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PubMed1988

High activities of NADP+-dependent isocitrate dehydrogenase and malic enzyme in rabbit lens epithelial cells.

Measurements were made of the activities of the NADP+-dependent isocitrate dehydrogenase, malic enzyme and glucose-6-phosphate dehydrogenase in cytosolic supernatants of whole lens and capsule-epithelium. The activities of all three NADP+-dependent enzymes were concentrated in the capsule-epithelium relative to the activities measured in the whole lens. These results show for the first time that the rabbit lens epithelial cells contain substantial activities of the NADP+-dependent isocitrate dehydrogenase and malic enzyme. It is proposed that these enzymes together with glucose-6-phosphate dehydrogenase are involved in the regulation of the intracellular level of glutathione during oxidative stress by providing the NADPH necessary for the activity of glutathione reductase.

Animals↗
PubMed1986

[NADP-dependent malate dehydrogenase from bovine adrenal cortex cytoplasm. Isolation and properties].

The NADP-dependent decarboxylating malate dehydrogenase was isolated from the cytoplasmic fraction of bovine adrenal cortex and purified 3530-fold by 3-fold ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Toyopearl 650 M and DEAE-Sephadex A-50 with subsequent two-fold gel filtration through Toyopearl HW-55. The specific activity of homogeneous enzyme preparations was equal to 60 U/mg protein with a 30% yield. The enzyme molecular weight as determined by gel filtration on Sephadex G-20 was 155000. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate malate dehydrogenase dissociated into two subunits with Mr 77000. The Arrhenius plot for the reaction rate showed a break at 30 degrees C. The values of activation energy and temperature coefficient above and below the breakpoint were equal to 45049 and 147188 J X mol-1; 1.68 and 2.63, respectively. Within the temperature range of 26-40 degrees C, malate dehydrogenase exhibited hyperbolic kinetics with respect to the substrate. At 30 degrees C, Km for malate was equal to 250 microM, whereas at 40 degrees C it was 130 microM. The curve for the dependence of the initial reaction velocity versus NADP concentration was S-shaped. The Hill coefficient was 1.4, which testifies to positive cooperativity of NADP interaction with malate dehydrogenase.

Adrenal Cortex↗
PubMed1981

[Vitamin A and NADP-dependent isocitrate dehydrogenase: difference in activity of mitochondrial and cytoplasmic isoenzyme fractions].

The effects of vitamin A excess on the activity of isoenzyme fractions of NADP-dependent isocitrate dehydrogenase from rat liver separated by polyacrylamide gel disc-electrophoresis was studied. Vitamin A was shown to activate mitochondrial isoenzyme of NADP-dependent isocitrate dehydrogenase and had but very slight effect on the cytoplasmic isoform. This effect was observed already after 24 hrs following the injection and was enhanced at increasing doses of the vitamin. The data obtained suggest that NADP-dependent isocitrate dehydrogenase is controlled by complex mechanisms. The possibility of independent regulation of activity of mitochondrial and cytoplasmic isoenzymes and a possible involvement of retinol in the regulation of activity of its mitochondrial isoform are postulated. It is assumed that vitamins, similar to hormones, can selectively influence individual isoenzymes.

Animals↗
PubMedDate not supplied

[The taxon-specific lens protein of the frog eye: its phylogenetic kinship with the proteins of the NADP-dependent reductase family].

Physicochemical and molecular-biological properties of the proteins from the family of NADP-dependent reductases are reviewed in the article. Physicochemical properties of aldehyde/aldosoreductases are well studied. Information on the genes, coding for these proteins has appeared recently as well. Comparison of the protein structures has revealed, that taxon-specific protein of the frog lens-rho-crystallin--is structurally related to the superfamily of NADP-dependent reductases, though it does not have an enzymatic activity. Sequence alignment reveals a set of clusters, conserved in all members of superfamily. Among them there are two highly conserved regions, providing for binding of NADP coenzyme. Secondary structure of the proteins is similar as well. All the members of superfamily predominantly have beta-sheets. Comparison of structural data for proteins, isolated from various organisms from bacterium to human, suggests phylogenetic relatedness of all the members of superfamily, including rho-crystallin. All the data presented enable to suppose, that rho-crystallin and other members of superfamily have a common ancestor gene. A set of successive duplications and mutations of the ancestor gene resulted in the appearance of rho-crystallin gene, that has lost the enzymatic activity and acquired the ability for tissue specific superexpression in the lens cells.

Amino Acid Sequence↗
PubMed1977

Heterozygosity of the sheep: Polymorphism of 'malic enzyme', isocitrate dehydrogenase (NADP+), catalase and esterase.

In contrast to other reports, it is found that the sheep has approximately as much enzyme variation as man. Most of the genetically interpretable enzyme variation in heart, liver, kidney and muscle from 52 sheep (Merinos or Merino crosses) is in the NADP-dependent dehydrogenases [two 'malic enzymes' and the supernatant isocitrate dehydrogenase (NADP+)] and in the esterases. Ten different loci for NAD-dependent dehydrogenases are electrophoretically monomorphic, as are five different NADH diaphorases from heart muscle and 15 different major proteins from skeletal muscle. It is highly statistically significant that NADP-dependent dehydrogenases and esterases are polymorphic but representatives of several other major classes of enzymes are not. The physiological significance of this polymorphism may be related to the role of these enzymes in growth and detoxication, sheep having been selected by man for faster growth, of wool or of carcass, and for grazing a wide variety of plants.

Animals↗
PubMed2001

Glutamate synthesis via photoreduction of NADP+ by photostable chlorophyllide coupled with polyethylene-glycol.

Chlorophyllide a was coupled with alpha-(3-aminopropyl)-omega-methoxypoly(oxyethylene) (PEG-NH2) to form a PEG-chlorophyllide conjugate through an acid-amide bond. The conjugate catalyzed the reduction of methylviologen in the presence of 2-mercaptoethanol. It also catalyzed the photoreduction of NADP+ or NAD+ in the presence of ascorbate as an electron donor and ferredoxin-NADP+ reductase as the coupling enzyme. Utilizing the reducing power of NADPH generated by PEG-chlorophyllide conjugate under illumination, glutamate was synthesized from 2-oxoglutarate and NH4+ in the presence of glutamate dehydrogenase. PEG-chlorophyllide conjugate was quite stable toward light illumination compared with chlorophyll a. The increase in the molecular weight of PEG in the PEG-chlorophyllide conjugates was accompanied by the enhancement of photostability of the conjugate and also by the increased solubility in the aqueous solution.

Carboxylic Ester Hydrolases↗
PubMed1989

Purification and characterization of the NADP-dependent glutamate dehydrogenase from Bacillus fastidiosus.

Ammonia assimilation in Bacillus fastidiosus proceeds via the NADP-dependent glutamate dehydrogenase. The enzyme, purified to homogeneity, is composed of identical subunits with a molecular weight of about 48,000 dalton. Presumably the enzyme is a hexamer. The enzyme is specific for NADP(H). The pH optima for the amination and deamination reactions are 7.7 and 8.6, respectively. The temperature optimum is 60 degrees C. Furthermore, temperature stability and apparent Km values for substrates of both the amination and deamination reactions were determined. Several metabolites were tested for their effect on the enzyme activity. Only malate and fumarate showed some inhibitory effect.

Bacillus↗
PubMed2005

Catalysis of NADH-->NADP+ transhydrogenation by adult Hymenolepis diminuta mitochondria.

Hymenolepis diminuta mitochondria catalyze nonenergy-linked and energy-linked NADH-->NADP(+) transhydrogenations, with the latter driven by electron-transport dependent NADH oxidation (electron transport-driven, ETD) or ATP hydrolysis (ATP-driven, ATPD). Using submitochondrial particles, NADH-->NADP(+) transhydrogenations were characterized further. ETD and ATPD reactions were enhanced by bovine serum albumin (BSA) and were inhibited by N,N'-dicyclohexylcarbodiimide (DCCD), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and niclosamide. The nonenergy-linked reaction was unaffected by these additives. Except for DCCD inhibition of the ATPD reaction, BSA mitigated inhibitor effects on energy-linked activities. BSA enhanced NADH oxidase (but not ATPase) activity. Although DCCD inhibited NADH oxidase and ATPase, BSA only lessened oxidase inhibition. With protonophores, an increase in NADH oxidase (but not ATPase) activity was suggested. Oxidase inhibition by rotenone was unaffected by BSA. The ATP-hydrolyzed/NADPH-formed for the ATPD reaction was almost unity. A model for H. diminuta energy-linked transhydrogenation is presented.

Animals↗
PubMed1987

Immunological studies of the binding protein for chloroplast ferredoxin-NADP+ reductase.

Monospecific rabbit antibodies against the ferredoxin-NADP+ reductase binding protein of spinach thylakoids were obtained and characterized. The immunoglobulin G (IgG) fraction gave single precipitation arcs with the purified antigen or with Triton X-100 extracts of thylakoids or the reductase binding protein complex. Antibodies against the flavoprotein behave similarly. Both antibodies agglutinated thylakoids and precipitated the diaphorase activity of a Triton X-100 extract of these membranes. Isolated Fab fragments of the IgG anti-binding protein inhibited NADP+ photoreduction in a time- and Fab concentration-dependent manner. The presence of ferredoxin diminished the rate of inhibition. In the light, the inactivation rate was higher than in dark and this effect was abolished in the presence of uncouplers. These results suggest that the binding protein is protruding from the thylakoids and could be sensing the proton gradient.

Carrier Proteins↗
PubMed1990

Tryptophan fluorescence studies of ferredoxin:NADP reductase indicate the presence of tryptophan in or near the ferredoxin binding site.

The tryptophan fluorescence properties of the flavoprotein ferredoxin:NADP reductase have been examined. Although not sensitive to changes in pH or salt concentration, the tryptophan fluorescence is affected by the presence of substrates for the flavoprotein. While NADP addition results in a slight quenching of the fluorescence, ferredoxin decreases the fluorescence by nearly 50%, suggesting the presence of tryptophan in or near the ferredoxin binding site. Titration of this effect gives a dissociation constant for the ferredoxin: flavoprotein complex which is similar to that obtained by spectral perturbations. This approach has also been used to demonstrate that a chemically modified ferredoxin which does not produce spectral perturbations when added to flavoprotein is capable of interacting with the flavoprotein although with a higher dissociation constant than for native ferredoxin.

Binding Sites↗
PubMed1992

Identification of arginyl residues involved in the binding of ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119 to its substrates.

Ferredoxin-NADP+ reductase from the cyanobacterium Anabaena sp. PCC 7119 was chemically modified by the alpha-dicarbonyl reagent phenylglyoxal. The studies of the inactivation by this compound, which is specific for arginyl residues, of both the diaphorase and NADPH-cytochrome c reductase activities, characteristic of the enzyme, are indicative of the involvement of at least one group of this kind in the binding site of NADP+ and a second one implicated in the interaction with ferredoxin. After specific cleavage of a FNR sample incubated with [7-14C]phenylglyoxal, two major labeled peptides were identified. The peptide which exhibited the higher degree of modification corresponded to residues 208-242. It contained four arginine residues but only two of them were the target of the modification: Arg224 and Arg233. Protection studies with protein substrates and sequence comparison with other reductases allow us to propose that these residues in Anabaena sp. PCC 7119 FNR must be involved in the interaction with the pyridine nucleotide. The second peptide corresponds to residues 75-103 and although it contains three arginine residues, Arg77 is the only one that exhibits the modification. This residue seems to be a key one in the interaction of this reductase with ferredoxin.

Amino Acid Sequence↗
PubMed1991

Transcriptional regulation of murine NADP(+)-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase.

The cytosolic NADP(+)-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase is ubiquitously expressed in all mouse tissues and cell lines examined. Northern analyses of the RNA indicated that there is an extensive variation in the levels of mRNA in different tissues. However, the gene is refractory to induction by serum, phorbol esters or growth factors in cultured fibroblasts. The mRNA of the NADP(+)-dependent trifunctional enzyme is stabilized post-transcriptionally by insulin-like growth factor-1.

3T3 Cells↗
PubMed2004

Titration of E. coli transhydrogenase domain III with bound NADP+ or NADPH studied by NMR reveals no pH-dependent conformational change in the physiological pH range.

A pH-titration 2D NMR study of Escherichia coli transhydrogenase domain III with bound NADP(+) or NADPH has been carried out, in which the pH was varied between 5.4 and 12. In this analysis, individual amide protons served as reporter groups. The apparent pK(a) values of the amide protons, determined from the pH-dependent chemical shift changes, were attributed to actual pK(a) values for several titrating residues in the protein. The essential Asp392 is shown to be protonated at neutral pH in both the NADP(+) and NADPH forms of domain III, but with a marked difference in pK(a) not only attributable to the charge difference between the substrates. Titrating residues found in loop D/alpha5 point to a conformational difference of these structural elements that is redox-dependent, but not pH dependent. The observed apparent pK(a) values of these residues are discussed in relation to the crystal structure of Rhodospirillum rubrum domain III, the solution structure of E. coli domain III and the mechanism of intact proton-translocating transhydrogenase.

Escherichia coli Proteins↗
PubMed1993

The role of cysteine residues of spinach ferredoxin-NADP+ reductase As assessed by site-directed mutagenesis.

To investigate the functional role of the cysteine residues present in the spinach ferredoxin-NADP+ oxidoreductase, we individually replaced each of the five cysteine residues with serine using site-directed mutagenesis. All of the mutant reductases were correctly assembled in Escherichia coli except for the C42S mutant protein. C114S and C137S mutant enzymes apparently showed structural and kinetic properties very similar to those of the wild-type reductase. However, C272S and C132S mutations yielded enzymes with a decreased catalytic activity in the ferredoxin-dependent reaction (14 and 31% of the wild type, respectively). Whereas the C132S was fully competent in the diaphorase reaction, the C272S mutant flavoprotein showed a 35-fold reduction in catalytic efficiency with respect to the wild-type enzyme (0.4 versus 14.28 microM-1 s-1) due to a substantial decrease of kcat. NADP+ binding by the C272S mutant enzyme was apparently quantitatively the same (Kd = 37 microM) but qualitatively different, as shown by the differential spectrum. Stopped-flow experiments showed that the enzyme-FAD reduction rate was considerably decreased in the C272S mutant reductase, along with a much lower yield of the charge-transfer transient species. It is inferred from these data that the charge transfer (FAD-NADPH) between the reductase and NADPH is required for hydride transfer from the pyridine nucleotide to flavin to occur with a rate compatible with catalysis.

Amino Acid Sequence↗
PubMed2003

Purification of recombinant non-phosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Streptococcus pyogenes expressed in E. coli.

Streprococcus pyogenes gapN was cloned and expressed by functional complementation of the Escherichia gap mutant W3CG. The IPTG-induced NADP non-phosphorylating GAPDH (GAPN) has been purified about 75.4 fold from E. coli cells, using a procedure involving conventional ammonium sulfate fractionation, anion-exchange chromatography, hydrophobic chromatography and hydroxyapatite chromatography. The purified protein was characterised: it's an homotetrameric structure with a native molecular mass of 224 kDa, have an acid pI of 4.9 and optimum pH of 8.5. Studies on the effect of assay temperature on enzyme activity revealed an optimal value of about 60 degrees C with activation energy of 51 KJ mole(-1). The apparent Km values for NADP and D-G3P or DL-G3P were estimated to be 0.385 +/- 0.05 and 0.666 +/- 0.1 mM, respectively and the Vmax of the purified protein was estimated to be 162.5 U mg(-1). The S. pyogenes GAPN was markedly inhibited by sulfydryl-modifying reagent iodoacetamide, these results suggest the participation of essential sulfydryl groups in the catalytic activity.

Chemistry, Physical↗
PubMed2000

NADP+-dependent glutamate dehydrogenase in the Antarctic psychrotolerant bacterium Psychrobacter sp. TAD1. Characterization, protein and DNA sequence, and relationship to other glutamate dehydrogenases.

The Antarctic psychrotolerant bacterium Psychrobacter sp. TAD1 contains two distinct glutamate dehydrogenases (GDH), each specific for either NADP+ or NAD+. This feature is quite unusual in bacteria, which generally have a single GDH. NADP+-dependent GDH has been purified to homogeneity and the gene encoding GDH has been cloned and expressed. The enzyme has a hexameric structure. The amino acid sequence determined by peptide and gene analyses comprises 447 residues, yielding a protein with a molecular mass of 49 285 Da. The sequence shows homology with hexameric GDHs, with identity levels of 52% and 49% with Escherichia coli and Clostridium symbiosum GDH, respectively. The coenzyme-binding fingerprint motif GXGXXG/A (common to all GDHs) has Ser at the last position in this enzyme. The overall hydrophilic character is increased and a five-residue insertion in a loop between two alpha-helices may contribute to the increase in protein flexibility. Psychrobacter sp. TAD1 GDH apparent temperature optimum is shifted towards low temperatures, whereas irreversible heat inactivation occurs at temperatures similar to those of E. coli GDH. The catalytic efficiency in the temperature range 10-30 degrees C is similar or lower than that of E. coli GDH. Unlike E. coli GDH the enzyme exhibits marked positive cooperativity towards 2-oxoglutarate and NADPH. This feature is generally absent in prokaryotic GDHs. These observations suggest a regulatory role for this GDH, the most crucial feature being the structural/functional properties required for fine regulation of activity, rather than the high catalytic efficiency and thermolability encountered in several cold-active enzymes.

Amino Acid Motifs↗
PubMed2001

Involvement of the flavin si-face tyrosine on the structure and function of ferredoxin-NADP+ reductases.

In ferredoxin-NADP(+) reductase (FNR), FAD is bound outside of an anti-parallel beta-barrel with the isoalloxazine lying in a two-tyrosine pocket. To elucidate the function of the flavin si-face tyrosine (Tyr-89 in pea FNR) on the enzyme structure and catalysis, we performed ab initio molecular orbital calculations and site-directed mutagenesis. Our results indicate that the position of Tyr-89 in pea FNR is mainly governed by the energetic minimum of the pairwise interaction between the phenol ring and the flavin. Moreover, most of FNR-like proteins displayed geometries for the si-face tyrosine phenol and the flavin, which correspond to the more negative free energy theoretical value. FNR mutants were obtained replacing Tyr-89 by Phe, Trp, Ser, or Gly. Structural and functional features of purified FNR mutants indicate that aromaticity on residue 89 is essential for FAD binding and proper folding of the protein. Moreover, hydrogen bonding through the Tyr-89 hydroxyl group may be responsible of the correct positioning of FAD and the substrate NADP(+)

Animals↗
PubMed1982

Arginyl residues of adrenodoxin reductase as the anion recognition site for 2'-phosphate group of NADP+1.

Adrenodoxin reductase from bovine adrenocortex was inactivated by arginine specific reagents, p-hydroxyphenylglyoxal, phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione. Inactivation of the enzyme caused by p-hydroxyphenylglyoxal obeyed pseudo-first-order kinetics and resulted in complete elimination of NADPH-ferricyanide reductase activity. The rate of inactivation increased with pH from 6.5 to 9.5. Ten out of 30-33 arginyl residues of the enzyme were modified, but residues essential to its enzymatic activity were less than 5. NADP+ strongly protected against inactivation by p-hydroxyphenylglyoxal, whereas NAD+ could afford only partial, weak protection. Furthermore, 2'-AMP and 2',5'-ADP afforded considerable protection but 5'-AMP did not. These data suggest that adrenodoxin reductase has essential arginyl residues which are crucial to the enzymatic activity as the recognition site for the negatively charged 2'-phosphate group of NADP+.

Animals↗