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PubMed2003

Crystal structure of the vertebrate NADP(H)-dependent alcohol dehydrogenase (ADH8).

The amphibian enzyme ADH8, previously named class IV-like, is the only known vertebrate alcohol dehydrogenase (ADH) with specificity towards NADP(H). The three-dimensional structures of ADH8 and of the binary complex ADH8-NADP(+) have been now determined and refined to resolutions of 2.2A and 1.8A, respectively. The coenzyme and substrate specificity of ADH8, that has 50-65% sequence identity with vertebrate NAD(H)-dependent ADHs, suggest a role in aldehyde reduction probably as a retinal reductase. The large volume of the substrate-binding pocket can explain both the high catalytic efficiency of ADH8 with retinoids and the high K(m) value for ethanol. Preference of NADP(H) appears to be achieved by the presence in ADH8 of the triad Gly223-Thr224-His225 and the recruitment of conserved Lys228, which define a binding pocket for the terminal phosphate group of the cofactor. NADP(H) binds to ADH8 in an extended conformation that superimposes well with the NAD(H) molecules found in NAD(H)-dependent ADH complexes. No additional reshaping of the dinucleotide-binding site is observed which explains why NAD(H) can also be used as a cofactor by ADH8. The structural features support the classification of ADH8 as an independent ADH class.

Alcohol Dehydrogenase↗
PubMed1975

Human granulocyte 6 phosphogluconate dehydrogenase. Purification by elective elution with NADP+, immunological and kinetic properties.

Human granulocyte 6 phosphogluconate dehydrogenase has been totally purified from a single patient with chronic granulocytic leukaemia. 48 mg of protein, of specific activity 20 IU per mg of protein, have been obtained in the course of three different steps only. The overall yield was 30 p. cent and the purification was 100 folds. Purified 6 phosphogluconate dehydrogenase was homogeneous when tested in acrylamide and acrylamide SDS gel electrophoresis or in immunodiffusion. The enzyme was immunologically identical in red blood cells, blood platelets and normal leukocytes. The fixation of both substrates, NADP-+ and 6 phosphogluconate, seemed to proceed through a non ordered mechanism. NADPH was an inhibitor strictly competitive with respect to NADP-+ and non competitive with respect to 6 phosphogluconate. 2-3 Diphosphoglycerate seemed to be able to bind on both the fixation sites of NADP-+ and 6 phosphogluconate. The inhibition by ATP was competitive with 6 phosphogluconate and non competitive with NADP-+. 6 phosphogluconate dehydrogenase was inactivated by SH reagents and was partially protected against this inactivation by both substrates. Both substrates protected the enzyme against thermal inactivation. The influence of ionic strength, pH and ions have been studied, and the results have been compared to those reported by other authors for erythrocyte enzyme.

Adenosine Triphosphate↗
PubMed1995

Identification of the NADP(H) binding site of rat liver microsomal 5 alpha-reductase (isozyme-1): purification of a photolabeled peptide corresponding to the adenine binding domain.

We have previously shown that [2'-32P]-2-azido-NADP+ is an effective probe of the NADP-(H) binding site of rat liver microsomal 5 alpha-reductase (5 alpha R-1) [Bhattacharyya et al. (1994) Steroids 59, 634-641]. PEG-fractionated (6.5%) detergent-solubilized preparations (40 mg) containing 5 alpha R-1 activity were UV-photolyzed with [32P]-2-azido-NADP+ and subjected to preparative gel electrophoresis on 8% SDS-PAGE. Fractions corresponding to the second major [32P]-labeled peak following the dye-front were analyzed by 10% SDS-PAGE and showed a single [32P]-labeled species with an apparent molecular mass of approximately 26 kDa (5 alpha R-1). TCA precipitation (13.6%) of the labeled fractions resulted in recovery of > 70% of the total radioactivity in the protein pellet. Trypsin digestion of the resuspended pellet followed by immobilized-Al3+ affinity chromatography indicated that > 90% of the radioactivity remained bound to the affinity column. The [32P]-2N3-NADP(+)-labeled peptide was eluted with potassium phosphate, concentrated, and further purified by reverse-phase (C8) HPLC. Sequence analysis of the purified peptide indicated that it consisted of 11 amino acids with the sequence N-L-R-K-P-G-E-T-G-Y-K, corresponding to residues 170-180 of the rat 5 alpha R-1 sequence [Andersson et al. (1989) J. Biol. Chem. 264, 16249-16255].

Adenine↗
PubMed2001

Structural basis for a change in substrate specificity: crystal structure of S113E isocitrate dehydrogenase in a complex with isopropylmalate, Mg2+, and NADP.

Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate and has negligible activity toward other (R)-malate-type substrates. The S113E mutant of IDH significantly improves its ability to utilize isopropylmalate as a substrate and switches the substrate specificity (k(cat)/K(M)) from isocitrate to isopropylmalate. To understand the structural basis for this switch in substrate specificity, we have determined the crystal structure of IDH S113E in a complex with isopropylmalate, NADP, and Mg(2+) to 2.0 A resolution. On the basis of a comparison with previously determined structures, we identify distinct changes caused by the amino acid substitution and by the binding of substrates. The S113E complex exhibits alterations in global and active site conformations compared with other IDH structures that include loop and helix conformational changes near the active site. In addition, the angle of the hinge that relates the two domains was altered in this structure, which suggests that the S113E substitution and the binding of substrates act together to promote catalysis of isopropylmalate. Ligand binding results in reorientation of the active site helix that contains residues 113 through 116. E113 exhibits new interactions, including van der Waals contacts with the isopropyl group of isopropylmalate and a hydrogen bond with N115, which in turn forms a hydrogen bond with NADP. In addition, the loop and helix regions that bind NADP are altered, as is the loop that connects the NADP binding region to the active site helix, changing the relationship between substrates and enzyme. In combination, these interactions appear to provide the basis for the switch in substrate specificity.

Amino Acid Substitution↗
PubMed1984

Phosphorus-31 nuclear magnetic resonance studies of the binding of nucleotides to NADP+-specific isocitrate dehydrogenase.

The interaction of the 2'-phosphate-containing nucleotides (NADP+, NADPH, 2'-phosphoadenosine 5'-diphosphoribose, and adenosine 2',5'-bisphosphate) with NADP+ -specific isocitrate dehydrogenase was studied by using 31P NMR spectroscopy. The separate resonances corresponding to free and bound nucleotides, characteristic for slow exchange of nuclei on the NMR time scale, were observed in the spectra of the enzyme (obtained in the presence of excess ligand) with NADP+ and NADPH in the absence and presence of Mg2+ and with 2'-phosphoadenosine 5'-diphosphoribose in the absence of metal or in the presence of the substrate magnesium isocitrate. The position of the 31P resonance of the bound 2'-phosphate group in these spectra is invariant (delta = 6) in the pH range 5-8, indicating that the pK of this group is much lower in the complexes with the enzyme than that (pK = 6.13) in the free nucleotides. The additional downfield shift of this resonance by 1.8 ppm beyond that (delta = 4.22) of the dianionic form of the 2'-phosphate in free nucleotides suggests interaction with a positively charged group(s) and/or distortion of P-O-P angles as the result of binding to the enzyme. A single resonance of 2'-phosphate was observed in the spectrum of the enzyme complex with 2'-phosphoadenosine 5'-diphosphoribose in the presence of Mg2+, with the chemical shift dependent on the nucleotide to enzyme ratio, characteristic for the fast exchange situation. Addition of metal does not perturb the environment of the 2'-phosphate in the complexes of NADP+ and NADPH with isocitrate dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

1H nuclear magnetic resonance studies of the conformation and environment of nucleotides bound to pig heart NADP+-dependent isocitrate dehydrogenase.

The binding of coenzymes, NADP+ and NADPH, and coenzyme fragments, 2'-phosphoadenosine 5'-(diphosphoribose), adenosine 2',5'-bisphosphate, and 2'-AMP, to pig heart NADP+-dependent isocitrate dehydrogenase has been studied by proton NMR. Transferred nuclear Overhauser enhancement (NOE) between the nicotinamide 1'-ribose proton and the 2-nicotinamide ring proton indicates that the nicotinamide-ribose bond assumes an anti conformation. For all nucleotides, a nuclear Overhauser effect between the adenine 1'-ribose proton and 8-adenine ring proton is observed, suggesting a predominantly syn adenine--ribose bond conformation for the enzyme-bound nucleotides. Transferred NOE between the protons at A2 and N6 is observed for NADPH (but not NADP+), implying proximity between adenine and nicotinamide rings in a folded enzyme-bound form of NADPH. Line-width measurements on the resonances of free nucleotides exchanging with bound species indicate dissociation rates ranging from less than 7 s-1 for NADPH to approximately 1600 s-1 for adenosine 2',5'-bisphosphate. Substrate, magnesium isocitrate, increases the dissociation rate for NADPH about 10-fold but decreases the corresponding rate for phosphoadenosine diphosphoribose and adenosine 2',5'-bisphosphate about 10-fold. These effects are consistent with changes in equilibrium dissociation constants measured under similar conditions. The 1H NMR spectrum of isocitrate dehydrogenase at pH 7.5 has three narrow peaks between delta 7.85 and 7.69 that shift with changes in pH and hence arise from C-4 protons of histidines. One of those, with pK = 5.35, is perturbed by NADP+ and NADPH but not by nucleotide fragments, indicating that this histidine is in the region of the nicotinamide binding site. Observation of nuclear Overhauser effects arising from selective irradiation at delta 7.55 indicates proximity of either a nontitrating histidine or an aromatic residue to the adenine ring of all nucleotides. In addition, selective irradiation of the methyl region of the enzyme spectrum demonstrates that the adenine ring is close to methyl side chains. The substrate magnesium isocitrate produces no observable differences in these protein--nucleotide interactions. The alterations in enzyme--nucleotide conformation that result in changes in affinity in the presence of substrate must involve either small shifts in the positions of amino acid side chains or changes in groups not visible in the proton NMR spectrum.

Adenine Nucleotides↗
PubMed1986

Kinetic and nuclear magnetic resonance study of the interaction of NADP+ and NADPH with chicken liver fatty acid synthase.

The ionic strength dependence of the second-order rate constant for the association of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and chicken liver fatty acid synthase was determined. This rate constant is 7.2 X 10(7) M-1 s-1 at zero ionic strength and 25 degrees C; the effective charge at the cofactor binding sites is +0.8. The conformations of nicotinamide adenine dinucleotide phosphate (NADP+) and NADPH bound to the beta-ketoacyl and enoyl reductase sites were determined from transferred nuclear Overhauser effect measurements. Covalent modification of the enzyme with pyridoxal 5'-phosphate abolished cofactor binding at the enoyl reductase site; this permitted the cofactor conformations at the beta-ketoacyl and enoyl reductase sites to be distinguished. For NADP+ bound to the enzyme, the conformation of the nicotinamide-ribose bond is anti at the enoyl reductase site and syn at the beta-ketoacyl reductase site; the adenine-ribose bond is anti, and the sugar puckers are C3'-endo. Nicotinamide-adenine base stacking was not detected. Structural models of NADP+ at the beta-ketoacyl and enoyl reductase sites were constructed by using the distances calculated from the observed nuclear Overhauser effects. Because of the overlap of the resonances of several nonaromatic NADPH protons with the resonances of HDO and ribose protons, less extensive structural information was obtained for NADPH bound to the enzyme. However, the conformations of NADPH bound to the two reductases are qualitatively the same as those of NADP+, except that the nicotinamide moiety of NADPH is closer to being fully anti at the enoyl reductase site.

Alcohol Oxidoreductases↗
PubMed1990

Crystal structures of Escherichia coli dihydrofolate reductase: the NADP+ holoenzyme and the folate.NADP+ ternary complex. Substrate binding and a model for the transition state.

The crystal structure of dihydrofolate reductase (EC 1.5.1.3) from Escherichia coli has been solved as the binary complex with NADP+ (the holoenzyme) and as the ternary complex with NADP+ and folate. The Bragg law resolutions of the structures are 2.4 and 2.5 A, respectively. The new crystal forms are nonisomorphous with each other and with the methotrexate binary complex reported earlier [Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650-13662]. In general, NADP+ and folate binding conform to predictions, but the nicotinamide moiety of NADP+ is disordered in the holoenzyme and ordered in the ternary complex. A mobile loop (residues 16-20) involved in binding the nicotinamide is also disordered in the holoenzyme. We report a detailed analysis of the binding interactions for both ligands, paying special attention to several apparently strained interactions that may favor the transition state for hydride transfer. Hypothetical models are presented for the binding of 7,8-dihydrofolate in the Michaelis complex and for the transition-state complex.

Catalysis↗
PubMed2002

Evaluation by mutagenesis of the roles of His309, His315, and His319 in the coenzyme site of pig heart NADP-dependent isocitrate dehydrogenase.

Sequence alignment predicts that His(309) of pig heart NADP-dependent isocitrate dehydrogenase is equivalent to His(339) of the Escherichia coli enzyme, which interacts with the coenzyme in the crystal structure [Hurley et al. (1991) Biochemistry 30, 8671-8688], and porcine His(315) and His(319) are close to that site. The mutant porcine enzymes H309Q, H309F, H315Q, and H319Q were prepared by site-directed mutagenesis, expressed in E. coli, and purified. The H319Q mutant has K(m) values for NADP, isocitrate, and Mn(2+) similar to those of wild-type enzyme, and V(max) = 20.1, as compared to 37.8 micromol of NADPH min(-1) (mg of protein)(-1) for wild type. Thus, His(319) is not involved in coenzyme binding and has a minimal effect on catalysis. In contrast, H315Q exhibits a K(m) for NADP 40 times that of wild type and V(max) = 16.2 units/mg of protein, with K(m) values for isocitrate and Mn(2+) similar to those of wild type. These results implicate His(315) in the region of the NADP site. Replacement of His(309) by Q or F yields enzyme with no detectable activity. The His(309) mutants bind NADPH poorly, under conditions in which wild type and H319Q bind 1.0 mol of NADPH/mol of subunit, indicating that His(309) is important for the binding of coenzyme. The His(309) mutants bind isocitrate stoichiometrically, as do wild-type and the other mutant enzymes. However, as distinguished from the wild-type enzyme, the His(309) mutants are not oxidatively cleaved by metal isocitrate, implying that the metal ion is not bound normally. Since circular dichroism spectra are similar for wild type, H315Q, and H319Q, these amino acid substitutions do not cause major conformational changes. In contrast, replacement of His(309) results in detectable change in the enzyme's CD spectrum and therefore in its secondary structure. We propose that His(309) plays a significant role in the binding of coenzyme, contributes to the proper coordination of divalent metal ion in the presence of isocitrate, and maintains the normal conformation of the enzyme.

Amino Acid Sequence↗
PubMed2006

Reduction of the pea ferredoxin-NADP(H) reductase catalytic efficiency by the structuring of a carboxyl-terminal artificial metal binding site.

Ferredoxin (flavodoxin)-NADP(H) reductases (FNRs) are ubiquitous flavoenzymes that deliver NADPH or low-potential one-electron donors (ferredoxin, flavodoxin, and adrenodoxin) to redox-based metabolisms in plastids, mitochondria, and bacteria. The FNRs from plants and most eubacteria constitute a unique family, the plant-type ferredoxin-NADP(H) reductases. Plastidic FNRs are quite efficient at sustaining the demands of the photosynthetic process. At variance, FNRs from organisms with heterotrophic metabolisms or anoxygenic photosynthesis display turnover numbers that are 20-100-fold lower than those of their plastidic and cyanobacterial counterparts. To gain insight into the FNR structural features that modulate enzyme catalytic efficiency, we constructed a recombinant FNR in which the carboxyl-terminal amino acid (Tyr308) is followed by an artificial metal binding site of nine amino acids, including four histidine residues. This added structure binds Zn2+ or Co2+ and, as a consequence, significantly reduces the catalytic efficiency of the enzyme by decreasing its kcat. The Km for NADPH and the Kd for NADP+ were increased 2 and 3 times, respectively, by the addition of the amino acid extension in the absence of Zn2+. Nevertheless, the structuring of the metal binding site did not change the Km for NADPH or the Kd for NADP+ of the FNR-tail enzyme. Our results provide experimental evidence which indicates that mobility of the carboxyl-terminal backbone region of the FNR, mainly Tyr308, is essential for obtaining an FNR enzyme with high catalytic efficiency.

Amino Acid Sequence↗
PubMed1999

Catalytic properties of hybrid complexes of the NAD(H)-binding and NADP(H)-binding domains of the proton-translocating transhydrogenases from Escherichia coli and Rhodospirillum rubrum.

Transhydrogenase couples reversible hydride transfer from NADH to NADP+ to proton translocation across the inner membrane in mitochondria and the cytoplasmic membrane in bacteria. The enzyme is composed of three parts. Domain I (dI) and domain III (dIII) are water soluble and contain the binding sites for NAD(H) and NADP(H), respectively; domain II (dII) spans the membrane. In the present investigation, dI from Rhodospirillum rubrum (rrI) and Escherichia coli (ecI), and dIII from R. rubrum (rrIII) and E. coli (ecIII) were overexpressed in E. coli and subsequently purified. Also, a preparation of a partially degraded E. coli transhydrogenase (ecbeta) was examined. Catalytic activities were analyzed in various dI+dIII and dI+ecbeta combinations. The abilities of the different dI+dIII combinations to catalyze cyclic transhydrogenation, i.e., the reduction of AcPyAD+ by NADH mediated via tightly bound NADP(H) in dIII, varied in the order: rrI+ecIII approximately rrI+rrIII > rrI+ecbeta >> ecI+ecIII; no measurable activities for ecI+rrIII and ecI+ecbeta were detected. Thus, rrI has a much greater apparent affinity than ecI for ecIII or rrIII or ecbeta. The pH dependences of the cyclic reaction seem to be determined by scalar protonation events on dI, both in rrI+rrIII and ecI+ecIII mixtures as well as in the wild-type R. rubrum and possibly in the E. coli enzyme. Higher reverse activities for rrI+ecbeta than for rrI+ecIII confirmed the regulatory role of dII for the association and dissociation rates of NADP(H).

Binding Sites↗
PubMed1999

The arginine 276 anchor for NADP(H) dictates fluorescence kinetic transients in 3 alpha-hydroxysteroid dehydrogenase, a representative aldo-keto reductase.

Fluorescence stopped-flow studies were conducted with recombinant rat liver 3 alpha-HSD, an aldo-keto reductase (AKR) that plays critical roles in steroid hormone inactivation, to characterize the binding of nicotinamide cofactor, the first step in the kinetic mechanism. Binding of NADP(H) involved two events: the fast formation of a loose complex (E.NADP(H)), followed by a conformational change in enzyme structure leading to a tightly bound complex (E.NADP(H)), which was observed as a fluorescence kinetic transient. Binding of NAD(H) was not characterized by a similar kinetic transient, implying a difference in the mode of binding of the two cofactors. Unlike previously characterized AKRs, the rates associated with the formation and decay of E.NADP(H) and E.NADP(H) were much faster than kcat for the oxidoreduction of various substrates, indicating that binding and release of cofactor is not rate-limiting overall in 3 alpha-HSD. Mutation of Arg 276, a highly conserved residue in AKRs that forms a salt bridge with the adenosine 2'-phosphate of NADP(H), resulted in large changes in Km and Kd for NADP(H) that were not observed with NAD(H). The loss in free energy associated with the increase in Kd for NADP(H) is consistent with the elimination of an electrostatic link. Importantly, this mutation abolished the kinetic transient associated with NADPH binding. Thus, anchoring of the adenosine 2'-phosphate of NADPH by Arg 276 appears to be obligatory for the fluorescence kinetic transients to be observed. The removal of Trp 86, a residue involved in fluorescence energy transfer with NAD(P)H, also abolished the kinetic transient, but mutation of Trp 227, a residue on a mobile loop associated with cofactor binding, did not. It is concluded that in 3 alpha-HSD, the time dependence of the change in Trp 86 fluorescence is due to cofactor anchoring, and thus, Trp 86 is a distal reporter of this event. Further, the loop movement that accompanies cofactor binding is spectrally silent.

3-Hydroxysteroid Dehydrogenases↗
PubMed1999

Kinetic evidence for the PsaE-dependent transient ternary complex photosystem I/Ferredoxin/Ferredoxin:NADP(+) reductase in a cyanobacterium.

A mutant of Synechocystis PCC 6803, deficient in psaE, assembles photosystem I reaction centers without the PsaE subunit. Under conditions of acceptor-side rate-limited photoreduction assays in vitro (with 15 microM plastocyanin included), using 100 nM ferredoxin:NADP(+) reductase (FNR) and either Synechocystis flavodoxin or spinach ferredoxin, lower rates of NADP(+) photoreduction were measured when PsaE-deficient membranes were used, as compared to the wild type. This effect of the psaE mutation proved to be due to a decrease of the apparent affinity of the photoreduction assay system for the reductase. In the psaE mutant, the relative petH (encoding FNR) expression level was found to be significantly increased, providing a possible explanation for the lack of a phenotype (i.e., a decrease in growth rate) that was expected from the lower rate of linear electron transport in the mutant. A kinetic model was constructed in order to simulate the electron transfer from reduced plastocyanin to NADP(+), and test for possible causes for the observed change in affinity for FNR. The numerical simulations predict that the altered reduction kinetics of ferredoxin, determined for the psaE mutant [Barth, P., et al., (1998) Biochemistry 37, 16233-16241], do not significantly influence the rate of linear electron transport to NADP(+). Rather, a change in the dissociation constant of ferredoxin for FNR does affect the saturation profile for FNR. We therefore propose that the PsaE-dependent transient ternary complex PSI/ferredoxin/FNR is formed during linear electron transport. Using the yeast two-hybrid system, however, no direct interaction could be demonstrated in vivo between FNR and PsaE fusion proteins.

Base Sequence↗
PubMed2000

Evaluation by site-directed mutagenesis of aspartic acid residues in the metal site of pig heart NADP-dependent isocitrate dehydrogenase.

Pig heart NADP-dependent isocitrate dehydrogenase requires a divalent metal cation for catalysis. On the basis of affinity cleavage studies [Soundar and Colman (1993) J. Biol. Chem. 268, 5267] and analysis of the crystal structure of E. coli NADP-isocitrate dehydrogenase [Hurley et al. (1991) Biochemistry 30, 8671], the residues Asp(253), Asp(273), Asp(275), and Asp(279) were selected as potential ligands of the divalent metal cation in the pig heart enzyme. Using a megaprimer PCR method, the Asp at each of these positions was mutated to Asn. The wild-type and mutant enzymes were expressed in Escherichia coli and purified. D253N has a specific activity, K(m) values for Mn(2+), isocitrate, and NADP, and also a pH-V(max) profile similar to those of the wild-type enzyme. Thus, Asp(253) is not involved in enzyme function. D273N has an increased K(m) for Mn(2+) and isocitrate with a specific activity 5% that of wild type. The D273N mutation also prevents the oxidative metal cleavage seen with Fe(2+) alone in the wild-type enzyme. As compared to wild type, D275N has greatly increased K(m) values for Mn(2+) and isocitrate, with a specific activity <0.1% that of wild type, and a large increase in pK(a) for the enzyme-substrate complex. D279N has only small increases in K(m) for Mn(2+) and isocitrate, but a specific activity <0.1% that of wild type and a major change in the shape of its pH-V(max) profile. These results suggest that Asp(273) and Asp(275) contribute to metal binding, whereas Asp(279), as well as Asp(275), is critical for catalysis. Asp(279) may function as the catalytic base. Using the Modeler program of Insight II, a structure for porcine NADP-isocitrate dehydrogenase was built based on the X-ray coordinates of the E. coli enzyme, allowing visualization of the metal-isocitrate site.

Amino Acid Sequence↗
PubMedDate not supplied

Formation of NADP(H) analogs of tobacco-specific nitrosamines in rat liver and pancreatic microsomes.

The metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), was examined in rat pancreatic microsomes. No pyridine N-oxidation or alpha-hydroxylation were observed in these preparations. However, one unidentified metabolite of NNAL (unknown A) and two unknown metabolites of NNK (unknowns B and C) were formed. These metabolites were also detected in rat liver microsomal incubations of NNK and NNAL. Studies using [5-3H]-NNK and [Me-3H]NNK demonstrated that the metabolites contained both the pyridyl and methyl portions of the parent compound. Similar results were obtained with NNAL. Formation of unknown C required active microsomes, NADP+, and an NADPH regenerating system. The regenerating system was not required for the formation of NNAL unknown metabolite A or NNK unknown metabolite B. Chemical characterization of unknowns A and B by NMR, UV, and electrospray ionization MS demonstrated that they are NADP+ analogs in which the nicotinamide portion has been replaced by NNAL or NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol adenosine dinucleotide phosphate [(NNAL)ADP+] and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone adenosine dinucleotide phosphate [(NNK)ADP+]. Unknown C was identified as (NNK)ADPH. Both (NNK)ADP+ and (NNK)ADPH were formed from NNK while only (NNAL)ADP+ was produced from NNAL. These NADP+ derivatives were also formed when porcine brain NAD+ glycohydrolase was incubated with NADP+ and NNK or NNAL. These results indicate that NNK and NNAL are substrates for rat liver and pancreatic microsomal NAD+ glycohydrolase-catalyzed transglycosylation reactions.

Adenosine Diphosphate↗
PubMed1998

Alternative splicing of a precursor-mRNA encoded by the Chlorella sorokiniana NADP-specific glutamate dehydrogenase gene yields mRNAs for precursor proteins of isozyme subunits with different ammonium affinities.

Chlorella sorokiniana has seven ammonium-inducible, chloroplastic NADP-specific glutamate dehydrogenase (NADP-GDH) isozymes composed of varying ratios of alpha- and beta-subunits. Southern blot and allele-specific PCR analyses indicate that the C. sorokiniana genome possesses a single 7178 bp nuclear NADP-GDH gene. cDNA cloning and sequencing, 5'-RACE-PCR analysis, and RNase protection analysis identified two NADP-GDH mRNAs that are identical with the exception of a 42 nt sequence located within the 5'-coding region of the longer mRNA. The 42 nt sequence, termed an auxon because it serves as an exon or intron, appears to undergo alternative splicing from the precursor mRNA by a process that is regulated by both nutritional and environmental signals. Depending upon whether the auxon is included or excluded in a mature mRNA, the gene can be considered to consist of 22 or 23 exons, respectively. The 2074 and 2116 nt mRNAs encode precursor proteins of 56,350 and 57,850 Da, respectively. The N-termini of the purified mature alpha- and beta-subunits were sequenced, identifying full-length subunits of 53,501 and 52,342 Da, respectively. The sequences of the subunits are identical except for an 11 amino acid extension at the N-terminus of the alpha-subunit. The alpha-subunit has an additional alpha-helical domain at its N-terminus compared with the beta-subunit. By correlating the abundances of the two mRNAs with the levels (and relative turnover rates) of the alpha- and beta-subunit antigens during induction in Chlorella, the larger mRNA is proposed to encode the larger subunit.

Alternative Splicing↗
PubMed1977

An examination of potential matrices for the affinity chromatography of NADP+-linked dehydrogenases with special reference to 6-phosphogluconate dehydrogenase.

1. 6-phosphogluconate dehydrogenase from sheep liver has been purified 350-fold by affinity chromatography with a final specific activity of 18 micronmol of NADP+/reduced min per mg of protein and an overall yield of greater than 40%. 2. A systematic investigation of potential ligands has been carried out: these included 6-phosphogluconate and NADP+, pyridoxal phosphate and several immobilized nucleotides. The results indicate that NADP+ is the most suitable ligand for the purification of 6-phosphogluconate dehydrogenase. 3. The effects of pH and alternative eluents have been examined in relation to the parameters known to affect the desorption phase of affinity chromatography; careful manipulation of the elution conditions permitted the separation of glucose 6-phosphate dehydrogenase, glutathione reductase and 6-phosphogluconate dehydrogenase from sheep liver on NADP+-Sepharose 4B. 4. A large-scale purification scheme for 6-phosphogluconate dehydrogenase is presented that uses the competitive inhibitors inorganic pyrophosphate and citrate as specific eluents.

Animals↗
PubMed1978

The selective retardation of NADP+-dependent dehydrogenases by immobilized procion red HE-3B.

The capacities of Procion Red HE-3B and Cibacron Blue F3G-A immobilized to Sepharose CL-4B and Matrex 201R for NAD+-, NADP+- and NAD(P)+-dependent dehydrogenases were measured. Procion Red HE-3B columns retarded NADP+-dependent dehydrogenases more effectively than NAD+-dependent dehydrogenases, whilst immobilized Cibacron Blue F3G-A retarded NAD+-dependent dehydrogenases more effectively than NADP+-dependent dehydrogenases. The capacity of procion Red HE-3B-Sepharose CL-4B for five dehydrogenases was highest in the region of 70nmol of immobilized ligand/ml of settled gel. The effects of using poly(ethyleneimine) as a spacer for both porous and pellicular supports were also examined. Four NADP+-dependent dehydrogenases were purified from yeast extract by using Procion Red HE-3B-Sepharose CL-4B. Two NAD+-dependent dehydrogenases were purified from the same source using Cibacron Blue F3G-A-Sepharose CL-4B. These results are discussed in relation to the use of immobilized Procion Red HE-3B to purify dehydrogenases. This immobilized dye's chromatograhic behaviour is compared with that of immobilized nucleotides. The most important feature of immobilized tirazine dyes seems to be their high operational capacities when compared with group-specific nucleotide adsorbents.

Chromatography, Affinity↗