Nicotinamide Adenine Dinucleotide Phosphate-malic Enzyme Research Articles

C4 Equivalent Decarboxylation Competence in Tropical Orchids

Tropical xero-epiphytic orchids undergo a photosynthetic mechanism called ‘Crassulacean Acid Metabolism’, a modification of Calvin–Benson cycle. This anabolic process aids in the long-term retention of organically fixed CO2, imprinting them as active sequestrators and fixers of gaseous CO2. The malate decarboxylation competence of enzymes—phosphoenolpyruvate carboxykinase (PEPCK), Nicotinamide Adenine Dinucleotide malic enzyme (NAD-ME) and Nicotinamide Adenine Dinucleotide Phosphate malic enzyme (NADP-ME) in xero-epiphytic orchids that concentrate CO2 by employing RuBisCO to embark on a carboxylation route is defined in this paper. Physiological parameters like photosynthetic rates, and quantum yield, accompanied by histomorphometry were analysed. Our study indicated PEPCK as the dominant decarboxylating enzyme in tropical orchids. The efficiency of PEPCK was reinforced by two additional malic enzymes, which are dependent on cofactors, namely Nicotinamide Adenine Dinucleotide and Nicotinamide Adenine Dinucleotide Phosphate. These results indicated the need to appraise the potential of tropical orchids as strategic plant contenders for CO2 triggered greenhouse effect mitigator.

Physiological responses of sub1A QTL under induced dehydration stress for varying days in rice

This study analysed the rice genotype with sub1A quantitative trait loci that may interact with ongoing exposure of dehydration. cv. Swarna Sub1 had more nutrients efficiency with increased membrane permeability than cv. Swarna. sub1A QTL with its expression to water / osmotic deficit, however, indirectly to impede the oxidative stress under dehydration might help cv. Swarna Sub1. At maximum dehydration cv. Swarna Sub1 had 1.12 fold excess electrolyte leakages than cv. Swarna under dehydration. cv. Swarna Sub1 had better Nicotinamide adenine dinucleotide phosphate-malic enzyme activity to secure carbon dioxide exchange. A proportional increase in enzyme activity all through dehydration stress maximize under light in cv. Swarna Sub1. At maximum dehydration cv. Swarna Sub1 at saturating substrate concentration was increased by 1.12 fold than other cultivar. The ratio of glutathione (GSH:GSSG) more depleted in cv. Swarna Sub1 through the dehydration period. cv. Swarna could be more promising to retrieve the activity by 1.80 fold than cv. Swarna Sub1 under maximum dehydration. Dehydroascorbate reductase activity was also maintained in cv. Swarna with 1.20 fold ahead than cv. Swarna Sub1 under same condition. As a biomarker of oxidative stress cv. Swarna Sub1 appeared to be less sensitive with the loss of protein oxidation, however, recorded with 25% less carbonyl content than cv. Swarna. Both the genotypes had scored a significant sensitivity with tissue specific distribution for reactive oxygen species as detected by histochemical assay.

Changes in secondary metabolites, organic acids and soluble sugars during the development of plum fruit cv. 'Furongli' (Prunus salicina Lindl).

Organic acids, sugars and pigments are key components that determine the taste and flavor of plum fruit. However, metabolism of organic acid and sugar is not fully understood during the development of plum fruit cv. 'Furongli'. Mature fruit of 'Furongli' has the highest content of anthocyanins and the lowest content of total phenol compounds and flavonoids. Malate is the predominant organic acid anion in 'Furongli' fruit, followed by citrate and isocitrate. Glucose was the predominant sugar form, followed by fructose and sucrose. Correlation analysis indicated that malate content increased with increasing phosphoenolpyruvate carboxylase (PEPC) activity and decreasing nicotinamide adenine dinucleotide-malate dehydrogenase (NAD-MDH) activity. Citrate and isocitrate content increased with increasing PEPC and aconitase (ACO) activities, respectively. Both acid invertase and neutral invertase had higher activities at the early stage than later stage of fruit development. Fructose content decreased with increasing phosphoglucoisomerase (PGI) activity, whereas glucose content increased with decreasing hexokinase (HK) activity. Dynamics in organic acid anions were not solely controlled by a single enzyme but regulated by the integrated activity of enzymes such as nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME), NAD-ME, PEPC, ACO and NADP-isocitrate dehydrogenase. Sugar metabolism enzymes such as PGI, invertase and HK may play vital roles in the regulation of individual sugar metabolic processes. © 2018 Society of Chemical Industry.

Temperature responses of Rubisco from Paniceae grasses provide opportunities for improving C3 photosynthesis.

Enhancing the catalytic properties of the CO2-fixing enzyme Rubisco is a target for improving agricultural crop productivity. Here, we reveal extensive diversity in the kinetic response between 10 and 37 °C by Rubisco from C3 and C4 species within the grass tribe Paniceae. The CO2 fixation rate (kcatc) for Rubisco from the C4 grasses with nicotinamide adenine dinucleotide (NAD) phosphate malic enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase (PCK) photosynthetic pathways was twofold greater than the kcatc of Rubisco from NAD-ME species across all temperatures. The declining response of CO2/O2 specificity with increasing temperature was less pronounced for PCK and NADP-ME Rubisco, which would be advantageous in warmer climates relative to the NAD-ME grasses. Modelled variation in the temperature kinetics of Paniceae C3 Rubisco and PCK Rubisco differentially stimulated C3 photosynthesis relative to tobacco above and below 25 °C under current and elevated CO2. Amino acid substitutions in the large subunit that could account for the catalytic variation among Paniceae Rubisco are identified; however, incompatibilities with Paniceae Rubisco biogenesis in tobacco hindered their mutagenic testing by chloroplast transformation. Circumventing these bioengineering limitations is critical to tailoring the properties of crop Rubisco to suit future climates.

Variations in nitrogen use efficiency reflect the biochemical subtype while variations in water use efficiency reflect the evolutionary lineage of C4 grasses at inter-glacial CO2.

C4 photosynthesis evolved multiple times in diverse lineages. Most physiological studies comparing C4 plants were not conducted at the low atmospheric CO2 prevailing during their evolution. Here, 24 C4 grasses belonging to three biochemical subtypes [nicotinamide adenine dinucleotide malic enzyme (NAD-ME), phosphoenolpyruvate carboxykinase (PCK) and nicotinamide adenine dinucleotide phosphate malic enzyme (NADP-ME)] and six major evolutionary lineages were grown under ambient (400 μL L(-1) ) and inter-glacial (280 μL L(-1) ) CO2 . We hypothesized that nitrogen-related and water-related physiological traits are associated with subtypes and lineages, respectively. Photosynthetic rate and stomatal conductance were constrained by the shared lineage, while variation in leaf mass per area (LMA), leaf N per area, plant dry mass and plant water use efficiency were influenced by the subtype. Subtype and lineage were equally important for explaining variations in photosynthetic nitrogen use efficiency (PNUE) and photosynthetic water use efficiency (PWUE). CO2 treatment impacted most parameters. Overall, higher LMA and leaf N distinguished the Chloridoideae/NAD-ME group, while NADP-ME and PCK grasses were distinguished by higher PNUE regardless of lineage. Plants were characterized by high photosynthesis and PWUE when grown at ambient CO2 and by high conductance at inter-glacial CO2 . In conclusion, the evolutionary and biochemical diversity among C4 grasses was aligned with discernible leaf physiology, but it remains unknown whether these traits represent ecophysiological adaptation.

Photosynthetic flexibility in maize exposed to salinity and shade

C4 photosynthesis involves a close collaboration of the C3 and C4 metabolic cycles across the mesophyll and bundle-sheath cells. This study investigated the coordination of C4 photosynthesis in maize plants subjected to two salinity (50 and 100mM NaCl) treatments and one shade (20% of full sunlight) treatment. Photosynthetic efficiency was probed by combining leaf gas-exchange measurements with carbon isotope discrimination and assaying the key carboxylases [ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC)] and decarboxylases [nicotinamide adenine dinucleotide phosphate malic enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase (PEP-CK)] operating in maize leaves. Generally, salinity inhibited plant growth and photosynthesis to a lesser extent than shade. Salinity reduced photosynthesis primarily by reducing stomatal conductance and secondarily by equally reducing Rubisco and PEPC activities; the decarboxylases were inhibited more than the carboxylases. Salinity increased photosynthetic carbon isotope discrimination (Δp) and reduced leaf dry-matter carbon isotope composition ((13)δ) due to changes in p i/p a (intercellular to ambient CO2 partial pressure), while CO2 leakiness out of the bundle sheath (ϕ) was similar to that in control plants. Acclimation to shade was underpinned by a greater downregulation of PEPC relative to Rubisco activity, and a lesser inhibition of NADP-ME (primary decarboxylase) relative to PEP-CK (secondary decarboxylase). Shade reduced Δp and ɸ without significantly affecting leaf (13)δ or p i/p a relative to control plants. Accordingly, shade perturbed the balance between the C3 and C4 cycles during photosynthesis in maize, and demonstrated the flexible partitioning of C4 acid decarboxylation activity between NADP-ME and PEP-CK in response to the environment. This study highlights the need to improve our understanding of the links between leaf (13)δ and photosynthetic Δp, and the role of the secondary decarboxylase PEP-CK in NADP-ME plants such as maize.

Leaf photosynthesis and carbohydrates of CO 2-enriched maize and grain sorghum exposed to a short period of soil water deficit during vegetative development

Among C 4 species, sorghum is known to be more drought tolerant than maize. The objective was to evaluate differences in leaf gas exchanges, carbohydrates, and two enzyme activities of these nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME) C 4 subtype monocots in response to water deficit and CO 2 concentration ([CO 2]). Maize and sorghum were grown in pots in sunlit environmental-controlled chambers. Treatments included well watered (WW) and water stressed (WS) (water withheld at 26 days) and daytime [CO 2] of 360 (ambient) and 720 (elevated) μmol mol −1. Midday gas exchange rates, concentrations of nonstructural carbohydrates, and activities of sucrose-phosphate synthase (SPS) and adenosine 5′-diphosphoglucose pyrophosphorylase (ADGP) were determined for fully expanded leaf sections. There was no difference in leaf CO 2 exchange rates (CER) between ambient and elevated [CO 2] control plants for both maize and sorghum. After withholding water, leaf CER declined to zero after 8 days in maize and 10 days for sorghum. Sorghum had lower stomatal conductance and transpiration rates than maize, which resulted in a longer period of CER under drought. Nonstructural carbohydrates of both control maize and sorghum were hardly affected by elevated [CO 2]. Under drought, however, increases in soluble sugars and decreases in starch were generally observed for maize and sorghum at both [CO 2] levels. For stressed maize and sorghum, decreases in starch occurred earlier and were greater at ambient [CO 2] than at elevated [CO 2]. For maize, drought did not meaningfully affect SPS activity. However, a decline in SPS activity was observed for drought-stressed sorghum under both [CO 2] treatments. There was an increase in ADGP activity in maize under drought for both [CO 2] treatments. Such a response in ADGP to drought, however, did not occur for sorghum. The generally more rapid response of maize than sorghum to drought might be related to the more rapid growth of leaf area of maize.

Evolution of C4 Phosphoenolpyruvate Carboxykinase in Grasses, from Genotype to Phenotype

C(4) photosynthesis is an adaptation over the classical C(3) pathway that has evolved multiple times independently. These convergences are accompanied by strong variations among the independent C(4) lineages. The decarboxylating enzyme used to release CO(2) around Rubisco particularly differs between C(4) species, a criterion used to distinguish three distinct biochemical C(4) subtypes. The phosphoenolpyruvate carboxykinase (PCK) serves as a primary decarboxylase in a minority of C(4) species. This enzyme is also present in C(3) plants, where it is responsible for nonphotosynthetic functions. The genetic changes responsible for the evolution of C(4)-specific PCK are still unidentified. Using phylogenetic analyses on PCK sequences isolated from C(3) and C(4) grasses, this study aimed at resolving the evolutionary history of C(4)-specific PCK enzymes. Four independent evolutions of C(4)-PCK were shown to be driven by positive selection, and nine C(4)-adaptive sites underwent parallel genetic changes in different C(4) lineages. C(4)-adaptive residues were also observed in C(4) species from the nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME) subtype and particularly in all taxa where a PCK shuttle was previously suggested to complement the NADP-ME pathway. Acquisitions of C(4)-specific PCKs were mapped on a species tree, which revealed that the PCK subtype probably appeared at the base of the Chloridoideae subfamily and was then recurrently lost and secondarily reacquired at least three times. Linking the genotype to subtype phenotype shed new lights on the evolutionary transitions between the different C(4) subtypes.

Identification of cold acclimation‐responsiveRhododendrongenes for lipid metabolism, membrane transport and lignin biosynthesis: importance of moderately abundant ESTs in genomic studies

We have previously analysed expressed sequence tags (ESTs) from non-acclimated (NA) and cold-acclimated (CA) Rhododendron leaves, and identified highly abundant complementary DNAs (cDNAs) possibly involved in cold acclimation. A potentially significant, but relatively unexplored, application of these EST data sets is the study of moderately abundant cDNAs, such as those picked only 1-3 times from each Rhododendron EST library containing approximately 430 ESTs. Using statistical tests and Northern blots, we established that the probability of differential expression of moderately abundant cDNAs based on the EST data is, indeed, a reasonably accurate predictor of their 'true' upregulation or downregulation as 11 out of 13 cDNAs (85%) studied fit this criterion. The analyses also revealed four aspects of cold acclimation in Rhododendron leaf tissues. Firstly, the concomitant upregulation of long-chain acyl-coenzyme A (acyl-CoA) synthetase, CTP:cholinephosphate cytidylyltransferase and delta-12 fatty acid desaturase in CA leaf tissues suggests that phospholipid biosynthesis and desaturation are important components of cold hardening in Rhododendron. Secondly, upregulation of plastidic nicotinamide adenine dinucleotide phosphatemalic enzyme (NADP-ME) in CA tissues suggests that malate is an important source of acetyl-CoA used for fatty acid biosynthesis during cold acclimation. Thirdly, down-regulation of plasma membrane intrinsic protein (PIP)2-1 aquaporin and upregulation of gated outward rectifying K+ channel (GORK) in CA tissues may be associated with the protection of overwintering leaves from freeze-induced cellular dehydration. Fourthly, upregulation of coumarate 3-hydroxylase may be associated with cell wall thickening in CA tissues. Physiological implications of these results, which reveal potentially novel regulations of cold acclimation in overwintering woody evergreens, are discussed. This work highlights the importance of also investigating low/moderately abundant ESTs (in addition to highly abundant ones) in genomic studies, in that it offers an effective strategy for identifying stress-related genes, especially when large-scale cDNA sequencing/microarray studies are not possible.

Development of biochemical specialization and organelle partitioning in the single‐cell C4 system in leaves of Borszczowia aralocaspica (Chenopodiaceae)

The terrestrial plant Borszczowia aralocaspica (Chenopodiaceae) has recently been shown to contain the entire C(4) photosynthesis mechanism within individual, structurally and biochemically polarized chlorenchyma cells rather than in a dual cell system, as has been the paradigm for this type of carbon fixation (Nature 414: 543-546, 2001). Analysis of carbon isotope composition and (14)CO(2) fixation shows that photosynthesis and growth of B. aralocaspica occurs through carbon acquired by C(4) photosynthesis. The development of this unique single-cell C(4) system in chlorenchyma cells was studied by analysis of young (0.2-0.3 cm length), intermediate (ca. 0.5-0.6 cm length), and mature leaves (ca. 3 cm length). The length of chlorenchyma cells approximately doubles from young to intermediate and again from intermediate to the mature leaf stage. In young chlorenchyma cells, there is a single type of chloroplast; the chloroplasts are evenly distributed throughout the cytosol, and all contain starch and rubisco. During leaf development, the activities of phosphoenolpyruvate carboxylase (PEPC; which is cytosolic), rubisco, and pyruvate,Pi dikinase (PPDK) increase on a chlorophyll basis. As leaves mature, chloroplasts differentiate into two distinct structural and biochemical types that are spatially separated into the proximal and distal parts of the cell (the proximal end being closest to the center of the leaf). The early stages of this polarization are observed in intermediate leaves, and the polarization is fully developed in mature leaves. The chloroplasts in the distal ends of the cell have reduced grana and little starch, while those at the proximal ends have well-developed grana and abundant starch. In mature leaves, PPDK is expressed in chloroplasts at the distal end of the cells, while rubisco and adenosine diphosphate glucose (ADPG) pyrophosphorylase are selectively expressed in chloroplasts at the proximal end of the cell. Mitochondrial polarization also occurs during development as nicotinamide-adenine dinucleotide phosphate-malic enzyme (NAD-ME) and the photorespiratory enzyme glycine decarboxylase are expressed in mature but not young leaves and are localized in mitochondria at the proximal end of the cells. The data show that single-cell C(4) develops from a single pool of identical organelles that develop differential biochemical functions and spatial partitioning in the cell during maturation.

Growth responses to elevated CO2 in NADP-ME, NAD-ME and PCK C4 grasses and a C3 grass from South Africa

The potential use of C4 biochemical subtypes [nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME), nicotinamide adenine dinucleotide-malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PCK)] as delimiters of plant functional types (PFTs) with distinct responses to rising atmospheric CO2 concentrations was investigated in South African grass species. Gas exchange and above-ground growth in ambient and elevated CO2 (360 and 660 µmol mol–1 , respectively) were determined in three NADP-ME species, two NAD-ME species, two PCK species and one C3 species, all excavated from the same field site. Plants were grown in open-top chambers in a greenhouse for 178 d. Net CO2 assimilation rates were only significantly increased in one NAD-ME species, but stomatal conductances decreased (in six out of eight species, by a mean of 46%) and instantaneous leaf water-use efficiency increased (in all species, by a mean of 89%) in elevated CO2. These responses did not differ between photosynthetic pathways. Parameters derived from photosynthetic CO2 and light response curves were also not differentially influenced by CO2 treatment between pathways. Gas exchange responses were generally poorly related to CO2 responsiveness. Significant increases in leaf growth and canopy leaf area in elevated CO2 were found in two NADP-ME species, whereas increases in non-leaf above-ground growth were measured in three species representing all three C4 subtypes. Growth responses in elevated CO2 were apparently not simply correlated with biochemical subtype characteristics, although the most significant responses (particularly at the leaf level) were found for the NADP-ME pathway. This result was more likely attributable to the significant positive correlation found between CO2 responsiveness of leaf growth and relative leaf regrowth potential of individual species, the latter being higher in the two responsive NADP-ME species. Therefore, categorisation of PFTs according to relative growth potential may be more appropriate for predictions of CO2 responsiveness in C4 grasses.

Differential Photosynthetic Acclimation Pattern to Limiting Growth-Irradiance in Two Types of C<sub>4</sub> Plants

Photosynthetic acclimation to reduced growth irradiances (650 and 200 µmol m-2 s-1) in Eleusine coracana (L.) Garten, a nicotinamide adenine dinucleotide-malic enzyme (NAD-ME) C4 species and Gomphrena globosa L., a nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME) C4 species were investigated. E. coracana plants acclimated in 4 and 8 d to 650 and 200 µmol m-2 s-1, respectively, whereas G. globosa plants took 8 and 10 d, respectively, to acclimate to the same irradiances. The acclimation to reduced irradiance was achieved in both species by greater partitioning of chlorophyll towards the light-harvesting antennae at the expense of functional components. However, magnitude of increase in the light-harvesting antenna was higher in E. coracana as compared to G. globosa. Superior photosynthetic acclimation to reduced irradiance in G. globosa was due to the smaller change in functions of the cytochrome b 6/f complex, photosystem (PS) 1 and PS2 leading to the higher carbon fixation rates compared to E. coracana.

Regulation of the expression of NADP-malic enzyme by UV-B, red and far-red light in maize seedlings.

The induction of nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME) in etiolated maize (Zea mays) seedlings by UV-B and UV-A radiation, and different levels of photosynthetically active radiation (PAR, 400-700 nm) was investigated by measuring changes in activity, protein quantity and RNA levels as a function of intensity and duration of exposure to the different radiations. Under low levels of PAR, exposure to UV-B radiation but not UV-A radiation for 6 to 24 h caused a marked increase in the enzyme levels similar to that observed under high PAR in the absence of UV-B. UV-B treatment of green leaves following a 12-h dark period also caused an increase in NADP-ME expression. Exposure to UV-B radiation for only 5 min resulted in a rapid increase of the enzyme, followed by a more gradual rise with longer exposure up to 6 h. Low levels of red light for 5 min or 6 h were also effective in inducing NADP-ME activity equivalent to that obtained with UV-B radiation. A 5-min exposure to far-red light following UV-B or red light treatment reversed the induction of NADP-ME, and this effect could be eliminated by further treatment with UV-B or red light. These results indicate that physiological levels of UV-B radiation can have a positive effect on the induction of this photosynthetic enzyme. The reducing power and pyruvate generated by the activity of NADP-ME may be used for respiration, in cellular repair processes and as substrates for fatty acid synthesis required for membrane repair.

Thiol-Dependent Regulation of Glycerate Metabolism in Leaf Extracts

We have recently reported that the activity of maize leaf glycerate kinase [EC 2.7.1.31] is regulated in vivo by the light/dark transition, possibly involving the ferredoxin/thioredoxin mechanism, and that the stimulating effect of light can be mimicked in vitro by incubation of crude leaf extract with reducing compounds (LA Kleczkowski, DD Randall 1985 Plant Physiol 79: 274-277). In the present study it was found that the time course of thiol activation of the enzyme was substantially dependent on the presence of some low molecular weight inhibitor(s) of activation found both in leaf extracts and mesophyll chloroplasts. Activity of glycerate kinase from maize as well as wheat leaves increased upon greening of etiolated plants and was correlated with the development of photosynthetic apparatus in these species. The maize enzyme was strongly activated by thiols at all stages of development from etiolated to green seedlings. Thiol activation of glycerate kinase was observed for a number of C(4) plants, notably of the nicotinamide adenine dinucleotide phosphate-malic enzyme type, with the strongest effect found for the enzyme from leaf extracts of maize and sorghum (10- and 8-fold activation, respectively). Among the C(3) species tested, only the enzyme from soybean leaves was affected under the same conditions (1.6-fold activation). This finding was reflected by an apparent lack of cross-reactivity between the enzyme from maize leaves and antibodies raised against purified spinach leaf glycerate kinase. We suggest that, in addition to its role as a final step of photorespiration in leaves, glycerate kinase from C(4) species may serve as a part of the facilitative diffusion system for the intercellular transport of 3-phosphoglycerate. Simultaneous operation of both the passive and the facilitative diffusion mechanisms of 3-phosphoglycerate transport in C(4) plants is postulated.

The Involvement of Aspartate and Glutamate in the Decarboxylation of Malate by Isolated Bundle Sheath Chloroplasts from Zea mays

Aspartate or glutamate stimulated the rate of light-dependent malate decarboxylation by isolated Zea mays bundle sheath chloroplasts. Stimulation involved a decrease in the apparent K(m) (malate) and an increased maximum velocity of decarboxylation. In the presence of glutamate other dicarboxylates (succinate, fumarate) competitively inhibited malate decarboxylation by intact chloroplasts with respect to malate with an apparent K(i) of about 6 millimolar. For comparison the K(i) for inhibition of nicotinamide adenine dinucleotide phosphate-malic enzyme from freshly lysed chloroplasts by these dicarboxylates was 15 millimolar. A range of compounds structurally related to aspartate stimulated malate decarboxylation by intact chloroplasts. K(a) values for stimulation at 5 millimolar malate were 1.7, 5, and 10 millimolar for l-glutamate, l-aspartate, and beta-methyl-dl-aspartate, respectively. Certain compounds, notably cysteic acid, which stimulated malate decarboxylation by intact chloroplasts inhibited malate decarboxylation by nicotinamide adenine dinucleotide phosphate-malic enzyme obtained from lysed chloroplasts and assayed under comparable conditions. It was concluded that aspartate, glutamate, and related compounds affect the transport of malate into the intact chloroplasts and that malate translocation does not take place on the general dicarboxylate translocator previously reported for higher plant chloroplasts.

Nicotinamide adenine dinucleotide phosphate-malic enzyme of rat liver. Purification, properties, and immunochemical studies.

Rat liver malic enzyme (EC 1.1.1.40) was purified from livers of rats fasted and refed a high sucrose diet containing 1% desiccated thyroid powder. The purification was accomplished by a six-step procedure. The specific activity of the purified enzyme was increased 181-fold above that of the initial high speed supernatant of liver extracts. Slight additional purification of malic enzyme was achieved with preparative disc electrophoresis. The specific activities of the purified rat liver malic enzyme from the least two steps were between 28.0 and 30.5 units per mg of protein. Homogeneity of the purified enzyme was determined by disc and starch gel electrophoresis as well as sedimentation velocity and sedimentation equilibrium studies. The molecular weight and S20, w values of rat liver malic enzyme are 268,000 and 10.2, respectively. Amino acid analysis based on milligram of protein hydrolyzed yielded higher amounts of leucine and glutamic acid but lower quantities of alanine and voline per subunit than the corresponding Escherichia coli enzyme...