Cytosolic NADP(+)-dependent isocitrate dehydrogenase protects macrophages from LPS-induced nitric oxide and reactive oxygen species

Macrophages activated by microbial lipopolysaccharides (LPS) produce bursts of nitric oxide and reactive oxygen species (ROS). Redox protection systems are essential for the survival of the macrophages since the nitric oxide and ROS can be toxic to them as well as to pathogens. Using suppression subtractive hybridization (SSH) we found that cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) is strongly upregulated by nitric oxide in macrophages. The levels of IDPc mRNA and of the corresponding enzymatic activity were markedly increased by treatment of RAW264.7 cells or peritoneal macrophages with LPS or SNAP (a nitric oxide donor). Over-expression of IDPc reduced intracellular peroxide levels and enhanced the survival of H2O2- and SNAP-treated RAW264.7 macrophages. IDPc is known to generate NADPH, a cellular reducing agent, via oxidative decarboxylation of isocitrate. The expression of enzymes implicated in redox protection, superoxide dismutase (SOD) and catalase, was relatively unaffected by LPS and SNAP. We propose that the induction of IDPc is one of the main self-protection mechanisms of macrophages against LPS-induced oxidative stress.     

Nad+-dependent isocitrate dehydrogenase from Arabidopsis thaliana. Characterization of two closely related subunits

Two cDNA clones which appear to encode different subunits of NAD⁺-dependent isocitrate dehydrogenase (IDH; EC 1.1.1.41) were identified by homology searches from the Arabidopsis EST database. These cDNA clones were obtained and sequenced; both encoded full-length messages and displayed 82.7% nucleotide sequence identity over the coding region. The deduced amino acid sequences revealed preprotein lengths of 367 residues, with an amino acid identity of 86.1%. Genomic Southern blot analysis showed distinct single-copy genes for both IDH subunits. Both IDH subunits were expressed as recombinant proteins in Escherichia coli, and polyclonal antibodies were raised to each subunit. The Arabidopsis cDNA clones were expressed in Saccharomyces cerevisiae mutants which were deficient in either one or both of the yeast NAD⁺-dependent IDH subunits. The Arabidopsis cDNA clones failed to complement the yeast mutations; although both IDH-I and IDH-II were expressed at detectable levels, neither protein was imported into the mitochondria.     

Changes in NADP+-linked isocitrate dehydrogenase during tomato fruit ripening

The activity of NADP⁺-specific isocitrate dehydrogenase (NADP⁺-IDH, EC 1.1.1.42) was investigated during the ripening of tomato (Lycopersicon esculentum Mill.) fruit. In the breaker stage, NADP⁺-IDH activity declined but a substantial recovery was observed in the late ripening stages when most lycopene synthesis occurs. These changes resulted in higher NADP⁺-IDH activity and specific polypeptide abundance in ripe than in green fruit pericarp. Most of the enzyme corresponded to the predominant cytosolic isoform which was purified from both green and ripe fruits. Fruit NADP⁺-IDH seems to be a dimeric enzyme having a subunit size of 48 kDa. The K _m values of the enzymes from green and ripe pericarp for NADP⁺, isocitrate and Mg²⁺ were not significantly different. The similar molecular and kinetic properties and chromatographic behaviour of the enzymes from the two kinds of tissue strongly suggest that the ripening process is not accompanied by a change in isoenzyme complement. The increase in NADP⁺-IDH in the late stage of ripening also suggests that this enzyme is involved in the metabolism of C₆ organic acids and in glutamate accumulation in ripe tissues.     

NADP+-dependent acetaldehyde dehydrogenase from Zymomonas mobilis

An NADP⁺-linked acetaldehyde dehydrogenase (EC 1.2.1.4) from the ethanol producing bacterium Zymomonas mobilis was purified 180-fold to homogeneity. The enzyme is a cytosolic protein with an isoelectric point of 8.0 and has an apparent molecular weight of 210000. It showed a single band in sodium dodecylsulfate gel electrophoresis with a molecular weight of 55000, which indicates that it consists of four probably identical subunits. The apparent K _m values for the substrate acetaldehyde were 57 M and for the cosubstrate NADP⁺ 579 M. The enzyme was almost inactive with NAD⁺ as cofactor. Several other aldehydes besides acetaldehyde were accepted as a substrate but not formaldehyde or trichloroacetaldehyde. In anaerobically grown cells of Zymomonas mobilis the enzyme showed a specific activity of 0.035 U/mg protein but its specific activity could be increased up to 0.132 U/mg protein by adding acetaldehyde to the medium during the exponential growth phase or up to 0.284 U/mg protein when cells were grown under aeration. The physiological role of the enzyme is discussed.Abbreviations ALD-DH acetaldehyde dehydrogenases from Z. mobilis - DTT dithiothreitol - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - SDS sodium dodecylsulfate Dedicated to Prof. Dr. H.-G. Schlegel, Universität Göttingen, on the occasion of his 65th birthday     

NADPH recycling systems in oxidative stressed pea nodules: a key role for the NADP<Superscript>+</Superscript>-dependent isocitrate dehydrogenase

The symbiosis between legumes and rhizobia is characterised by the formation of dinitrogen-fixing root nodules. In natural conditions, nitrogen fixation is strongly impaired by abiotic stresses which generate over-production of reactive oxygen species. Since one of the nodule main antioxidant systems is the ascorbate–glutathione cycle, NADPH recycling that is involved in glutathione reduction is of great relevance under stress conditions. NADPH is mainly produced by glucose 6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) from the oxidative pentose phosphate pathway, and also by NADP⁺-dependent isocitrate dehydrogenase (ICDH; EC 1.1.1.42). In this work, 10 μM paraquat (PQ) was applied to pea roots in order to determine the in vivo relationship between oxidative stress and the activity of the NADPH-generating enzymes in nodules. Whereas G6PDH and 6PGDH activities remained unchanged, a remarkable induction of ICDH gene expression and a dramatic increase of the ICDH activity was observed during the PQ treatment. These results support that ICDH has a key role in NADPH recycling under oxidative stress conditions in pea root nodules.     

The isolation and characterization of a mutant allele at a new X-linked locus,mex, affecting NADP+-dependent enzymes inDrosophila melanogaster

The isolation and characterization of mutant alleles in a regulatory gene affecting NADP⁺-dependent enzymes are described. The locus,mex, is at position 26.5 ± 0.74 on the X chromosome ofDrosophila melanogaster. The newly isolated mutant allele,mex ¹, is recessive to either themex allele found in Oregon-R wild-type individuals or that found in thecm v parental stock in which the new mutants were induced. Themex ¹ mutant allele is associated with statistically significant decreases in malic enzyme (ME) specific activity and ME specific immunologically cross-reacting material (ME-CRM) in newly emerged adult males. During this same developmental stage in males, the NADP⁺-dependent isocitrate dehydrogenase specific activity increases to statistically significant levels. Females of themex ¹ mutant strain show statistically significant elevated levels of the pentose phosphate shunt enzymes, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Isoelectric focusing and thermolability comparisons of the active ME from mutant and control organisms indicate that the enzyme is the same. Developmental profiles ofmex ¹ and control strains indicate that this mutant allele differentially modulates the levels of ME enzymatic activity and ME-CRM during development. This work was supported by an Operating Grant from the Natural Sciences and Engineering Research Council of Canada to M.M.B.     

Shifted Cytosolic NADP+-Dependent Isocitrate Dehydrogenase on 2-D Gel in the Brain of Genetically Epileptic El Mice

We observed a spot on two-dimensional (2-D) gel in the epileptic mutant strain El mice with a similar molecular weight but with a different isoelectric point of approximately 0.2, compared with its mother strain ddY mice. The collected protein from the El mice was identified as cytosolic NADP⁺-dependent isocitrate dehydrogenase by internal amino acid sequencing. The enzyme is known to be maximally active during the development of the brain and to play an important role in NADPH production for fatty acids and cholesterol synthesis. In addition, alterations in cholesterol synthesis early in the development of the mammalian brain have been reported to lead to chronic epilepsy. The results in the present study therefore suggest that cytosolic NADP⁺-dependent isocitrate dehydrogenase might be involved in the epileptogenesis of the El mouse.     

Characterization of NADP+-dependent isocitrate dehydrogenase isozymes from a psychrophilic bacterium,Colwellia psychrerythraea strain 34H

NADP⁺-dependent isocitrate dehydrogenase (IDH) isozymes of a psychrophilic bacterium, Colwellia psychrerythraea strain 34H, were characterized. The coexistence of monomeric and homodimeric IDHs in this bacterium was confirmed by Western blot analysis, the genes encoding two monomeric (IDH-IIa and IDH-IIb) and one dimeric (IDH-I) IDHs were cloned and overexpressed in Escherichia coli, and the three IDH proteins were purified. Both of the purified IDH-IIa and IDH-IIb were found to be cold-adapted enzymes while the purified IDH-I showed mesophilic properties. However, the specific activities of IDH-IIa and IDH-IIb were lower even at low temperatures than that of IDH-I. Therefore, IDH-I was suggested to be important for the growth of this bacterium. The results of colony formation of E. coli transformants carrying the respective IDH genes and IDH activities in their crude extracts indicated that the expression of the IDH-IIa gene is cold-inducible in the E. coli cells.     

Overexpression of the NADP+-specific isocitrate dehydrogenase gene (icdA) in citric acid-producing Aspergillus niger WU-2223L

In the tricarboxylic acid (TCA) cycle, NADP⁺-specific isocitrate dehydrogenase (NADP⁺-ICDH) catalyzes oxidative decarboxylation of isocitric acid to form α-ketoglutaric acid with NADP⁺ as a cofactor. We constructed an NADP⁺-ICDH gene (icdA)-overexpressing strain (OPI-1) using Aspergillus niger WU-2223L as a host and examined the effects of increase in NADP⁺-ICDH activity on citric acid production. Under citric acid-producing conditions with glucose as the carbon source, the amounts of citric acid produced and glucose consumed by OPI-1 for the 12-d cultivation period decreased by 18.7 and 10.5%, respectively, compared with those by WU-2223L. These results indicate that the amount of citric acid produced by A. niger can be altered with the NADP⁺-ICDH activity. Therefore, NADP⁺-ICDH is an important regulator of citric acid production in the TCA cycle of A. niger. Thus, we propose that the icdA gene is a potentially valuable tool for modulating citric acid production by metabolic engineering.     

6-Phosphogluconate dehydrogenase from Drosophila melanogaster. I. Purification and properties of the a isozyme

6-Phosphogluconate dehyrogenase is evident at all developmental stages of Drosophila melanogaster. The activity level is highest in early third instar larvae and declines to a lower, but relatively constant, level at all later stages of development. The enzyme is localized in the cytosolic portion of the cell. The A-isozymic form of 6-phosphogluconate dehydrogenase was purified to homogeneity and has a molecular weight of 105,000. The enzyme is a dimer consisting of subunits with molecular weights of 55,000 and 53,000. For the oxidative decarboxylation of 6-phosphogluconate the K_m for substrate is 81 µm while that for NADP⁺ is 22.3 µm. The optimum pH for activity is 7.8 while the optimum temperature is 37 C.This work was supported by National Research Council of Canada Grant A5860 and by the University of Calgary Research Policy and Grants Committee.     

Characterization and regulation of NADP+-isocitrate dehydrogenase from Saccharopolyspora erythraea

NADP⁺-Isocitrate dehydrogenase (ICDH) activity was detected in cell-free extracts of Saccharopolyspora erythraea CA340, an erythromycin producer. Apparent K _m values for dl-isocitrate and NADP⁺ were 0.14 M and 0.026 M, respectively. ATP, ADP, GTP, citric acid, oxaloacetate, -ketoglutarate, glyoxalate and glyoxalate plus oxaloacetate, each at 1 mM concentration, caused 50, 20 10, 50, 25, 60, 20 and 50% inhibition of ICDH activity, respectively. Phosphoenolpyruvate, fructose 1,6-diphosphate and pyruvate had no effect. ICDH specific activity profile was growth-associated and activity with dextrose or fructose as sole carbon source, was twice of that obtained with lactose.     

NAD+-依赖型异柠檬酸脱氢酶的结构和功能研究进展

NAD^ —依赖型异柠檬酸脱氢酶是一个核编码线粒体酶,参与三羧酸循环,负责催化异柠檬酸氧化脱羧成α-酮戊二酸,是循环路径中的限速酶。目前在酶学性质、亚基组成、基因克隆、蛋白组装与转运,以及功能等方面开展了许多研究。本文就这些方面的新进展进行综述。     

Crystal structure studies of NADP dependent isocitrate dehydrogenase from Thermus thermophilus exhibiting a novel terminal domain

NADP⁺ dependent isocitrate dehydrogenase (IDH) is an enzyme catalyzing oxidative decarboxylation of isocitrate into oxalosuccinate (intermediate) and finally the product α-ketoglutarate. The crystal structure of Thermus thermophilus isocitrate dehydrogenase (TtIDH) ternary complex with citrate and cofactor NADP⁺ was determined using X-ray diffraction method to a resolution of 1.80 Å. The overall fold of this protein was resolved into large domain, small domain and a clasp domain. The monomeric structure reveals a novel terminal domain involved in dimerization, very unique and novel domain when compared to other IDH’s. And, small domain and clasp domain showing significant differences when compared to other IDH’s of the same sub-family. The structure of TtIDH reveals the absence of helix at the clasp domain, which is mainly involved in oligomerization in other IDH’s. Also, helices/beta sheets are absent in the small domain, when compared to other IDH’s of the same sub family. The overall TtIDH structure exhibits closed conformation with catalytic triad residues, Tyr144-Asp248-Lys191 are conserved. Oligomerization of the protein is quantized using interface area and subunit–subunit interactions between protomers. Overall, the TtIDH structure with novel terminal domain may be categorized as a first structure of subfamily of type IV.     

Genetic Variants Affecting Phenoloxidase Activity in Drosophila melanogaster

In Drosophila melanogaster, two new variants affecting the activity of phenoloxidase were found in natural populations at Gomel in Belorussia and at Krasnodar in Russia. Prophenoloxidases, A ₁ and A ₃ , in these variants had the same mobilities on native electrophoresis as the wild type. However, enzymatic activities in their activated states were much lower than in the wild type, whereas the existence of prophenoloxidase proteins was demonstrated. Egg-to-adult and relative viabilities in the variants did not decrease at temperatures between 18 and 29°C. Genetic analyses indicated that the genes showing the phenotype of variants are new alleles of Mox and Dox-3 on the second chromosome.     

Recent origin for a thermostable alcohol dehydrogenase allele ofDrosophila melanogaster

Summary The nucleotide sequence of theFast-Chateau Douglas isolate of the thermostable alcohol dehydrogenase allele is compared with the sequences of theSlow andFast alleles ofDrosophila melanogaster. Conceptual translation of theFChD sequence indicates that the thermostable polypeptide has the diagnostic FAST amino acid replacement at residue 192 and an additional replacement of serine for proline at residue 214. This suggests aFast origin for the thermostableAdh allele. However, some of the biochemical properties of the FCHD protein resemble those of the SLOW rather than the FAST polypeptides. The serine for proline replacement confers upon the thermostable polypeptide substrate specificities and some kinetic parameters similar to the SLOW protein. The same replacement substitution within the third coding exon also appears to alter the ADH protein concentration to a level similar to the SLOW polypeptide and the probable effect is at the level of mRNA concentration. The low level of nucleotide sequence variation, other than that leading to the amino acid substitution, suggests a recent origin for the thermostable allele. The time since divergence of theFChD sequence fromFast is estimated to be approximately 260,000–470,000 years.     

X-linked mutations that give rise to overproduction of glucose-6-phosphate dehydrogenase in Drosophila melanogaster

Two X-linked mutations that give rise to overproduction of glucose-6-phosphate dehydrogenase (G6PD) were found among the progenies of isogenic strains which had been subjected to selection for high G6PD activity. Mapping of the high-activity factor in these mutants was carried out using car Zw ^B sw males of low G6PD activity. As a result, the factor mapped 0.02–0.04 unit to the left of the Zw locus. The amount of the G6PD gene was also quantitated utilizing a cloned G6PD gene as a probe, but no significant difference was found between the mutants and low-G6PD activity flies which shared the same X, second, and third chromosomes with the mutants. These findings are consistent with our notion that the mutations might be regulatory mutations, possibly resulting from the insertion of a novel class of transposable genetic elements.This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.     

Drosophila melanogaster alcohol dehydrogenase: An electrophoretic study of the AdhS,AdhF, and AdhUF alleloenzymes

The nature and the interconversion of the three multiple forms Adh-5, Adh-4, and Adh-3 of the purified alleloenzymes Adh^S, Adh^F, and Adh^UF from the fruitflyDrosophila melanogaster have been examined. The experiments show that these multiple forms differ from those in crude extracts of flies homozygous at the Adh locus. On electrophoresis in a starch gel containing NAD or NADH, of purified Adh^S which consists of the three Adh forms S-5, S-4, and S-3, five enzymatically active zones appear. This contrasts with the single active zone that arises with crude extracts. Of the five zones that appear with purified enzyme, S-5 gives rise to one, while the other four zones come from the two minor forms S-4 and S-3. The occurrence of the three multiple forms Adh-5, Adh-4, and Adh-3 for each of the purified alleloenzymes is considered due to Adh-5 and, in the case of Adh-4 and Adh-3, deamidation of Adh-5, with the Adh-3 fraction also containing some reversible modified Adh-5. Of the labile amides, at least one must be located in the coenzyme binding region with deamidation preventing coenzyme binding. Pure NAD does not convert Adh-5 to Adh-3 and Adh-1. To produce conversion, the presence of either acetone or butanone along with NAD is necessary. Increased amounts of either acetone or butanone result in increased conversion. In contrast to this, none of the carbonyl compounds cyclohexanone, (+)- and (−)-verbenone, acetaldehyde, acrolein, or crotonaldehyde produces conversion. The ketone group binds to the alcohol binding site in the enzyme-NAD complex. Conversion is considered due to the ketone group binding to a nucleophilic amino acid residue and forming a bridge to the C-4 of the nicotinamide moiety of NAD.     

NADP+ glyceraldehyde-3-phosphate dehydrogenase in soybeans [Glycine max (L.) Merr.]: genetics and developmental expression

Summary Chloroplastic (NADP⁺) glyceraldehyde-3-phosphate dehydrogenase (E.C. 1.2.1.9) catalyzes the second reaction in photosynthesis after the fixation of carbon by RuBisCO. Chloroplast-bound (NADP⁺) G3PDH was resolved in soybean by starch gel electrophoresis using l-histidine-citrate buffer (pH 5.7). Histochemical staining revealed zymogram patterns indicative of a tetramer. A survey of soybean genotypes revealed differences in zymogram patterns between the principal cytoplasmic sources of the northern and southern US germplasms. In the soybean pedigree, an allelic frequency shift toward a five-banded pattern was observed. G3PDH polymorphism was due to allele associated with gene expression at the slow locus. No linkage was found between the slow locus of (NADP⁺) G3PDH and AC02, AC03, AC04, ACP, DIA1, IDH1, IDH2, PGM1, and PGM3. Developmental expression in the above-ground tissues was identical, whereas roots as a rule did not express (NADP⁺) G3PDH activity. The importance of chloroplast-bound (NADP⁺) G3PDH in photo-synthesis and its interesting mode of inheritance warrants further exploration of this enzyme in soybean.Technical contribution no. 3293 of the South Carolina Agricultural Experiment Station, Clemson University     

Progressive redistribution of alcohol dehydrogenase during vitellogenesis in Drosophila melanogaster: characterization of ADH-positive bodies in mature oocytes

Summary The use of monoclonal antibodies against Drosophila alcohol dehydrogenase (ADH) provides a powerful tool in the analysis of the tissue and temporal patterns of Adh gene expression. Immunocytochemical techniques at the light- and electron-microscopic levels have been used to determine the distribution of ADH in the ovarian follicles of D. melanogaster during oogenesis. In the early stages of oogenesis, small amounts of ADH are detectable in the cystocytes. At the beginning of vitellogenesis (S₇), ADH appears to be located mainly in the nurse cells. From stage S₉ onwards, the ADH protein is evenly distributed in the ooplasm until the later stages of oogenesis (S_13–14), when multiple ADH-positive bodies of varying size appear in the ooplasm. This change in distribution is a result of the compartmentalization of the ADH protein within the glycogen yolk or -spheres. Yolk becomes enclosed within the lumen of the primitive gut during embryonic development, and thus our results suggest a mechanism for the transfer of maternally-inherited enzymes to the gut lumen via yolk spheres.