Energetic aspects of the light activation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase

The light energy requirements for photoactivation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase were studied in a reconstituted chloroplast system. This system comprised isolated pea thylakoids, ferredoxin (Fd), ferredoxin-thioredoxin reductase (FTR) thioredoxin_m and _f (Td_m, Td_f) and the photoactivatable enzyme. Light-saturation curves of the photoactivation process were established with once washed thylakoids which did not require the addition of Td for light activation. They exhibited a plateau at 10 W·m^–2 under nitrogen and 50 W·m^–2 under air, while NADP photoreduction was saturated at 240 W·m^–2. Cyclic and pseudocyclic phosphorylations saturated at identical levels as enzyme photoactivations. All these observations suggested that the shift of the light saturation plateau towards higher values under air was due to competing oxygen-dependent reactions. With twice washed thylakoids, which required Td for enzyme light-activation, photophosphorylation was stimulated under N₂ by the addition of the components of the photoactivation system. Its rate increased with increasing Td concentrations, just as did the enzyme photoactivation rate, while varying the target enzyme concentration had only a weak effect. Considering that Td concentrations were in a large excess over target enzyme concentrations, it may be assumed that the observed ATP synthesis was essentially dependent on the rate of Td reduction.Under air, Fd-dependent pseudo-cyclic photophosphorylation was not stimulated by the addition of the other enzyme photoactivation components, suggesting that an important site of action of O₂ was located at the level of Fd.Abbreviations Fd ferredoxin - FBPase fructose-1,6-bisphosphatase - FTR ferredoxin-thioredoxin reductase - LEM light effect mediator - NADP-MDH NADP-malate dehydrogenase - Td thioredoxin     

Molecular basis for the special properties of proteins and enzymes from Halobacterium marismortui

Abstract Three proteins from Halobacterium marismortui , malate dehydrogenase (hMDH), glutamate dehydrogenase (hGDH) and ferredoxin (hFD) were purified and characterized with respect to their molecular masses, amino acid composition and, for hFD only, primary structure. Striking features of halophilic proteins are: the high excess of acidic over basic residues; acidic clusters in the sequence. Low-salt concentration causes inactivation and changes in structural parameters of hMDH and hGDH. Reactivation of hMDH involves long-lived stable intermediates. The salt concentration optimum of enzymic activity is independent of salt nature. The high capacity of halophilic proteins to retain water and salt is due to unique molecular properties, studied by physico-chemical techniques.     

Developmental regulation of expression of the malate synthase gene in transgenic plants

The cucumber malate synthase (MS) gene, including 1856 bp of 5 non-trnascribed sequence, has been transferred into Petunia (Mitchell) and Nicotiana plumbaginifolia plants using an Agrobacterium binary vector. The transferred gene is found in variable copy number in different transformants, and is stably transmitted in each case as a single Mendelian character. Transgene mRNA accumulates in the seedling during the first three days of germination, then declines in amount as the cotyledons emerge from the seed. The decline is more pronounced in light-grown seedlings than in dark-grown seedlings. Expression of the MS transgene is also detected at a low level in petals of transformed Petunia plants. In these respects the pattern of MS gene expression is similar in cucumber and in trnasformed plants, showing that the transferred DNA fragment contains a functional MS gene. A 1076 bp fragment of 5 sequence was linked to the -glucuronidase reporter gene and transferred into Nicotiana, where it was shown to direct temporal and spatial patterns of expression similar to that of the complete MS gene. However, histochemical localisation of -glucuronidase activity demonstrated that the chimaeric gene is expressed not only in cotyledons of transgenic plants, but also in endosperm and some hypocotyl cells during early germination. The relevance of these findings to the control of malate synthase gene expression is discussed.     

Anaerobic energy metabolism in the oligochaeteLumbriculus variegatus Müller

Summary Anoxia tolerance, glycogen degradation, free amino acid pool, adenylate energy charge and the accumulation and excretion of end products were monitored inLumbriculus variegatus Müller throughout 48 h of anoxia. A transition period lasting about 4 h could be distinguished from subsequent events during which malate, present in high amounts in the resting animals, is utilized, probably by conversion to succinate. Up to the 12th hour of anoxia there is an increase in concentration of free amino acids, except aspartate. Glutamate increases rapidly during the first half hour but decreases thereafter. Beginning with the second hour of anoxia the alanine concentration increases at the same rate glutamate concentration decreases, but the source of nitrogen during the first hour is unknown. It is argued that the nitrogen required for the synthesis of some of the amino acids is ultimately derived from proteolysis. After about 3 h of anoxia propionate and acetate are synthesized. At first these acids accumulate in the tissues, but after 4–6 h they are excreted into the surrounding medium. Acetate is excreted over the whole experimental period at a constant rate, whereas the excretion rate of propionate decreases slowly with time. The propionate/acetate ratio is in excess of 2. Classic malate dismutation is by far the most important mechanism in the maintenance of redox balance. Depletion of glycogen stores appears to play an important role in determining anoxic survival time. Due to extremely low activity of PEPCK the ratio of the specific activities of PK and PEPCK is very high. Further, the kinetic properties of pyruvate kinase do not support the assumption of a shift of the glycolytic carbon flow at the PEP level.Abbreviations PK Pyruvate kinase - PEPCK phosphoenolpyruvate carboxykinase - PEP phospho(enol)pyruvate - FBP fructose-1,6-bisphosphate - AEC adenylate energy charge - EMP-scheme Embden-Meyerhof-Parnas scheme of glycolysis - f _w fresh body weight - dw dry body weight     

The Compatibility of d-Pinitol and 1d-1-O-Methyl-Muco-Inositol with Malate Dehydrogenase Activity

Pinitol (1d -3-O-methyl-chiro-inositol) and 1d -1-O-methyl-muco-inositol, two cyclitols wide-spread in the plant kingdom, were isolated from plant sources in order to test their compatibility with malate dehydrogenase activity. Both compounds had no inhibitory effect on malate dehydrogenase from Rhizophora mangle in a range of 100 to 1000 mol . m^?3. Their influence on malate dehydrogenase activity from different plant sources (Rh. mangle L., Mesembryanthemum crystallinum L., Cicer arietinum L. and Spinacia oleracea L.) was also small and similar to that observed for a number of well established compatible solutes (e.g. proline, glycine betaine). A possible role of cyclitols as cryoprotectants or radical scavengers is discussed.     

Influence of mechanical impedance on root exudation of maize seedlings at two development stages

Studies were undertaken to evaluate the effects of mechanical impedance on root exudation by maize (Zea mays L., var Dea) and to examine the importance of these effects in relation to the stage of plant development. Plants were grown under sterile and hydroponic conditions. Mechanical impedance was simulated using glass beads of 1 mm diameter. This treatment was compared with a control without beads. Results demonstrated that plant growth was influenced by mechanical impedance. Mechanical impedance markedly affected the growth of the shoot, whether this was measured as leaf area or total dry matter. Besides increasing root/shoot biomass ratios, mechanical impedances also stimulated root exudation of organic and inorganic compounds. Stressed plants lost more nitrogenous compounds than control plants. Otherwise, the percentage of released carbon decreased. Depending on the developmental stage of the plant, there was a large variation in the magnitude and time course on mechanical impedance effects. The effects of mechanical impedance persist and accentuate with time.     

Rhizodeposition under ambient and elevated CO2 levels

As global CO₂ levels rise, can soils store more carbon and so buffer atmospheric CO₂ levels? Answering this question requires a knowledge of the rates of C inputs to soil and of CO₂ outputs via decomposition. Below-ground inputs from roots are a major component of the C flow into soils but are still poorly understood. In this article, new techniques for measuring rhizodeposition are reviewed and discussed and the need for cross-comparisons between methods is identified. One component of rhizodeposition, root exudation, is examined in more detail and evidence is presented which suggests that current estimates of exudate flow into soils are incorrect. A mechanistic mathematical model is used to explore how exudate flows might change under elevated CO₂.     

Amino acid concentrations and selected enzyme activities in rat auditory,olfactory, and visual systems

Homogenates of specific brain regions of three sensory systems (auditory, olfactory, and visual) were prepared from pigmented Long-Evans Hooded rats and assayed for amino acid concentrations and activities of glutaminase, aspartate aminotransferase (total, cytosolic, and, by difference, mitochondrial), malate dehydrogenase, lactate dehydrogenase, and choline acetyltransferase. Comparing the quantitative distributions among regions revealed significant correlations between AAT and aspartate, between glutaminase and glutamate, between glutamate and glutamine, and between AAT plus glutaminase, or glutaminase alone, and the sum of aspartate, glutamate, and GABA, suggesting a metabolic pathway involving the synthesis of a glutamate pool as precursor to aspartate and GABA. Of the inhibitory transmitter amino acids, GABA concentrations routinely exceeded those of glycine, but glycine concentrations were relatively high in brainstem auditory structures.     

Genetics and polymorphism of a duplicated malate dehydrogenase (MDH) locus in the grasshopperOxya japonica japonica (Orthoptera:Acrididae:Oxyinae)

A biochemical genetic study of the enzyme malate dehydrogenase (MDH) was conducted in the grasshopperOxya j. japonica. Analysis of MDH electrophoretic variation in this species of grasshopper shows that one of the two autosomal loci for MDH in grasshoppers, the Mdh-2 locus, controlling the anodal set of MDH isozymes, is duplicated. Results of breeding studies confirm this and the observed polymorphism at theMdh-2 locus in the two populations ofOxya j. japonica studied can be attributed to three forms of linked alleles at the duplicated locus in equilibrium in both populations. In this respect, all individuals of this species possess heterozygous allelic combinations at the duplicatedMdh-2 locus, which may account for the spread of the duplicated locus in the populations of this species of grasshopper.This research was supported by a grant (Vote F) from the University of Malaya, Kuala Lumpur.