Altered Redox Potential Research Articles

1H and 15N resonance assignments and solution secondary structure of oxidized Desulfovibrio vulgaris flavodoxin determined by heteronuclear three-dimensional NMR spectroscopy

Sequence-specific 1H and 15N resonance assignments have been made for all 145 non-prolyl residues and for the flavin cofactor in oxidized Desulfovibrio vulgaris flavodoxin. Assignments were obtained by recording and analyzing 1H-15N heteronuclear three-dimensional NMR experiments on uniformly 15N-enriched protein, pH 6.5, at 300 K. Many of the side-chain resonances have also been assigned. Observed medium-and long-range NOEs, in combination with 3JNH alpha coupling constants and 1HN exchange data, indicate that the secondary structure consists of a five-stranded parallel beta-sheet and four alpha-helices, with a topology identical to that determined previously by X-ray crystallographic methods. One helix, which is distorted in the X-ray structure, is non-regular in solution as well. Several protein-flavin NOEs, which serve to dock the flavin ligand to its binding site, have also been identified. Based on fast-exchange into 2H2O, the 1HN3 proton of the isoalloxazine ring is solvent accessible and not strongly hydrogen-bonded in the flavin binding site, in contrast to what has been observed in several other flavodoxins. The resonance assignments presented here can form the basis for assigning single-site mutant flavodoxins and for correlating structural differences between wild-type and mutant flavodoxins with altered redox potentials.

Contrasts in interleukin-1 and interleukin-2 activity in alcoholic hepatitis and cirrhosis.

To investigate whether disordered immune function, as shown by abnormalities in lymphokine production, is present in alcoholic liver disease, interleukin-1 and interleukin-2 activity were assayed in a group of patients with acute alcoholic hepatitis in the absence of underlying cirrhosis, and a group of patients with inactive alcoholic cirrhosis. Activities of both IL-1 and IL-2 in alcoholic hepatitis were similar to those of normal individuals, although in abstinent patients with alcoholic cirrhosis, IL-1 activity was increased and IL-2 activity decreased. Lymphocyte transformation in response to PHA in patients with alcoholic hepatitis was significantly impaired when compared with normal controls, and addition of exogenous IL-2 did not correct this impaired response over a wide range of concentrations of both PHA and IL-2. These observations suggest the underlying defects in cell mediated immunity in acute alcoholic hepatitis, as assessed by blast transformation, could be fundamentally different from those of alcoholic cirrhosis and could be secondary to the metabolic effects of acetaldehyde or altered redox potentials on the behaviour of proliferating cells.

A modified flavodoxin with altered redox potentials is less efficient in electron transfer to nitrogenase

The flavodoxins of the Azotobacter vinelandii wild-type and a mutant strain TZN 200 have been studied. Although the primary structure of the two proteins is the same, the ability of the mutant flavodoxin to donate electrons to nitrogenase is reduced by 75%. One reason may be the raised mid-point potential of −435 mV for the semiquinone/hydroquinone couple in the mutant flavodoxin. The respective redox potential for the wild-type flavodoxin was found to be −480 mV. As shown by paper chromatography and light absorption spectroscopy, the structure of FMN is modified in the TZN 200 flavodoxin.

Lactate-mediated changes in growth morphology and transformation frequency of irradiated C3h [formula omitted] cells

Treatment of mammalian cells with lactate or inhibitors of glycolysis alters their radiation response, particularly in the low dose region of the dose response curve. The occurrence of both high lactate levels and high glycolytic metabolism in tumours is well known and therefore the effect of lactate on a cell line sensitive to radiation induced transformation was examined using a single exposure to Cobalt 60 gamma rays as the carcinogen challenge. The results indicate that cells treated with 5mM lactate before irradiation exhibit changes in morphology and growth rate and that the transformation frequency is increased by three to ten fold following 24 hours lactate treatment just prior to irradiation. Examination of radiation survival curves showed a positive correlation between transformation frequency and size of the shoulder, but increasing transformation frequency was associated with a decrease in Do. A mechanism involving altered Redox potential in lactate treated cells is suggested. The results are discussed in terms of their possible significance for radiotherapy.

The reaction of benzoquinone with amines and proteins

It has been shown that benzoquinone will form 1,4 addition products with amines, amino acids, and proteins. The substituted quinones produced via this reaction have altered redox potentials. The quinone amine adduct can be reduced by ascorbate and oxidized by oxygen to produce hydrogen peroxide. This sequence of reactions can be conveniently monitored by following the oxygen consumption in the presence of excess ascorbate. Under appropriate experimental conditions the rate of oxygen consumption is proportional to the concentration of the quinoae adduct. Since benzoquinone alone will not react in this way, the reaction between the amines and benzoquinone can be followed by determining the rate of ascorbic acid oxidation. With this assay, it was shown that the glycine will react with benzoquinone at a very slow rate below pH 7.0, exhibiting an optimum rate at pH 8. All amino acids seemed to react with the benzoquinone in the same manner independent of their side chains. The reaction can be made an assay for an amino acid, and with this procedure quantities of glycine as low as 1 × 10 −7 m, could be readily detected. Both mono- and di-substituted benzoquinone amine products were formed. Proteins reacted with the benzoquinone in a manner similar to that described for the simple amino acids. The reaction with cytochrome c was studied in some detail. By using radioactively-labeled benzoquinone, it was shown that cytochrome c reacted readily to form a product containing two to four moles of benzoquinone per mole of cytochrome c. The position of the benzoquinone was such that it was not accessible to the heme groups, and did not cause denaturation or alteration of the structure of the cytochrome c. Other proteins vary with regard to their ability to form the quinone adduct, but all form adducts which are capable of catalyzing ascorbate oxidation. It has been suggested that quinone adducts of the type described here may be important in biological systems.