Regulation of NADP-malate dehydrogenase in C4 plants: effect of varying NADPH to NADP ratios and thioredoxin redox state on enzyme activity in reconstituted systems

Activation and inactivation of NADP-malate dehydrogenase purified from Zea mays leaves were followed in a reconstituted system provided with thioredoxin poised in various redox states with dithiothreitol. The initial rate of activation or inactivation of NADP-malate dehydrogenase was proportional to the concentration of reduced or oxidized thioredoxin, respectively. The rate of inactivation was about 16 times that for activation at pH 7.4. Both activities increased when the pH was increased from 7.4 to 8.0. The redox potentials (E'0, pH 7) for the dithiol-disulfide systems of thioredoxin and NADP-malate dehydrogenase were estimated to be about -0.30 and -0.33 V, respectively. As would be predicted from these values, high proportions of active malate dehydrogenase were developed only in the presence of very high ratios of reduced to oxidized thioredoxin. Similarly, when pyridine nucleotide was included, a high degree of activation of malate dehydrogenase was only observed with high NADPH/NADP ratios. These results confirm predictions based on models developed in earlier studies that the NADPH to NADP ratio as well as the thioredoxin redox state may be critical in determining the level of NADPH-malate dehydrogenase activity in vivo.     

Effect of amino acids on choline uptake and phosphatidylcholine biosynthesis in the isolated hamster heart

Choline uptake by the hamster heart has been shown to be enhanced by exogenous glycine. In this study, the effect of neutral, basic, and acidic amino acids on choline uptake was assessed. Hamster hearts were perfused with labelled choline, and in the presence of L-alanine, L-serine, or L-phenylalanine (greater than or equal to 0.1 mM), choline uptake was enhanced 20-38%. L-Arginine, L-lysine, L-aspartate, and L-glutamate did not influence choline uptake. The rate of phosphatidylcholine biosynthesis was unaffected by all amino acids tested. Enhancement of choline uptake by neutral amino acids was not additive or dose dependent but required a concentration threshold. The enhancement of choline uptake by neutral amino acids was not influenced by preperfusion with the same amino acid. Exogenous choline had no effect on the uptake of amino acids. We postulate that choline and the neutral amino acids are not cotransported and modulation of choline uptake is facilitated by direct interaction of the neutral amino acids with the choline transport system.     

Transport of 3-phosphoglyceric acid,phosphoenolpyruvate, and inorganic phosphate in maize mesophyll chloroplasts,and the effect of 3-phosphoglyceric acid on malate and phosphoenolpyruvate production

Maize mesophyll chloroplasts loaded with radioactively labeled 3-phosphoglycerate or phosphoenolpyruvate exchange these compounds for externally provided inorganic phosphate, 3-phosphoglycerate, phosphoenolpyruvate, and dihydroxyacetone phosphate. These exchanges are inhibited by pyridoxal phosphate. 3-Phosphoglycerate uptake, which leads to accumulation of this substance in the stroma, is competitively inhibited by inorganic phosphate and phosphoenolpyruvate. These results are consistent with the transport of 3-phosphoglycerate, phosphoenolpyruvate, inorganic phosphate, and dihydroxyacetone phosphate being mediated by a common carrier (the phosphate translocator). The activation energy of 3-phosphoglycerate uptake as determined from its temperature dependence is 19.5 kcal (4–15 °C). In isolated chloroplasts malate and phosphoenolpyruvate production from oxalacetate and pyruvate, respectively, is inhibited by 3-phosphoglycerate, the extent of inhibition being dependent on the relative concentrations of inorganic phosphate and 3-phosphoglycerate. We propose that 3-phosphoglycerate from bundle-sheath cells may serve as a feedback regulator of mesophyll cell photosynthesis.     

Association of NADP- and NAD-linked malic enzyme acitivities in Zea mays: relation to C4 pathway photosynthesis.

Two of the three metabolic subtypes of species utilizing C₄-pathway photosynthesis are defined by high activities of either NADP malic enzyme (NADP malic enzyme type) or a coenzyme A (CoA)- and acetyl-CoA-activated NAD malic enzyme (NAD malic enzyme type). These enzymes function to decarboxylate malate as an integral part of the photosynthetic process. Leaves of NADP malic enzyme-type species also contain significant NAD-dependent malic enzyme activity. The purpose of the present study was to examine the nature and photosynthetic role of this activity. With Zea mays, this NAD-dependent activity was found to vary widely in fresh leaf extracts. Incubating extracts at 25 °C resulted in a disproportionate increase in NAD activity so that the final ratio of NADP to NAD activity was always about 5. Strong evidence was provided that the NADP and NAD malic enzyme activities in Z. mays extracts were catalyzed by the same enzyme. These activities remained associated during purification and were coincident after polyacrylamide gel electrophoresis. The pH optimum for NAD-dependent activity was about 7.1, compared with 8.3 for NADP malic enzyme activity. Other properties of the NAD-dependent activity are described, a particularly notable feature being the inhibition of this activity by less than 1 μm NADP and NADPH. Evidence is provided that the NADP malic enzyme of several other NADP malic enzyme-type C₄ species also has associated activity toward NAD. We concluded that the NAD-dependent malic enzyme activity would have no significant function in photosynthesis.     

The roles of malate and aspartate in C4 photosynthetic metabolism of Flaveria bidentis (L.)

In C₄ grasses belonging to the NADP-malic enzyme-type subgroup, malate is considered to be the predominant C₄ acid metabolized during C₄ photosynthesis, and the bundle sheath cell chloroplasts contain very little photosystem-II (PSII) activity. The present studies showed that Flaveria bidentis (L.), an NADP-malic enzyme-type C₄ dicotyledon, had substantial PSII activity in bundle sheath cells and that malate and aspartate apparently contributed about equally to the transfer of CO₂ to bundle sheath cells. Preparations of bundle sheath cells and chloroplasts isolated from these cells evolved O₂ at rates between 1.5 and 2 mol · min^–1 · mg^–1 chlorophyll (Chl) in the light in response to adding either 3-phosphoglycerate plus HCO ₃ ^– or aspartate plus 2-oxoglutarate. Rates of more than 2 mol O₂ · min^–1 · mg^–1 Chl were recorded for cells provided with both sets of these substrates. With bundle sheath cell preparations the maximum rates of light-dependent CO₂ fixation and malate decarboxylation to pyruvate recorded were about 1.7 mol · min^–1 · mg^–1 Chl. Compared with NADP-malic enzyme-type grass species, F. bidentis bundle sheath cells contained much higher activities of NADP-malate dehydrogenase and of aspartate and alanine aminotransferases. Time-course and pulse-chase studies following the kinetics of radiolabelling of the C-4 carboxyl of C₄ acids from ¹⁴CO₂ indicated that the photosynthetically active pool of malate was about twice the size of the aspartate pool. However, there was strong evidence for a rapid flux of carbon through both these pools. Possible routes of aspartate metabolism and the relationship between this metabolism and PSII activity in bundle sheath cells are considered.Abbreviations DHAP dihydroxyacetone phosphate - NADP-ME(-type) NADP-malic enzyme (type) - NADP-MDH NADP-malate dehydrogenase - OAA oxaloacetic acid - 2-OG 2-oxoglutarate - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - Pi orthophosphate - Ru5P ribulose 5-phosphate     

Food availability affects the maternal transfer of androgens and antibodies into eggs of a colonial seabird

Mothers can improve the quality of their offspring by increasing the level of certain components in their eggs. To examine whether or not mothers increase deposition of such components in eggs as a function of food availability, we food-supplemented black-legged kittiwake females (Rissa tridactyla) before and during egg laying and compared deposition of androgens and antibodies into eggs of first and experimentally induced replacement clutches. Food-supplemented females transferred lower amounts of androgens and antibodies into eggs of induced replacement clutches than did non-food-supplemented mothers, whereas first clutches presented no differences between treatments. Our results suggest that when females are in lower condition, they transfer more androgens and antibodies into eggs to facilitate chick development despite potential long-term costs for juveniles. Females in prime condition may avoid these potential long-term costs because they can provide their chicks with more and higher quality resources.     

Phospholipid scramblase-3 regulates cardiolipin de novo biosynthesis and its resynthesis in growing HeLa cells

PLS3 (phospholipid scramblase-3) is a new member of the family of phospholipid scramblases and transports CL (cardiolipin) from the inner to the outer mitochondrial membrane. In the present paper we examined whether changing the levels of functional PLS3 in HeLa cells altered de novo CL biosynthesis and its resynthesis. HeLa cells overexpressing PLS3 or expressing a disrupted PLS3 (F258V) or control were incubated with [1,3-3H]glycerol and radioactivity incorporated into CL was determined. CL biosynthesis from [1,3-3H]glycerol was increased 1.8-fold in PLS3 cells and 2.1-fold in F258V cells compared with control. This was due to a 64% (P<0.05) and 2.6-fold (P<0.05) elevation in CL synthase activity in PLS3 and F258V cells respectively, compared with control, and not due to changes in phosphatidylglycerolphosphate synthase activity. The increase in CL synthase activity in these cells was due to an increase in its mRNA expression. In contrast, resynthesis of CL from [1-14C]linoleic acid was reduced 52% (P<0.05) in PLS3 and 45% (P<0.05) in F258V cells compared with control and this was due to a reduction in mitochondrial monolysocardiolipin acyltransferase activity. Although protein levels of mitochondrial monolysocardiolipin acyltransferase were unaltered, activity and mRNA expression of endoplasmic reticulum monolysocardiolipin acyltransferase was upregulated in PLS3 and F258V cells compared with controls. These data indicate that the CL resynthesis in HeLa cells is sensitive to the mitochondrial localization of CL and not the level of the reacylating enzymes. Alterations in functional PLS3 levels in PLS3 or F258V cells did not affect the mitochondrial decarboxylation of phosphatidylserine to phosphatidylethanolamine indicating that the biosynthetic changes to CL were specific for this mitochondrial phospholipid. We hypothesize that the cardiolipin resynthesis machinery in the cell 'senses' altered levels of CL on mitochondrial membranes and that de novo CL biosynthesis is up-regulated in HeLa cells as a compensatory mechanism in response to altered movement of mitochondrial CL. The results identify PLS3 as a novel regulator of CL de novo biosynthesis and its resynthesis.     

Public information needs after the poisoning of Alexander Litvinenko with polonium-210 in London: cross sectional telephone survey and qualitative analysis

Objectives To identify public perceptions of the risk to health after the poisoning of Alexander Litvinenko with polonium-210 (²¹⁰Po) in London and to assess the impact of public health communications.Design Cross sectional telephone survey and qualitative interviews.Setting London, United Kingdom.Participants 1000 people completed the cross sectional survey and 86 potentially exposed people completed the qualitative interviews.Main outcome measures Perception of risk to personal health after the ²¹⁰Po incident. Qualitative interviews were analysed with an emphasis on information needs.Results 11.7% of the survey sample (n=117) perceived their health to be at risk. Aside from personal variables the main predictors of perceived risk to health were believing that the incident was related to terrorism (odds ratio 2.7, 95% confidence interval 1.5 to 4.6) rather than to espionage, that it was targeted at the wider public rather than one person (5.9, 3.2 to 10.9), and that it could affect people who had not been in the contaminated area (3.2, 2.1 to 5.1). Participants in the qualitative interviews were generally satisfied with the information they had received, although they would have preferred more information about their individual risk of exposure, the results of their urine tests, and the health implications of the incident.Conclusions Perceptions of the public that the ²¹⁰Po incident in London in 2006 was related to espionage helped to reassure them that the risks to personal health were low. In the event of future incidents it is important to ensure that detailed, comprehensible information about the risks of any exposure is available.     

Carbon mineralization and distribution of nutrients within different particle-size fractions of commercially produced olive mill pomace

Composting is a realistic option for disposal of olive mill pomace (OMP) by making it suitable as a soil amendment for organic farming. The chemical and physical characteristics and contribution of particle-size fractions to total nutrients and carbon mineralization of seven commercial composts of OMP (COMP) were investigated. Higher proportions of manure, co-composted with OMP, reduced the organic matter (OM), total carbon and C:N ratio of the product, but increased the content of nutrients and fine particles. The fine particles had higher nutrient contents, but less OM and carbon and, unlike larger particles, did not exhibit any phytotoxicity. Less than 1.5% of added carbon was mineralized in whole compost, but a lower rate was found with larger particles. Separation of COMP by particle size fractionation and application as a soil conditioner is recommended for better optimization of COMP with the <1 mm fraction providing the higher quality compost.