Variations in the activity of glutathione reductase and the cellular glutathione content in relation to sensitivity to methylviologen in Escherichia coli.

To study the function of glutathione reductase and glutathione in Escherichia coli the coding sequence of the bacterial glutathione reductase gene (gor gene) was cloned into the vector pBR322, and the gor gene was expressed under the control of the promoter of the tetracycline-resistance gene (tet gene) in different Escherichia coli strains. Cells of the gor-mutant strain SG5 containing the vector pBR322 (SG5:pBR322) had no detectable glutathione reductase activity and a significantly lower total glutathione (GSH + GSSG) content relative to control cells of the strain JM101 (JM101: pBR322). The gor mutant cells were less sensitive to inhibition by methylviologen (as defined by changes in growth) than cells of the strain JM101. Elevated levels of both glutathione reductase activity and the total glutathione content (GSH + GSSG) were found when the gor gene was expressed in cells of the gor-mutant strain SG5 (SG5:pJIK1). Thus the activity of glutathione reductase is essential in order to maintain a high glutathione content. Furthermore, cells of the strain SG5: pJIK1 showed an increased sensitivity to methylviologen compared to cells of the gor mutant containing the vector pBR322 alone without the cloned gor gene insert (SG5:pBR322). In all experiments, the glutathione pool (GSH + GSSG) of bacterial cells was 90% reduced. In methylviologen-sensitive sodB mutant cells lacking iron superoxide dismutase activity (QC773:pBR322) overexpression of the cloned gor gene resulted in an elevated level of glutathione reductase activity which partially protected sodB mutant cells (QC773:pJIK1) against methylviologen toxicity. In sodB mutant cells expressing the gor gene (QC773:pJIK1) protection by glutathione reductase was, however, less effective than protection provided by expression of the iron superoxide dismutase gene (sodB gene) in these mutant cells (QC773:pJIK2). In sodA mutant cells lacking manganese superoxide dismutase activity but expressing the cloned gor gene (QC772:pJIK1) increased cellular glutathione reductase activity did not provide protection against methylviologen.

Nitrite uptake into intact pea chloroplasts : I. Kinetics and relationship with nitrite assimilation.

The uptake of nitrite into intact pea chloroplasts was observed and its relationship with internal nitrite reduction was assessed. Net nitrite uptake exhibited saturation kinetics and an alkaline pH preference. This evidence questioned the accepted major role for neutral HNO(2) permeation and its reported influence on photosynthesis. The possible involvement of a nitrite permeation channel or transport protein is discussed. Net nitrite uptake curves were closely comparable with those for nitrite reduction within the chloroplast. Net nitrite uptake into chloroplasts was profoundly influenced by darkness, incubation temperature, and plant nitrate nutrition. Of several inorganic salts tested, sulfite was the only anion to exhibit a distinct inhibition of nitrite uptake. In contrast, nitrite uptake could be stimulated by the presence of certain cations, particularly at acidic pH values. It was concluded that nitrite uptake was closely related to stromal pH, internal nitrite accumulation, and nitrite reduction. The possible dependence of nitrite reduction on nitrite uptake rather than electron transport is discussed. External ATP and NAD(P)H did not significantly affect net nitrite uptake. This suggested that cytoplasmic ATP or reductant could not directly support nitrite uptake and, possibly, nitrite assimilation.

Nitrite Uptake into Intact Pea Chloroplasts : II. Influence of Electron Transport Regulators, Uncouplers, ATPase and Anion Uptake Inhibitors and Protein Binding Reagents.

The relationship between net nitrite uptake and its reduction in intact pea chloroplasts was investigated employing electron transport regulators, uncouplers, and photophosphorylation inhibitors. Observations confirmed the dependence of nitrite uptake on stromal pH and nitrite reduction but also suggested a partial dependance upon PSI phosphorylation. It was also suggested that ammonia stimulates nitrogen assimilation in the dark by association with stromal protons. Inhibition of nitrite uptake by N-ethylmaleimide and dinitrofluorobenzene could not be completely attributed to their inhibition of carbon dioxide fixation. Other protein binding reagents which inhibited photosynthesis showed no effect on nitrite uptake, except for p-chlormercuribenzoate which stimulated nitrite uptake. The results with N-ethylmaleimide and dinitrofluorobenzene tended to support the proposed presence of a protein permeation channel for nitrite uptake in addition to HNO(2) penetration. On the basis of a lack of effect by known anion uptake inhibitors, it was concluded that the nitrite uptake mechanism was distinct from that of phosphate and chloride/sulfate transport.

Control of nitrate and nitrite assimilation by carbohydrate reserves, adenosine nucleotides and pyridine nucleotides in leaves of Zea mays L. under dark conditions.

The rate of in-vivo nitrate reduction by leaf segments of Zea mays L. was found to decline during the second hour of dark anaerobic treatment. On transfer to oxygen the capacity to reduce nitrate under dark conditions was restored. These observations led to the proposal that nitrate reductase is a regulatory enzyme with ADP acting as a negative effector. The effect of ADP on the invitro activity of nitrate reductase and the changes in the in-vivo adenylate pool under dark-N2 and dark-O2 were investigated. It was found that ADP inhibited the activity of partially purified nitrate reductase. Similarly, the in-vivo anaerobic inhibition of nitrate reduction was associated with a build-up of ADP in the leaf tissue. Under anaerobic conditions nitrite accumulated and on transfer to oxygen the accumulated nitrite was reduced. To explain this phenomenon the following hypothesis was proposed and tested. Under anaerobic conditions the supply of reducing equivalents for nitrite reduction in the plastid becomes restricted and nitrite accumulates as a consequence. On transfer to oxygen this restriction is removed and nitrite disappears. This capacity to reduce accumulated nitrite was found to be dependent on the carbohydrate status of the leaf tissue.

The effect of oxygen on nitrate and nitrite assimilation in leaves of Zea mays L. under dark conditions.

The assimilation of nitrate under dark-N2 and dark-O2 conditions in Zea mays leaf tissue was investigated using colourimetric and (15)N techniques for the determination of organic and inorganic nitrogen. Studies using (15)N indicated that nitrate was assimilated under dark conditions. However, the rate of nitrate assimilation in the dark was only 28% of the rate under non-saturating light conditions. No nitrite accumulated under dark aerobiosis, even though nitrate reduction occurred under these conditions. The pattern of nitrite accumulation in leaf tissue in response to dark-N2 conditions consisted of three phases: an initial lag phase, followed by a period of rapid nitrite accumulation and finally a phase during which the rate of nitrite accumulation declined. After a 1-h period of dark-anaerobiosis, both nitrate reduction and nitrite accumulation declined considerably. However, when O2 was supplied, nitrate reduction was stimulated and the accumulated nitrite was rapidly reduced. Anaerobic conditions stimulated nitrate reduction in leaf tissue after a period of dark-aerobic pretreatment.

The occurrence of both C3 and C 4 photosynthetic characteristics in a single Zea mays plant.

The activities of the carboxylating enzymes ribulose-1,5-biphosphate (RuBP) carboxylase and phosphoenolpyruvate (PEP) carboxylase in leaves of three-week old Zea mays plants grown under phytotron conditions were found to vary according to leaf position. In the lower leaves the activity of PEP carboxylase was lower than that of RuBP carboxylase, while the upper leaves exhibited high levels of PEP carboxylase. Carbon dioxide compensation points and net photosynthetic rates also differed in the lower and upper leaves. Differences in the fine structure of the lowermost and uppermost leaves are shown. The existence of both the C3 and C4 photosynthetic pathways in the same plant, in this and other species, is discussed.