The steady-state internal redox state (NADH/NAD) reflects the external redox state and is correlated with catabolic adaptation in Escherichia coli.
Escherichia coli MC4100 was grown in anaerobic glucose-limited chemostat cultures, either in the presence of an electron acceptor (fumarate, nitrate, or oxygen) or fully fermentatively. The steady-state NADH/NAD ratio depended on the nature of the electron acceptor. Anaerobically, the ratio was highest, and it decreased progressively with increasing midpoint potential of the electron acceptor. Similarly, decreasing the dissolved oxygen tension resulted in an increased NADH/NAD ratio. As pyruvate catabolism is a major switch point between fermentative and respiratory behavior, the fluxes through the different pyruvate-consuming enzymes were calculated. Although pyruvate formate lyase (PFL) is inactivated by oxygen, it was inferred that the in vivo activity of the enzyme occurred at low dissolved oxygen tensions (DOT +info)
Utilization of electrically reduced neutral red by Actinobacillus succinogenes: physiological function of neutral red in membrane-driven fumarate reduction and energy conservation.
Neutral red (NR) functioned as an electronophore or electron channel enabling either cells or membranes purified from Actinobacillus succinogenes to drive electron transfer and proton translocation by coupling fumarate reduction to succinate production. Electrically reduced NR, unlike methyl or benzyl viologen, bound to cell membranes, was not toxic, and chemically reduced NAD. The cell membrane of A. succinogenes contained high levels of benzyl viologen-linked hydrogenase (12.2 U), fumarate reductase (13.1 U), and diaphorase (109.7 U) activities. Fumarate reductase (24.5 U) displayed the highest activity with NR as the electron carrier, whereas hydrogenase (1.1 U) and diaphorase (0.8 U) did not. Proton translocation by whole cells was dependent on either electrically reduced NR or H2 as the electron donor and on the fumarate concentration. During the growth of Actinobacillus on glucose plus electrically reduced NR in an electrochemical bioreactor system versus on glucose alone, electrically reduced NR enhanced glucose consumption, growth, and succinate production by about 20% while it decreased acetate production by about 50%. The rate of fumarate reduction to succinate by purified membranes was twofold higher with electrically reduced NR than with hydrogen as the electron donor. The addition of 2-(n-heptyl)-4-hydroxyquinoline N-oxide to whole cells or purified membranes inhibited succinate production from H2 plus fumarate but not from electrically reduced NR plus fumarate. Thus, NR appears to replace the function of menaquinone in the fumarate reductase complex, and it enables A. succinogenes to utilize electricity as a significant source of metabolic reducing power. (+info)
Metabolic behaviour and cleavage capacity in the amphibian egg.
During the winter season the full grown Bufo arenarum oocyte shows the metabolic behaviour characteristic of differentiated tissues of the same species. Due to seasonal variations, during the amplexus period, it acquires the metabolic behaviour of the segmenting egg. Short-time-induced ovulations (5-6 h) determine germinal vesicle breakdown immediately before the expulsion of the oocyte, without modifying the ovarian metabolism of the same. The incidence of the operative type of metabolism upon their capacity to cleave after insemination and needle pricking, has been studied in coelomic oocytes, which have attained nuclear maturation and have not experienced oviducal secretion effects. The results obtained indicate that the segmenting capacity of the egg is attained only when, through biochemical modifications, the oocyte acquires the metabolic behaviour characterizing embryonic cells. It is postulated that the metabolic changes observed in the oocyte constitue a fundamental aspect of cytoplasmic maturation. (+info)
Functioning of DcuC as the C4-dicarboxylate carrier during glucose fermentation by Escherichia coli.
The dcuC gene of Escherichia coli encodes an alternative C4-dicarboxylate carrier (DcuC) with low transport activity. The expression of dcuC was investigated. dcuC was expressed only under anaerobic conditions; nitrate and fumarate caused slight repression and stimulation of expression, respectively. Anaerobic induction depended mainly on the transcriptional regulator FNR. Fumarate stimulation was independent of the fumarate response regulator DcuR. The expression of dcuC was not significantly inhibited by glucose, assigning a role to DcuC during glucose fermentation. The inactivation of dcuC increased fumarate-succinate exchange and fumarate uptake by DcuA and DcuB, suggesting a preferential function of DcuC in succinate efflux during glucose fermentation. Upon overexpression in a dcuC promoter mutant (dcuC*), DcuC was able to compensate for DcuA and DcuB in fumarate-succinate exchange and fumarate uptake. (+info)
Structure of the Escherichia coli fumarate reductase respiratory complex.
The integral membrane protein fumarate reductase catalyzes the final step of anaerobic respiration when fumarate is the terminal electron acceptor. The homologous enzyme succinate dehydrogenase also plays a prominent role in cellular energetics as a member of the Krebs cycle and as complex II of the aerobic respiratory chain. Fumarate reductase consists of four subunits that contain a covalently linked flavin adenine dinucleotide, three different iron-sulfur clusters, and at least two quinones. The crystal structure of intact fumarate reductase has been solved at 3.3 angstrom resolution and demonstrates that the cofactors are arranged in a nearly linear manner from the membrane-bound quinone to the active site flavin. Although fumarate reductase is not associated with any proton-pumping function, the two quinones are positioned on opposite sides of the membrane in an arrangement similar to that of the Q-cycle organization observed for cytochrome bc1. (+info)
Electrophysiological effects of LU111995 on canine hearts: in vivo and in vitro studies.
We studied the electrophysiological effects of LU111995 (1-15 mg/kg p.o.) in conscious dogs with chronic atrioventricular block and ventricular pacing at 50 to 130 beats/min. LU111995 had no effects on idioventricular rhythm, QRS duration, and ventricular conduction time. It significantly prolonged Q-T interval (by 5-8%) and effective refractory period (ERP) (by 5-12%) with the maximal effect at 4 h after a 10 mg/kg dose. At 10 and 15 mg/kg, it increased the ERP/Q-T ratio. In vitro, the effects of LU111995 (1 x 10(-7) to 1 x 10(-5) M) on action potentials of Purkinje fibers (PFs) and M cells were studied at cycle lengths (CL) of 300 to 2000 ms. It had no effects on maximum diastolic potential and action potential amplitude in either tissue. High concentrations induced a moderate, rate-independent decrease of Vmax in M cells. In PFs and M cells, it produced reverse use-dependent lengthening of action potential duration (APD). In PFs at long CL, the drug exhibited a biphasic concentration-dependent effect on APD: maximum prolongation (by 26% at a CL of 2000 ms) was attained at 1 x 10(-6) M, and a decrease of APD occurred at higher concentrations. In M cells, the maximum effect on APD occurred at 3 x 10(-6) M. Early afterdepolarizations were seen in 50% of M cell preparations but only at CL of 2000 ms. Triggered activity did not occur. In summary, LU111995 prolongs the Q-T interval to a limited degree and is not arrhythmogenic over the physiological range of CLs. (+info)
Microbial utilization of electrically reduced neutral red as the sole electron donor for growth and metabolite production.
Electrically reduced neutral red (NR) served as the sole source of reducing power for growth and metabolism of pure and mixed cultures of H2-consuming bacteria in a novel electrochemical bioreactor system. NR was continuously reduced by the cathodic potential (-1.5 V) generated from an electric current (0.3 to 1.0 mA), and it was subsequently oxidized by Actinobacillus succinogenes or by mixed methanogenic cultures. The A. succinogenes mutant strain FZ-6 did not grow on fumarate alone unless electrically reduced NR or hydrogen was present as the electron donor for succinate production. The mutant strain, unlike the wild type, lacked pyruvate formate lyase and formate dehydrogenase. Electrically reduced NR also replaced hydrogen as the sole electron donor source for growth and production of methane from CO2. These results show that both pure and mixed cultures can function as electrochemical devices when electrically generated reducing power can be used to drive metabolism. The potential utility of utilizing electrical reducing power in enhancing industrial fermentations or biotransformation processes is discussed. (+info)
Characterization of two members of a novel malic enzyme class.
The Gram-negative bacterium Rhizobium meliloti contains two distinct malic enzymes. We report the purification of the two isozymes to homogeneity, and their in vitro characterization. Both enzymes exhibit unusually high subunit molecular weights of about 82 kDa. The NAD(P)(+) specific malic enzyme [EC 18.104.22.168] exhibits positive co-operativity with respect to malate, but Michaelis-Menten type behavior with respect to the co-factors NAD(+) or NADP(+). The enzyme is subject to substrate inhibition, and shows allosteric regulation by acetyl-CoA, an effect that has so far only been described for some NADP(+) dependent malic enzymes. Its activity is positively regulated by succinate and fumarate. In contrast to the NAD(P)(+) specific malic enzyme, the NADP(+) dependent malic enzyme [EC 22.214.171.124] shows Michaelis-Menten type behavior with respect to malate and NADP(+). Apart from product inhibition, the enzyme is not subjected to any regulatory mechanism. Neither reductive carboxylation of pyruvate, nor decarboxylation of oxaloacetate, could be detected for either malic enzyme. Our characterization of the two R. meliloti malic enzymes therefore suggests a number of features uncharacteristic for malic enzymes described so far. (+info)