiting and thus will decrease the amount of inhibitor of the other required for half-maximal inhibition. These two types of “coupling relationships” between steps were designated as “sequential” and “fixed,” respectively. The results indicate the existence of freely diffusible intermediates (sequential coupling relationships) between the succinate transport and succinate dehydrogenase reactions, between the succinate dehydrogenase and cytochromebc 1 reactions, and between the cytochromesbc 1 andaa 3 reactions. Uncoupling respiration from phosphorylation results in the coupling relationship between thebc 1 andaa 3 reactions becoming partially fixed. This change is accompanied by marked decreases in the degrees to which thebc 1 andaa 3 reactions limit the overall reaction and appears to account for the large uncoupler-induced releases of inhibition at the levels of thebc 1 andaa 3 reactions observed previously by others. It is suggested that cytochromec is the freely diffusible intermediate between thebc 1 andaa 3 reactions and that the uncoupler-induced changes occur as a result of formation of functional and highly efficient supercomplexes between cytochromec and the cytochromesbc 1 andaa 3 complexes.
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  • 2
    ISSN: 1573-6881
    Keywords: (Na + K)-ATPase ; pyridoxal phosphate ; chemical modification ; conformational changes ; enzyme kinetics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract Reaction of a dog kidney (Na + K)-ATPase with pyridoxal phosphate, followed by borohydride reduction, reduced the catalytic activity when measured subsequently. The time course of inactivation did not follow a first-order process, and certain characteristics of the residual enzymatic activity were modified. Moreover, various catalytic activities were diminished differently: Na-ATPase activity was largely spared, K-phosphatase activity was diminished only by half that of the (Na + K)-ATPase, whereas (Na + K)-CTPase and Na-CTPase activities were diminished more. ATP, ADP, CTP, nitrophenyl phosphate, and Pi all protected against inactivation. Increasing salt concentrations increased inactivation, but KCl slowed and NaCl hastened inactivation when compared with choline chloride. Occupancy of certain substrate or cation sites seemed more crucial than selection of conformational states. For the residual (Na + K)-ATPase activity theK 0.5 for K+ was lower and theK 0.5 for Na+ higher, while the sensitivities to ouabain, oligomycin, and dimethylsulfoxide were diminished; for the residual K-phosphatase activity theK 0.5 for K+ was unchanged, the sensitivity to ouabain and oligomycin diminished, but the stimulation by dimethylsulfoxide increased. These properties cannot be wholly accommodated by assuming merely shifts toward either of the two major enzyme conformations.
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  • 3
    ISSN: 1573-6881
    Keywords: H+-ATPase ; proton conduction ; amino acid modification
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract In this paper a detailed study of the effect of nitration of tyrosine residues by tetranitromethane on H+ conduction and other reactions catalyzed by the H+-ATPase complex in phosphorylating submitochondrial particles, uncoupled particles, and the purified complex is presented. Tetranitromethane treatment of submitochondrial particles results in marked inhibition of ATP hydrolysis, ATP-33Pi exchange, and proton conduction by the H+-ATPase complex. These effects are caused by nitration of tyrosine residues of H+-ATPase complex as shown by the appearance of the absorption peak at 360 nm (specific for nitrotyrosine formation) and inhibition of ATP hydrolysis and ATP-33Pi exchange in the complex purified from tetranitromethane-treated particles. H+ conduction in phospholipid vesicles inlaid with F0 is also inhibited by tetranitromethane treatment. These observations indicate that tyrosine residue(s) of F0 are critically involved in energy-linked proton translocation in the ATP-ase complex.
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    ISSN: 1573-6881
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    ISSN: 1573-6881
    Keywords: crystal ; membrane proteins ; diffraction ; negative staining ; freeze fracture ; phospholipids ; ligand
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract After many years of discouraging failures, it is now possible to crystallize the intrinsic membrane proteins and to obtain structural information from diffraction studies on the crystals. The strategy for the crystallization consists of depletion of boundary phospholipids from the protein and complex formation with specific ligands.
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  • 6
    ISSN: 1573-6881
    Keywords: Ca2+-ATPase ; affinity-purification ; antibodies ; sidedness ; erythrocyte ; membrane ; nitrophenylphosphatase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract Antibodies raised in rabbits against the purified erythrocyte membrane Ca2+ pumping ATPase were affinity-purified using an ATPase-Sepharose column. Addition of a few molecules of the purified antibody per molecule of ATPase was sufficient to inhibit the ATPase activity. Extensively washed ghosts or preincubated pure ATPase sometimes develop an appreciable Mg2+-ATPase activity. In such cases, the antibodies inhibited the Mg2+-ATPase as well as the Ca2+-ATPase. This is consistent with the hypothesis that a portion of the Mg2+-ATPase activity of ghosts is derived from the Ca2+-ATPase. When nitrophenylphosphatase activity was observed, both Mg2+ - and Ca2+-stimulated activities were observed. Only the Ca2+ activity was inhibited by the antibodies, confirming that this activity is due to the Ca2+ pump, and suggesting that the Mg2+-nitrophenylphosphatase is due to a separate enzyme. Amounts of antibody comparable to those which inhibited the Ca2+-ATPases had no effect on the Na+-K+-ATPase; 4-fold higher amounts of antibody significantly stimulated the Na+-K+-ATPase, but this effect of the antibody was not specific: Immunoglobulins from the nonimmune serum also significantly stimulated the Na+-K+-ATPase. In resealed erythrocyte membranes, antibodies incorporated into the ghosts inactivated the Ca2+-ATPase, while antibodies added to the outside had no significant effect.
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  • 7
    ISSN: 1573-6881
    Keywords: Regulation of state 3 oxidation by cytochromec ; decrease in H2O2 generation ; α-glycerophosphate dehydrogenase ; cytochromec in mitochondria ; stress heat ; propylthiouracil treatment ; starvation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract Exposure of rats to higher environmental temperature (36–37°C) decreased the capacity of their kidney mitochondria to oxidize succinate. The decrease was corrected on the addition of exogenous cytochromec. Kidney mitochondria of heat-exposed animals showed decreased rates of H2O2 generation when α-glycerophosphate, but not succinate, was used as electron donor. These mitochondria also showed decreased activity of α-glycerophosphate dehydrogenase but not of succinate dehydrogenase. The content of cytochromec in kidney mitochondria of heat-exposed animals was low even though the concentration of the pigment in the whole tissue did not decrease. Starvation as well as administration of an antithyroid agent like propylthiouracil simulated some of the effects of heat exposure on kidney mitochondria, but the cytochromec-dependent reversal of inhibition of oxidation was obtained only in heat exposure.
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  • 8
    ISSN: 1573-6881
    Keywords: H+-ATPase ; beef heart mitochondria ; sulfhydryl groups ; maleimides ; Pi-ATP exchange ; proton translocation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract Electron transport particles and purified H+-ATPase (F1-Fo) vesicles from beef heart mitochondria have been treated with two classes of thiol reagent, viz. membrane-impermeable organomercurials and a homologous series ofN-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. The organomercurials inhibited ATP-Pi exchange and one of them (p-chloromercuribenzoate) inhibited ATPase activity. Of the maleimide series (AMx), AM10 and AM11 inhibited both ATP-Pi exchange and ATP-driven membrane potential, but not ATPase activity. The other members of the series were essentially inactive.N-Ethylmaleimide was intermediate in its efficacy. Passive H+ conductance through the membrane sector Fo was 50% blocked by AM10, slightly blocked by AM2 andN-ethylmaleimide, and unaffected by the other members of the AMx series. The data imply that one -SH near the membrane surface and one -SH about 12 Å from the surface are functional in proton translocation through the H+-ATPase.
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  • 9
    ISSN: 1573-6881
    Keywords: Transmembranous NADH-dehydrogenase ; enzyme kinetics ; human erythrocyte ; plasma membrane
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract Evidence is presented for a transmembranous NADH-dehydrogenase in human erythrocyte plasma membrane. We suggest that this enzyme is responsible for the ferricyanide reduction by intact cells. This NADH-dehydrogenase is distinctly different from the NADH-cytochromeb 5 reductase on the cytoplasmic side of the membrane. Pretreatment of erythrocytes with the nonpenetrating inhibitor diazobenzene sulfonate (DABS) results in a 35% loss of NADH-ferricyanide reductase activity in the isolated plasma membrane. Since NADH and ferricyanide are both impermeable, the transmembrane enzyme can only be assayed in open membrane sheets with both surfaces exposed, and not in closed vesicles. The transmembrane dehydrogenase has affinity constants of 90 µM for NADH and 125 µM for ferricyanide. It is inhibited byp-chloromercuribenzoate, bathophenanthroline sulfonate, and chlorpromazine.
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  • 10
    ISSN: 1573-6881
    Keywords: H+-ATPase complex ; assembly defect ; Saccharomyces cerevisiae ; mitochondrial biogenesis ; membrane association
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract We have investigated the extent to which the assembly of the cytoplasmically synthesized subunits of the H+-ATPase can proceed in a mtDNA-less (rho°) strain of yeast, which is not capable of mitochondrial protein synthesis. Three of the membrane sector proteins of the yeast H+-ATPase are synthesized in the mitochondria, and it is important to determine whether the presence of these subunits is essential for the assembly of the imported subunits to the inner mitochondrial membrane. A monoclonal antibody against the cytoplasmically synthesized β-subunit of the H+-ATPase was used to immunoprecipitate the assembled subunits of the enzyme complex. Our results indicate that the imported subunits of the H+-ATPase can be assembled in this mutant, into a defective complex which could be shown to be associated with the mitochondrial membrane by the analysis of the Arrhenius kinetics of the mutant mitochondrial ATPase activity.
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