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PubMed1996

2'-Phospho-cyclic ADP-ribose, a calcium-mobilizing agent derived from NADP.

Cyclic adenosine diphosphoribose (cADPR), a metabolite of NAD, appears to modulate changes in intracellular free Ca2+ levels by activation of ryanodine-sensitive Ca2+ channels. We report here that an ADPR cyclase purified from Aplysia californica readily catalyzes the conversion of NADP to 2'-phospho-cyclic adenosine diphosphoribose (2'-P-cADPR), cyclized at N-1 of the adenine moiety. An enzyme from canine spleen previously shown to contain NAD glycohydrolase, ADPR cyclase, and cADPR hydrolase activities also utilized NADP and 2'-P-cADPR as substrates. The apparent Km value for NADP was 1.6 microM compared with 9.9 microM for NAD, and the Vmax with NADP was twice that with NAD, indicating that 2'-P-cADPR is a likely metabolite in mammalian cells. 2'-P-cADPR was as active as cADPR in eliciting Ca2+ release from rat brain microsomes, but was unable to elicit Ca2+ release following conversion to 2'-P-ADPR by the action of canine spleen NAD glycohydrolase. 2'-P-cADPR and 1-D-myo-inositol 1,4,5-trisphosphate (IP3) appear to act by distinct mechanisms as microsomes desensitized to IP3 still released Ca2+ in response to 2'-P-cADPR and vice versa. Also, inhibition of IP3-induced Ca2+ release by heparin had no effect on release by 2'-P-cADPR. Both 2'-P-cADPR and cADPR appear to act by a similar mechanism based on similar kinetics of Ca2+ release, similar dose-response curves, cross-desensitization, and partial inhibition of release by procaine. The results of this study suggest that 2'-P-cADPR may function as a new component of Ca2+ signaling and a possible link between NADP metabolism and Ca2+ homeostasis.

ADP-ribosyl Cyclase↗
PubMed1998

The NADP-reducing hydrogenase of Desulfovibrio fructosovorans: evidence for a native complex with hydrogen-dependent methyl-viologen-reducing activity.

The NADP-reducing hydrogenase of Desulfovibrio fructosovorans represents a novel class of [Fe] hydrogenases which is encoded by the well-characterized hndABCD operon containing the genes hndA, hndB, hndC, and hndD. Expression of this operon, monitored by measuring the NADP-reducing activity, was found to be maximum during the exponential phase of growth on fructose and then decreased when the concentration of the carbon and energy source became limiting. The optimum pH for the H2-driven NADP reduction was 8, and the apparent K(m) and Vmax were determined to be 0.09 mM and 13 x 10(-3) u/mg, respectively. Heterologous expression of the hnd genes in Escherichia coli was carried out to raise antisera against the different subunits of the NADP-reducing hydrogenase. The antisera were used to detect the four subunits in cell extract of D. fructosovorans after separation by SDS- and native PAGE. The four subunits of the NADP-reducing hydrogenase were demonstrated to be associated in a complex which exhibited H2-driven methyl viologen reduction. Furthermore, on native gel, a form lacking HndD, with no hydrogen-dependent methyl viologen reductase activity was also shown to be present in D. fructosovorans.

Bacterial Proteins↗
PubMed1996

Expression of pig heart mitochondrial NADP-dependent isocitrate dehydrogenase in Escherichia coli.

Pig heart mitochondrial NADP-specific isocitrate dehydrogenase is the most extensively studied among the mammalian isocitrate dehydrogenases. The 1.2-kbp cDNA encoding this porcine mitochondrial NADP-specific enzyme has now been inserted into an expression vector, pMAL-c2, to be expressed as a fusion protein with maltose binding protein. Initially, the vector was constructed with a cleavage site for protease Factor Xa between the maltose binding protein and isocitrate dehydrogenase; however, since Factor Xa was also found to digest isocitrate dehydrogenase, a thrombin recognition site was substituted. The fusion protein was expressed in Escherichia coli by IPTG induction at 25 degrees C, and was separated from the endogenous E. coli isocitrate dehydrogenase by affinity chromatography on an amylose resin which adsorbs maltose binding protein and its fusion products. Cleavage of the fusion protein with thrombin generated pig heart NADP-specific isocitrate dehydrogenase, which was purified to homogeneity by affinity chromatography on Matrex Gel Red-A resin and gel filtration by FPLC. A 41-fold increase in specific activity to 37 enzyme units/mg with an approximate yield of 34% for the expressed enzyme was achieved by this purification procedure. This enzyme exhibits a single band (M(r) = 46,600) on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and, under standard assay conditions, has a Km for DL-isocitrate of 7.74 +/- 0.18 microM and a Km for NADP+ of 6.63 +/- 1.34 microM. These values are similar to the Kms measured for the enzyme purified from pig heart. The amino-terminal sequence of the expressed enzyme is identical with that of authentic porcine enzyme and distinguishable from the E. coli enzyme at 17 of the 18 residues determined. We conclude that this expression and purification system yields pure pig heart mitochondrial NADP-specific isocitrate dehydrogenase and should allow generation of wild-type and mutant enzymes in amounts suitable for their biochemical characterization and comparison.

ATP-Binding Cassette Transporters↗
PubMed1979

Ontogeny, cell distribution, and the physiological role of NADP-malic enxyme in Drosophila melanogaster.

NADP-malic enzyme (NADP-ME) (E.C. 1.1.1.40) is situated in the cytosol of Drosophila melanogaster. Both the tissue activity and CRM level of NADP-ME parallel changes in the dosage of a gene, Men+, located in region 87C2-3 to 87D1-2 of the third chromosome. The tissue activity of NADP-ME is very high in early third instar larvae, providing about 33% of the NADPH at this life stage. The tissue activity declines during pupal development but increases as the adult ages. The concentration of NADP-ME CRM and tissue activity are coordinately increased in third instar larvae by dietary carbohydrate and decreased by dietary lipid.

Animals↗
PubMed2006

Regulation of mitochondrial NADP-isocitrate dehydrogenase in rat heart during ischemia.

The changes in the regulation of at mitochondrial NADP-isocitrate dehydrogenase (NADP-ICDH) in a rat heart during have been analysed. Increase of enzyme activity in the cytosol and mitochondria of the heart ischemia was detected. Catalytic properties of the mitochondrial NADP-ICDH at norm and pathology have been compared on homogeneous enzyme preparations. Enzyme from the normoxic and ischemic heart showed the same electrophoretical mobility and molecular mass. Enzyme isolated from the ischemic heart mitochondria demonstrated higher activation energy and lower thermal stability. NADP-isocitrate dehydrogenase at the normoxic and ischemic conditions exhibited different Km for substrates and regulatory behaviour in relation to ATP, ADP, 2-oxoglutarate, citrate, malate, reduced and oxidised glutathione. The inhibitory effect of the Fe2+ and H2O2 mixture associated with the generation of hydroxyl radicals was lower in the ischemic enzyme. We hypothesise that the specific features of regulation behaviour of NADP-ICDH from the ischemic tissues permits the enzyme to supply NADPH to the glutathione reductase/glutathione peroxidase system.

Animals↗
PubMed1984

Enzyme regulation in C4 photosynthesis: mechanism of activation of NADP-malate dehydrogenase by reduced thioredoxin.

The mechanism of activation of thioredoxin-linked NADP-malate dehydrogenase was investigated by using 14C-iodoacetate and 14C-dansylated thioredoxin m, and Sepharose affinity columns (thioredoxin m, NADP-malate dehydrogenase) as probes to monitor enzyme sulfhydryl status and enzyme-thioredoxin interaction. The data indicate that NADP-malate dehydrogenase, purified to homogeneity from corn leaves, is activated by a net transfer of reducing equivalents from thioredoxin m, reduced by dithiothreitol, to enzyme disulfide groups, thereby yielding oxidized thioredoxin m and reduced enzyme. The appearance of new sulfhydryl groups that accompanies the activation of NADP-malate dehydrogenase appears to involve a structural change that is independent of the formation of a stable complex between the enzyme and reduced thioredoxin m. The data are consistent with the conclusion that oxygen promotes deactivation of NADP-malate dehydrogenase through oxidation of SH groups on reduced thioredoxin and on the reduced (activated) enzyme.

Bacterial Proteins↗
PubMed1983

The subcellular location of isozymes of NADP-isocitrate dehydrogenase in tissues from pig, ox and rat.

Antibodies against purified NADP-isocitrate dehydrogenase from pig liver cytosol and pig heart were raised in rabbits. The purified enzymes from these sources are different proteins, as demonstrated by differences in electrophoretic mobility and absence of crossreactivity by immunotitration and immunodiffusion. The NADP-isocitrate dehydrogenase in the soluble supernatant homogenate fraction from pig liver, kidney cortex, brain and erythrocyte hemolyzate was identical with the purified enzyme from pig liver cytosol, as determined by electrophoretic mobility and immunological techniques. The enzyme in extracts of mitochondria from pig heart, kidney, liver and brain was identical with the purified pig heart enzyme by the same criteria. However, the 'mitochondrial' isozyme was the major component also in the soluble supernatant fraction of pig heart homogenate. The 'cytosolic' isozyme accounted for only 1-2% of total NADP-isocitrate dehydrogenase in pig heart, as determined by separation of the isozymes with agarose gel electrophoresis and immunotitration. The mitochondrial isozyme was also the predominant NADP-isocitrate dehydrogenase in porcine skeletal muscle. The ratio of cytosolic/mitochondrial isozyme for porcine whole tissue extract, determined by immunotitration, was about 2 for liver and 1 for kidney cortex and brain. The distribution of isozymes in cell homogenate fractions from ox and rat tissues corresponded to that observed in organs of porcine origin. The mitochondrial and cytosolic isozymes from ox and rat tissues exhibited crossreactivity with the antibodies against the pig heart and pig liver cytosol enzyme, respectively, and the electrophoretic migration patterns were similar qualitatively to those found for the isozymes in porcine tissues. Nevertheless, there were species specific differences in the characteristics of each of the corresponding isozymes. NAD-isocitrate dehydrogenase was not inhibited by the antibodies, confirming that the protein is distinct from that of either isozyme of NADP-isocitrate dehydrogenase.

Animals↗
PubMed1995

Cloning, sequencing and functional expression of a cDNA encoding a NADP-dependent malic enzyme from human liver.

This work reports the structure of a cDNA (ME) encoding a human malic enzyme (ME) (malate NADP oxidoreductase, EC 1.1.1.40) elucidated by joining several overlapping fragments amplified by PCR from human hepatic cDNA or from cDNA libraries. The full-length cDNA has an open reading frame (ORF) of 1719 bp that encodes a 572-amino-acid protein of 64 113 Da, similar to the native monomeric, cytosolic, NADP-dependent ME isolated from human liver. The comparison of the structure of this cDNA with that of the human mitochondrial NAD(P)-dependent ME (EC 1.1.1.39) shows a homology of 63%, suggesting that these two forms originated from the same gene. The expression of the cDNA in Escherichia coli as a translational fusion (glutathione S-transferase::ME) protein yielded a product of the predicted mass. The recombinant protein shows NADP-dependent malate oxidoreductase activity and is virtually inactive with NAD. It also shows other distinct features of the native cytosolic NADP-dependent ME, like Mn2+ dependence, similar substrate (Km = 117 microM) and cofactor affinity (Km = 2 microM) constants, and a lack of allosteric regulation. In human proliferative cells, the NADP-dependent ME activity is poorly expressed and barely inducible by thyroid hormones.

Amino Acid Sequence↗
PubMed1977

Affinity chromatography of the Neurospora NADP-specific glutamate dehydrogenase, its mutational variants and hybrid hexamers.

The synthesis of an affinity adsorbent, 8-(6-aminohexyl)aminoadenosine 2'-phosphate-Sepharose 4B, is described. The assembly of the 2'-AMP ligand and the hexanediamide spacer arm was synthesized in free solution before its attachment to the Sepharose matrix. This adsorbent retarded the hexameric NADP-specific glutamate dehydrogenase of Neurospora crassa, showing a capacity for this enzyme similar to that of comparable coenzyme-analogue adsorbents for other dehydrogenases. The enzyme was eluted either at pH 6.8 in a concentration gradient of NADP+, or at pH 8.5 in the presence of NADP+ in concentration gradients of either dicarboxylates or NaCl. Anomalous effects of dicarboxylates in facilitating elution are discussed. 2'-AMP and its derivatives, 8-bromoadenosine 2'-phosphate and 8-(l-aminohexyl)aminoadenosine 2'-phosphate, which were used in the synthesis of the adsorbent, all acted as enzyme inhibitors competitive with NADP+. The chromatographic properties of the wild-type enzyme were compared with those of mutationally modified variants containing defined amino acid substitutions. This approach was used to assess the biospecificity of adsorption and elution and the contribution of non-specific binding. The adsorbent showed a low capacity for the enzyme from mutant am1 (Ser-336 replaced by Phe), a variant that has a localized defect in NADP binding, but an otherwise almost normal conformation, suggesting that non-specific interactions are at most weak. The enzyme from mutant am3, a variant modified in a conformational equilibrium, was fully retarded by the adsorbent, but showed a significantly earlier elution position than the wild-type enzyme. This is consistent with measurements in free solution that showed the am3 enzyme to have a higher Ki for 2'-AMP than the wild-type enzyme. The enzyme from mutant am19 was eluted as two distinct peaks at both pH 6.8 and 8.5. The adsorbent was used to separate hybrid hexamers constructed in vitro by a freeze-thaw procedure from pairs of purified variants. Several chromatographically distinct peaks of differing enzymological properties were purified from each hybridization mixture in quantities of up to a few milligrams, and represented distinct species of hybrid hexamers differing in subunit ratio.

Adenosine Monophosphate↗
PubMed1983

Photosynthetic electron transport from water to NADP driven by photosystem II in inside-out chloroplast vesicles.

It is now widely held that the light-induced noncyclic (linear) electron transport from water to NADP(+) requires the collaboration in series of the two photosystems that operate in oxygen-evolving cells: photosystem II (PSII) photooxidizes water and transfers electrons to photosystem I (PSI); PSI photoreduces ferredoxin, which in turn reduces NADP(+) (the Z scheme). However, a recently described alternative scheme envisions that PSII drives the noncyclic electron transport from water to ferredoxin and NADP(+) without the collaboration of PSI, whose role is limited to cyclic electron transport [Arnon, D. I., Tsujimoto, H. Y. & Tang, G. M.-S. (1981) Proc. Natl. Acad. Sci. USA 78, 2942-2946]. Reported here are findings at variance with the Z scheme and consistent with the alternative scheme. Thylakoid membrane vesicles were isolated from spinach chloroplasts by the two-phase aqueous polymer partition method. Vesicles, originating mainly from appressed chloroplast membranes that are greatly enriched in PSII, were turned inside-out with respect to the original sidedness of the membrane. With added plastocyanin, ferredoxin, and ferredoxin-NADP(+) reductase, the inside-out vesicles enriched in PSII gave a significant photoreduction of NADP(+) with water as electron donor, under experimental conditions that appear to exclude the participation of PSI.

Journal Article↗
PubMed2000

Crystal structure of paprika ferredoxin-NADP+ reductase. Implications for the electron transfer pathway.

cDNA of Capsicum annuum Yolo Wonder (paprika) has been prepared from total cellular RNA, and the complete gene encoding paprika ferredoxin-NADP(+) reductase (pFNR) precursor was sequenced and cloned from this cDNA. Fusion to a T7 promoter allowed expression in Escherichia coli. Both native and recombinant pFNR were purified to homogeneity and crystallized. The crystal structure of pFNR has been solved by Patterson search techniques using the structure of spinach ferredoxin-NADP(+) reductase as search model. The structure was refined at 2.5-A resolution to a crystallographic R-factor of 19.8% (R(free) = 26.5%). The overall structure of pFNR is similar to other members of the ferredoxin-NADP(+) reductase family, the major differences concern a long loop (residues 167-177) that forms part of the FAD binding site and some of the variable loops in surface regions. The different orientation of the FAD binding loop leads to a tighter interaction between pFNR and the adenine moiety of FAD. The physiological redox partners [2Fe-2S]-ferredoxin I and NADP(+) were modeled into the native structure of pFNR. The complexes reveal a protein-protein interaction site that is consistent with existing biochemical data and imply possible orientations for the side chain of tyrosine 362, which has to be displaced by the nicotinamide moiety of NADP(+) upon binding. A reasonable electron transfer pathway could be deduced from the modeled structures of the complexes.

Amino Acid Sequence↗
PubMed1982

The regulation of NADP-linked isocitrate dehydrogenase in Aspergillus nidulans.

The regulation of NADP-linked isocitrate dehydrogenase (NADP-IDH) has been studied in wild-type and mutant strains of Aspergillus nidulans. In the wild-type strain studied, the levels of NADP-IDH vary in a similar way to those of acetamidase, acetyl-CoA synthase, isocitrate lyase and malate synthase under all growth conditions used. Similarly, fac mutants, which are altered in the regulation of these enzymes of acetate utilization, are affected in NADP-IDH levels in a parallel fashion, as are cre mutants, which show altered carbon catabolite repression of this group of enzymes. Possible functions of the NADP-IDH enzyme are considered.

Acetate-CoA Ligase↗
PubMed1995

A NADP-glutamate dehydrogenase mutant of the petit-negative yeast Kluyveromyces lactis uses the glutamine synthetase-glutamate synthase pathway for glutamate biosynthesis.

The activities of the enzymes involved in ammonium assimilation and glutamate biosynthesis were determined in wild-type and NADP-glutamate dehydrogenase (GDH) null mutant strains of Kluyveromyces lactis. The specific NADP-GDH activity from K. lactis was fivefold lower than that found in Saccharomyces cerevisiae. The glutamine synthetase (GS) and glutamate synthase (GOGAT) activities were similar to those reported in S. cerevisiae. The NADP-GDH null mutant was obtained by transforming the uraA strain MD2/1 with a linearized integrative yeast vector harbouring a 390 bp fragment of the NADP-GDH structural gene. This mutant grew as well as the parent strain on ammonium, but showed GS and GOGAT activities higher that those found in the wild-type strain, implying that the GS-GOGAT pathway could play a leading role in glutamate biosynthesis in K. lactis. Southern blotting analysis of K. lactis chromosomes separated by contour-clamped homogeneous electric field electrophoresis, indicated that the NADP-GDH structural gene is localized on chromosome VI.

Amino Acid Sequence↗
PubMed1997

NADP-Malate Dehydrogenase in the C4 Plant Flaveria bidentis (Cosense Suppression of Activity in Mesophyll and Bundle-Sheath Cells and Consequences for Photosynthesis).

Flaveria bidentis, a C4 dicot, was transformed with sorghum (a monocot) cDNA clones encoding NADP-malate dehydrogenase (NADP-MDH; EC 1.1.1.82) driven by the cauliflower mosaic virus 35S promoter. Although these constructs were designed for over-expression, many transformants contained between 5 and 50% of normal NADP-MDH activity, presumably by cosense suppression of the native gene. The activities of a range of other photosynthetic enzymes were unaffected. Rates of photosynthesis in plants with less than about 10% of normal activity were reduced at high light and at high [CO2], but were unaffected at low light or at [CO2] below about 150 [mu]L L-1. The large decrease in maximum activity of NADP-MDH was accompanied by an increase in the activation state of the enzyme. However, the activation state was unaffected in plants with 50% of normal activity. Metabolic flux control analysis of plants with a range of activities demonstrates that this enzyme is not important in regulating the steady-state flux through C4 photosynthesis in F. bidentis. Cosense suppression of gene expression was similarly effective in both the mesophyll and bundle-sheath cells. Photosynthesis of plants with very low activity of NADP-MDH in the bundle-sheath cells was only slightly inhibited, suggesting that the presence of the enzyme in this compartment is not essential for supporting maximum rates of photosynthesis.

Journal Article↗
PubMed1978

Glyceraldehyde-3-Phosphate Dehydrogenase (NADP) from Sinapis alba L: Reversible Association of the Enzyme with a Protein Factor as Controlled by Pyridine Nucleotides in Vitro.

Aggregation of glyceraldehyde-3-P dehydrogenase (NADP) (EC 1.2.1.13) from Sinapis alba seedlings during gel filtration on Sepharose 6B is dependent on the presence of a fraction ("binding fraction") which can be separated from the enzyme by precipitation with 55% ammonium sulfate. Association of the enzyme with this binding fraction is NAD-dependent whereas NADP(+) causes release. Dithioerythritol (2 mM) has no influence on these reversible processes.Binding fractions, partially purified by ammonium sulfate and acetone fractionation, were submitted to dodecylsulfate-polyacrylamide gel electrophoresis. They always contain one or two dominant polypeptides with apparent molecular weights 42,000 and 58,000. The 42,000 polypeptide comigrates during dodecylsulfate electrophoresis with the corresponding subunit of the enzyme. It comprises up to 70% of the total protein in partially purified binding fractions and can be regarded as a major protein in seedling extracts.The differential transport behavior of glyceraldehyde-3-P dehydrogenase (NADP) on Sephadex G-200 in the presence of NAD(+) and NADP(+) can be used as a simple and effective purification procedure. The enzyme isolated in this way has an isoelectric point of about 4.5 and maintains under all tested conditions a heterogeneous subunit composition of at least three different polypeptide chains (apparent molecular weights, 39,000, 42,000, 43,000).The present data suggest that NAD(P)-controlled aggregation of glyceraldehyde-3-P dehydrogenase (NADP) from Sinapis alba L. is due primarily to enzyme association with a separate binding fraction rather than to enzyme polymerization. It is possible that a major component of this binding fraction, the 42,000 polypeptide, consists of "surplus" nonactive enzyme subunits, which self-associate and interact with the NAD-conformer of the enzyme.

Journal Article↗
PubMed1980

Effect of Light on Chemical Modification of Chloroplast Ferredoxin-NADP Reductase.

Chemical modification of spinach chloroplasts by phenylglyoxal and dansyl chloride resulted in inhibition of NADP photoreduction. The rate of inactivation was higher with both reagents when modification was carried out in the light with methylviologen or phenazine methosulfate present. Uncouplers prevent the effect of light. Electron transport from water to methylviologen was not affected by the modifiers.The presence of 10 millimolar NADP completely protected the membrane-bound reductase against inactivation by phenylglyoxal. With lower concentrations, protection was higher in the light than in the dark. The apparent dissociation constants of the enzyme-substrate complex for NADP were 0.9 and 0.1 millimolar for the dark and light inactivation, respectively. Inactivation of NADP photoreduction by dansyl chloride was completely prevented by ferredoxin, but only partially by nucleotides.The diaphorase activity was inhibited in chloroplasts modified by phenylglyoxal, but not when modified by dansyl chloride.The results suggest that energizing thylakoid membranes by light induces a conformational change in membrane-bound ferredoxin-NADP reductase, and that the reductase is an allotopic enzyme.

Journal Article↗
PubMed1988

Adenine Nucleotide Levels, the Redox State of the NADP System, and Assimilatory Force in Nonaqueously Purified Mesophyll Chloroplasts from Maize Leaves under Different Light Intensities.

Recently, a nonaqueous fractionation method of obtaining highly purified mesophyll chloroplasts from maize leaves was established. This method is now used to determine adenine nucleotide levels, the redox states of the NADP system, Pi levels and dihydroxyacetone phosphate/3-phosphoglycerate ratios in mesophyll chloroplasts of Zea mays L. leaves under different light intensities. The sum of the ATP, ADP, and AMP levels was estimated to be 1.4 millimolar and the ATP/ADP ratio was 1 in the dark and 2.5 to 4 in the light. The adenine nucleotides were equilibrated by adenylate kinase. The total concentration of NADP(H) in the chloroplasts was 0.3 millimolar in the dark and 0.48 millimolar in the light. The ratio of NADPH/NADP was 0.1 to 0.18 in the dark and 0.23 to 0.48 in the light. The Pi level was estimated to be 20 millimolar in the dark and 10 to 17 millimolar in the light. The 3-phosphoglycerate reducing system was under thermodynamic equilibrium in the light. The calculated assimilatory forces were 8 per molar and 40 to 170 per molar in the dark and the light, respectively. There was no relationship between the degree of activation of pyruvate, Pi dikinase, and adenylate energy charge, or ATP/ADP ratio or ADP level under various light intensities. Only a weak relationship was found between the degree of activation of NADP-malate dehydrogenase and the NADPH/NADP ratio or NADP(H) level with increasing light intensity. A possible regulatory mechanism which is responsible for the regulation of activation of pyruvate,Pi dikinase and NADP-malate dehydrogenase is discussed.

Journal Article↗
PubMed1992

Characterization and expression of a NADP-malic enzyme cDNA induced by salt stress from the facultative crassulacean acid metabolism plant, Mesembryanthemum crystallinum.

The facultative halophyte and crassulacean acid-metabolism plant, Mesembryanthemum crystallium shifts from C3 photosynthesis to crassulacean acid metabolism when exposed to high-salt or drought conditions. To study the molecular basis of this metabolic transition, the expression of NADP(+)-dependent malic enzyme (NADP-ME), which catalyzes the decarboxylation of malate to release pyruvate and CO2, has been investigated. The complete nucleotide sequence of a full-length cDNA clone was determined and found to contain a single open reading frame encoding a 585-amino-acid polypeptide of 64284 Da. The ice plant (M. crystallinum) NADP-ME shares amino acid identities in the range 72.5-79.0% when compared to other higher-plant enzymes and is more closely related to C3 rather than C4 forms of the enzyme. Genomic Southern-blot analysis of ice-plant DNA indicates that NADP-ME is encoded by a small gene family. Steady-state transcript levels increase 8-10-fold in response to salt stress in the leaves. Transcript levels in roots are extremely low and are unaffected by salt-stress treatment. Nuclear run-on experiments, using isolated nuclei from leaf tissue, confirm that the accumulation of NADP-ME transcripts is, in part, the result of increased transcription of this gene during salt stress.

Amino Acid Sequence↗