Activated bleomycin appears to have two more oxidizing equivalents than the Fe(III).bleomycin to which it spontaneously decays. Activated bleomycin reacts with NADH and thio-NADH, two-electron reductants, and with KI, a one-electron reductant, to yield Fe(III).bleomycin. The observed stoichiometries were 0.85 +/- 0.07 eq of thio-NADH oxidized or 1.5 +/- 0.25 eq of KI oxidized per mole of activated bleomycin. None of these reactions requires the presence of a redox mediator, as does the reduction of Fe(III).bleomycin by NADH or thio-NADH. The oxidations of both pyridine nucleotide coenzymes and of KI are inhibited by DNA, the usual bleomycin target. (+info)
Streptomycin accumulation by Bacillus subtilis requires both a membrane potential and cytochrome aa3.
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Cytochrome aa3 concentrations in the cytoplasmic membrane of Bacillus subtilis were altered by growth conditions, and the effects on the membrane potential (delta psi) in whole cells were measured. When cytochrome aa3 was absent, the magnitude of delta psi was not diminished by comparison with the delta psi measured in cells containing normal cytochrome aa3 concentrations. In addition, the energy-dependent uptake of proline and glutamate was comparable at both cytochrome aa3 concentrations. However, in the cytochrome aa3-deficient cell preparation, accumulation of the aminoglycoside antibiotic streptomycin was much lower than that of the cytochrome aa3-sufficient cells. When cells were cultured under conditions that stimulated higher than normal concentrations of cytochrome aa3, delta psi was also increased, and enhanced streptomycin accumulation was observed. Phenazine methosulfate-ascorbate was used both in delta psi measurements and in uptake studies to provide high rates of electron transport and maximal delta psi values. These results, taken together with those previously published (A. S. McEnroe and H. W. Taber, Antimicrob. Agents Chemother. 26:507-512, 1984) suggest that the uptake of streptomycin by B. subtilis requires adequate levels both of delta psi and cytochrome aa3. (+info)
Phosphoribosyl pyrophosphate and phosphoribosyl pyrophosphate synthetase in rat mammary gland. Changes in the lactation cycle and effects of diabetes, insulin and phenazine methosulphate.
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Changes in the tissue content of phosphoribosyl pyrophosphate (PPRibP), glucose 6-phosphate, ribose 5-phosphate (Rib5P), RNA and DNA, of the activity of PPRibP synthetase (EC 2.7.6.1) and the conversion of [1-14C]- and [6-14C]-glucose into 14CO2 were measured at mid-lactation in the normal and diabetic rat and in pregnancy, lactation and mammary involution in the normal rat. The PPRibP, glucose 6-phosphate and Rib5P contents increase during pregnancy and early lactation to reach a plateau value at mid-lactation, before falling sharply during weaning. The PPRibP content, PPRibP synthetase activity and flux of glucose through the oxidative pentose phosphate pathway (PPP) all change in parallel during the lactation cycle. Similarly, after 3 and 5 days duration of streptozotocin-induced diabetes, ending on day 10 of lactation, there were parallel declines in PPRibP content, PPRibP synthetase and PPP activity. The effect of streptozotocin was prevented by pretreatment with nicotinamide and partially reversed by insulin administration. Addition of insulin to lactating rat mammary-gland slices incubated in vitro significantly raised the PPRibP content (+47%) and the activity of the PPP (+40%); phenazine methosulphate, which gives a 2-fold increase in PPP activity, raised the PPRibP content of lactating mammary gland slices by approx. 3-fold. It is concluded that Rib5P, generated in the oxidative segment of the PPP, is an important determinant of PPRibP synthesis in the lactating rat mammary gland and that insulin plays a central role in the regulation of the bioavailability of this precursor of nucleotide and nucleic acid synthesis. (+info)
Chloroplast ATP synthase contains one single copy of subunit delta that is indispensable for photophosphorylation.
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F0F1 ATP synthases synthesize ATP in their F1 portion at the expense of free energy supplied by proton flow which enters the enzyme through their channel portion F0. The smaller subunits of F1, especially subunit delta, may act as energy transducers between these rather distant functional units. We have previously shown that chloroplast delta, when added to thylakoids partially depleted of the coupling factor CF1, can reconstitute photophosphorylation by inhibiting proton leakage through exposed coupling factor CF0. In view of controversies in the literature, we reinvestigated two further aspects related to subunit delta, namely (a) its stoichiometry in CF0CF1 and (b) whether or not delta is required for photophosphorylation. By rocket immunoelectrophoresis of thylakoid membranes and calibration against purified delta, we confirmed a stoichiometry of one delta per CF0CF1. In CF1-depleted thylakoids photophosphorylation could be reconstituted not only by adding CF1 and subunit delta but, surprisingly, also by CF1 (-delta). We found that the latter was attributable to a contamination of CF1 (-delta) preparations with integral CF1. To lesser extent CF1 (-delta) acted by complementary rebinding to CF0 channels that were closed because they contained delta [CF0(+delta)]. This added catalytic capacity to proton-tight thylakoid vesicles. The ability of subunit delta to control proton flow through CF0 and the absolute requirement for delta in restoration of photophosphorylation suggest an essential role of this small subunit at the interface between the large portions of ATP synthase: delta may be part of the coupling site between electrochemical, conformational and chemical events in this enzyme. (+info)
Glucose oxidation in the chick cornea: effect of diamide on the pentose shunt.
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Chick embryo corneas (stages 38 and 45) have been used to study variations in pentose shunt activity following the use of a glutathione-specific oxidizing agent, diamide, and a sulfydryl blocking agent, N-ethylmaleimide (NEM). Shunt activity was measured by the ratio of radiolabeled carbon 1 (14C-1) of glucose to radiolabeled carbon 6 (14C-6) of glucose derived as expired 14CO2. Diamide and NEM were both found to increase pentose shunt activity relative to glycolysis, although by different means. Diamide appeared to exert its effect by oxidizing glutathione and creating a demand for higher shunt activity to facilitate glutathione reduction by NADPH. Both C-1 and C-6 oxidation were increased, but C-1 oxidation was increased to a much greater extent. In contrast, NEM decreased both C-1 and C-6 oxidation, with C-6 preferentially affected. Thus NEM appears to preferentially inhibit the enzymatic machinery of the glycolytic-tricarboxylic acid cycle pathway and acts as an effective metabolic stress on the cornea. Our data suggest that the pentose shunt in the cornea may serve as an important alternative pathway under conditions of metabolic stress for glucose utilization and the production of energy (ATP) in the corneal cells. (+info)
Unexpected additional mode of energization of amino-acid transport into Ehrlich cells.
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Ehrlich cells treated with dinitrophenol and iodoacetate rapidly recover their 30-sec uptake of 2-(methyl-amino)-isobutyrate on treatment with 0.1 mM phenazine methosulfate + 20 mM sodium ascorbate before they begin to recover from the severely depressed ATP levels and alkali-ion gradients. Addition of 10 mM pyruvate also restores uptake of methylaminoisobutyrate before the alkali-ion gradients rise. This restoration is prevented by rotenone, but rotenone does not handicap restoration by phenazine methosulfate/ascorbate. Na+-independent uptake of 2-aminonorbornane-2-carboxylate by Ehrlich cells is affected the same way. Quinacrine almost completely suppresses uptake of methylaminoisobutyrate within the 30-sec uptake test, even when ATP levels are sustained by pyruvate and alkali-ion gradients are not depressed. Ouabain prevents restoration of both Na+-dependent and Na+-independent amino-acid transport by phenazine methosulfate/ascorbate or pyruvate. We interpret these results to indicate that amino-acid transport can be energized not only by known means, but also by reducing equivalents, which presumably reach the plasma membrane in the form of NADH from the mitochondria when the source of energy is pyruvate. In support of this hypothesis, the distribution of methylaminoisobutyrate between plasma membrane vesicles and their supporting media was influenced in the predictable way by NADH, quinacrine, and an uncoupling agent, proceeding on the assumption that more of the vesicles had the everted rather than the natural orientation. (+info)
Reduction of methemoglobin by cobaltocytochrome c catalyzed by mediators.
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The reduction of methemoglobin by cobaltocytochrome c (Cocyt c) has been measured using nine mediators of different half-reduction potentials, Em, 7. The rate increases with the increase of Em, 7 for the mediator but dropped precipitously when it becomes more positive than the Em, 7 for the methemoglobin/hemoglobin couple. The reaction is most efficient with phenzaine methosulfate, therefore it was studied in detail. The reaction is first order in the concentrations of Cocyt c and phenazine methosulfate. The average second-order rate constant for Cocyt c + phenazine methosulfate (M) k1 leads to Cocyt c+ M-. is 2.9 x 10(4) M-1 s-1 at 25 degrees C, 0.1 M phosphate pH 7.0. There is a slight negative temperature dependence of k1 at low temperature; at higher temperatures the process has deltaH not equal to approximately 27 kJ mol-1 and deltaS not equal to approxmately - 75 J mol-1 K-1. The effect of anions reflects the dependence of Em, 7 for the methemoglobin/hemoglobin couple with various anions. There is no significant effect on k1 by the addition of inositol hexakisphosphate. The variation of k1 with pH is complicated. The experimental rate constants are compared with values calculated with the theory of nonadiabatic multiphonon process of electron tunneling. (+info)
Alcohol dehydrogenases from a facultative methylotrophic bacterium.
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Alcohol-oxidizing enzymes of the facultative methylotroph PAR were investigated after growth of the bacteria on methanol and ethanol. During methanol growth only a phenazine methosulfate-linked alcohol dehydrogenase was detected. This enzyme had broad specificity for primary alcohols and was also capable of oxidation of secondary alcohols. It had a molecular weight of 112,000, was composed of two subunits of equal molecular weight, and showed an absolute requirement for ammonium ion for activation. During ethanol growth this enzyme was absent and was replaced by a typical nicotinamide adenine dinucleotide-linked alcohol dehydrogenase of molecular weight 150,000. The latter enzyme also had broad specificity but could not oxidize methanol. This enzyme was not found during methanol growth. These data show that the organism has two distinctly separate mechanisms for oxidation of alcohols. (+info)