Molecular structure of malate synthase and structural changes upon ligand binding to the enzyme

Malate synthase has a molecular weight of about 170 000 as shown by ultracentrifugation, sucrose gradient centrifugation, and thin layer gel-chromatography. High dilution, extremes of pH, succinylation, and treatment with sodium dodecylsulfate suggest the enzyme to be a tetramer. The CD spectrum is typical for a globular protein with moderate helical content (～30 %), and shows anomalous Cotton effects at 250–290 nm. Binding of substrates (acetyl-CoA, glyoxylate) or the substrate analog pyruvate causes slight conformational changes which are reflected in alterations of the CD bands in the range of aromatic absorption; binding of Mg²⁺ causes no structural effects, suggesting the metal ion to be involved in enzymatic catalysis rather than structural alterations.     

Malate dehydrogenase of spinach: Substrate kinetics of different forms

Sephadex G-200 gel filtration of an ammonium sulfate fraction, containing the bulk of NAD-dependent malate dehydrogenase, yields forms of differing MW. Both Mg²⁺ and NADH stabilize the 127000 daltons MW form. K⁺, or incubation with dithioerythritol, cause splitting and partial reaggregation, resulting in MWs ranging between 35000 and 180000 daltons. Chromatography in the presence of dithioerythritol and NADH results in an enzyme with a non-linear reaction rate at low substrate concentrations. Plots of initial velocity vs substrate and cofactor concentration respectively are characterized by two slopes of positive cooperativity separated by an intermediary plateau of negative cooperativity. Gel chromatography in the presence of Mg²⁺ or K⁺ or drastic dilution of the enzyme results in an enzyme with linear reaction rates also at low substrate concentration. Its kinetics are consistent with the view that the enzyme undergoes conformational changes when the substrate concentration is varied.     

Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport

Long-chain nonesterified (“free”) fatty acids (FFA) can affect the mitochondrial generation of reactive oxygen species (ROS) in two ways: (i) by depolarisation of the inner membrane due to the uncoupling effect and (ii) by partly blocking the respiratory chain. In the present work this dual effect was investigated in rat heart and liver mitochondria under conditions of forward and reverse electron transport. Under conditions of the forward electron transport, i.e. with pyruvate plus malate and with succinate (plus rotenone) as respiratory substrates, polyunsaturated fatty acid, arachidonic, and branched-chain saturated fatty acid, phytanic, increased ROS production in parallel with a partial inhibition of the electron transport in the respiratory chain, most likely at the level of complexes I and III. A linear correlation between stimulation of ROS production and inhibition of complex III was found for rat heart mitochondria. This effect on ROS production was further increased in glutathione-depleted mitochondria. Under conditions of the reverse electron transport, i.e. with succinate (without rotenone), unsaturated fatty acids, arachidonic and oleic, straight-chain saturated palmitic acid and branched-chain saturated phytanic acid strongly inhibited ROS production. This inhibition was partly abolished by the blocker of ATP/ADP transfer, carboxyatractyloside, thus indicating that this effect was related to uncoupling (protonophoric) action of fatty acids. It is concluded that in isolated rat heart and liver mitochondria functioning in the forward electron transport mode, unsaturated fatty acids and phytanic acid increase ROS generation by partly inhibiting the electron transport and, most likely, by changing membrane fluidity. Only under conditions of reverse electron transport, fatty acids decrease ROS generation due to their uncoupling action.     

The cellular basis of guard cell sensing of rising CO2

Numerous studies conducted on both whole plants and isolated epidermes have documented stomatal sensitivity to CO₂. In general, CO₂ concentrations below ambient stimulate stomatal opening, or an inhibition of stomatal closure, while CO₂ concentrations above ambient have the opposite effect. The rise in atmospheric CO₂ concentrations which has occurred since the industrial revolution, and which is predicted to continue, will therefore alter rates of transpirational water loss and CO₂ uptake in terrestrial plants. An understanding of the cellular basis for guard cell CO₂ sensing could allow us to better predict, and perhaps ultimately to manipulate, such vegetation responses to climate change. However, the mechanisms by which guard cells sense and respond to the CO₂ signal remain unknown. It has been hypothesized that cytosolic pH and malate levels, cytosolic Ca²⁺ levels, chloroplastic zeaxanthin levels, or plasma-membrane anion channel regulation by apoplastic malate are involved in guard cell perception and response to CO₂. In this review, these hypotheses are discussed, and the evidence for guard cell acclimation to prevailing CO₂ concentrations is also considered.     

Age-dependent changes of the ion content and the circadian leaf movement period in thePhaseolus pulvinus

The circadian movement of the lamina of primary leaves ofPhaseolus coccineus L. depends on circadian changes of the K⁺, Cl^- and (depending on the Cl^- availability) malate content in the swelling and shrinking motor cells of the laminar pulvinus. After sowing in soil, the laminar pulvinus develops within about 26 days. When the leaves emerge from the soil (about 6 days after sowing) and the pulvinus starts with the diurnal movement (about 9 days after sowing) the pulvinar dimensions are about half of those of the mature pulvinus. The anatomical structure, however, is basically the same as in the developed pulvinus. In soil-grown plants, the K⁺, Cl^- and malate content as well as the period length of the circadian leaf movement rhythm change in the developing pulvinus. In the embryo of the dry seed, the Cl^- content is low (0.03 mmol g^-1 DW), the K⁺ content, however, 22-fold higher than the Cl^- content. When the leaves emerge from the soil, the pulvinar K⁺ and Cl^- content is the same as in the whole embryo of the dry seed. In the developing pulvinus the K⁺ content increases by a factor of 2 and the Cl^- content by a factor of 41 in the mature pulvinus. The pulvinar malate content increases between the 6th and 10th day from about 40 to 180Μmol g^-1 DW, then decreases until the 17th day and remains thereafter on a low level (around 80 Μmol g^-1 DW). These results indicate that the Cl^- availability increases in the developing pulvinus with age. It explains furthermore why in young leaves malate was found as counterion to K⁺ in the osmotic leaf movement motor, in older ones, however, Cl^-. The circadian leaf movement starts 9 days after sowing. The period length decreases during the development of the pulvinus from 31.3 to 28.6 h in leaves of intact soil-grown plants. In leaves which were cut from the plants and immersed with their petioles in distilled water, the age dependent decrease of the period length is also found. However, the period lengths are shorter by more than 1 h than in the leaves of intact plants. The increasing Cl^- availability in the developing pulvinus does not seem to be the cause for the age dependent shortening of the period length, because the period length in 22 days old Cl^- deprived pulvini is the same as in 22 days old pulvini with a high Cl^- content.     

Adjustment of growth, starch turnover, protein content and central metabolism to a decrease of the carbon supply when Arabidopsis is grown in very short photoperiods

Arabidopsis was grown in a 12, 8, 4 or 3 h photoperiod to investigate how metabolism and growth adjust to a decreased carbon supply. There was a progressive increase in the rate of starch synthesis, decrease in the rate of starch degradation, decrease of malate and fumarate, decrease of the protein content and decrease of the relative growth rate. Carbohydrate and amino acids levels at the end of the night did not change. Activities of enzymes involved in photosynthesis, starch and sucrose synthesis and inorganic nitrogen assimilation remained high, whereas five of eight enzymes from glycolysis and organic acid metabolism showed a significant decrease of activity on a protein basis. Glutamate dehydrogenase activity increased. In a 2 h photoperiod, the total protein content and most enzyme activities decreased strongly, starch synthesis was inhibited, and sugars and amino acids levels rose at the end of the night and growth was completely inhibited. The rate of starch degradation correlated with the protein content and the relative growth rate across all the photoperiod treatments. It is discussed how a close coordination of starch turnover, the protein content and growth allows Arabidopsis to avoid carbon starvation, even in very short photoperiods.     

Influence of tamoxifen on gluconeogenesis and glycolysis in the perfused rat liver

The actions of tamoxifen, a selective estrogen receptor modulator used in chemotherapy and chemo-prevention of breast cancer, on glycolysis and gluconeogenesis were investigated in the isolated perfused rat liver. Tamoxifen inhibited gluconeogenesis from both lactate and fructose at very low concentrations (e.g., 5 μM). The opposite, i.e., stimulation, was found for glycolysis from both endogenous glycogen and fructose. Oxygen uptake was unaffected, inhibited or stimulated, depending on the conditions. Stimulation occurred in both microsomes and mitochondria. Tamoxifen did not affect the most important key-enzymes of gluconeogenesis, namely, phosphoenolpyruvate carboxykinase, pyruvate carboxylase, fructose 1,6-bisphosphatase and glucose 6-phosphatase. Confirming previous observations, however, tamoxifen inhibited very strongly NADH- and succinate-oxidase of freeze–thawing disrupted mitochondria. Tamoxifen promoted the release of both lactate dehydrogenase (mainly cytosolic) and fumarase (mainly mitochondrial) into the perfusate. Tamoxifen (200 μM) clearly diminished the ATP content and increased the ADP content of livers in the presence of lactate with a diminution of the ATP/ADP ratio from 1.67 to 0.79. The main causes for gluconeogenesis inhibition are probably: (a) inhibition of energy metabolism; (b) deviation of intermediates (malate and glucose 6-phosphate) for the production of NADPH required in hydroxylation and demethylation reactions; (c) deviation of glucosyl units toward glucuronidation reactions; (d) secondary inhibitory action of nitric oxide, whose production is stimulated by tamoxifen; (e) impairment of the cellular structure, especially the membrane structure. Stimulation of glycolysis is probably a compensatory phenomenon for the diminished mitochondrial ATP production. The multiple actions of tamoxifen at relatively low concentrations can represent a continuous burden to the overall hepatic functions during long treatment periods.