Aldose reductase,NADPH and NADP+ in normal,galactose-fed and diabetic rat lens

NADPH and NADP⁺ levels were measured in rat lens from normal controls, from galactose-fed and diabetic rats during the first week of cataract formation.The level of NADPH in normal rat lens was determined to be 12.3 ± 0.4 nmol/g wet weight, and that of NADP⁺ 4.6 ± 0.2 nmol/g wet weight. In early cataract formation NADPH levels decreased rapidly during the first 2 days and then remained stable at 76% of control for galactose-fed and 84% for diabetic rats. NADP⁺ levels increased by 38% of control for galactose-fed and 54% for diabetic rats. Calculated NADPH/NADP⁺ ratios dropped from 3.36 ± 0.21 to 1.86 ± 0.16 in galactose fed rats, and from 2.81 ± 0.15 to 1.61 ± 0.16 in diabetic rats (P < 0.001 for both experimental groups). These data are consistent with rapid NADPH oxidation during onset of lens cataracts. No significant changes in aldose reductase enzymatic activity levels were observed in either the galactosemic or the diabetic rats during the times measured.     

Inhibition of adrenodoxin reductase by NADP+ and NADPH

Adrenodoxin reductase (EC 1.18.1.2) catalyzes the oxidation of NADPH by 1.4-benzoquinone. The catalytic constant of this reaction at pH 7.0 is equal to 25-28 s-1. NADP+ acts as the mixed-type nonlinear inhibitor of enzyme increasing Km of NADPH and decreasing catalytic constant. NADP+ and NADPH act as mutually exclusive inhibitors relative to reduced adrenodoxin reductase. The patterns of 2',5'-ADP inhibition are analogous to that of NADP+. These data support the conclusion about the existence of second nicotinamide coenzyme binding centre in adrenodoxin reductase.     

Aldose reductase deficiency protects sugar-induced lens opacification in rats

Aldose reductase (AKR1B1), which catalyzes the reduction of glucose to sorbitol and lipid aldehydes to lipid alcohols, has been shown to be involved in secondary diabetic complications including cataractogenesis. Rats have high levels of AKR1B1 in lenses and readily develop diabetic cataracts, whereas mice have very low levels of AKR1B1 in their lenses and are not susceptible to hyperglycemic cataracts. Studies with transgenic mice that over-express AKR1B1 indicate that it is the key protein for the development of diabetic complications including diabetic cataract. However, no such studies were performed in genetically altered AKR1B1 rats. Hence, we developed siRNA-based AKR1B1 knockdown rats (ARKO) using the AKR1B1-siRNA-pSuper vector construct. Genotyping analysis suggested that more than 90% of AKR1B1 was knocked down in the littermates. Interestingly, all the male animals were born dead and only 3 female rats survived. Furthermore, all 3 female animals were not able to give birth to F1 generation. Hence, we could not establish an AKR1B1 rat knockdown colony. However, we examined the effect of AKR1B1 knockdown on sugar-induced lens opacification in ex vivo. Our results indicate that rat lenses obtained from AKR1B1 knockdown rats were resistant to high glucose-induced lens opacification as compared to wild-type (WT) rat lenses. Biochemical analysis of lens homogenates showed that the AKR1B1 activity and sorbitol levels were significantly lower in sugar-treated AKR1B1 knockdown rat lenses as compared to WT rat lenses treated with 50mM glucose. Our results thus confirmed the significance of AKR1B1 in the mediation of sugar-induced lens opacification and indicate the use of AKR1B1 inhibitors in the prevention of cataractogenesis.     

Adrenodoxin reductase. Properties of the complexes of reduced enzyme with NADP+ and NADPH.

Anaerobic reduction of the flavoprotein adrenodoxin reductase with NADPH yields a spectrum with long wavelength absorbance, 750 nm and higher. No EPR signal is observed. This spectrum is produced by titration of oxidized adrenodoxin reductase with NADPH, or of dithionite-reduced adrenodoxin reductase with NADP+. Both titrations yield a sharp endpoint at 1 NADP(H) added per flavin. Reduction with other reductants, including dithionite, excess NADH, and catalytic NADP+ with an NADPH generating system, yields a typical fully reduced flavin spectrum, without long wavelength absorbance. The species formed on NADPH reduction appears to be a two-electron-containing complex, with a low dissociation constant, between reduced adrenodoxin reductase and NADP+, designated ARH2-NADP+. Titration of dithionite-reduced adrenodoxin reductase with NADPH also produces a distinctive spectrum, with a sharp endpoint at 1 NADPH added per reduced flavin, indicating formation of a four-electron-containing complex between reduced adrenodoxin reductase and NADPH. Titration of adrenodoxin reductase with NADH, instead of NADPH, provides a curved titration plot rather than the sharp break seen with NADPH, and permits calculation of a potential for the AR/ARH2 couple of -0.291 V, close to that of NAD(P)H (-0.316 V). Oxidized adrenodoxin reductase binds NADP+ much more weakly (Kdiss=1.4 X 10(-5) M) than does reduced adrenodoxin reductase, with a single binding site. The preferential binding of NADP+ to reduced enzyme permits prediction of a more positive oxidation-reduction potential of the flavoprotein in the presence of NADP+; a change of about + 0.1 V has been demonstrated by titration with safranine T. From this alteration in potential, a Kdiss of 1.0 X 10(-8) M for binding of NADP+ to reduced adrenodoxin reductase is calculated. It is concluded that the strong binding of NADP+ to reduced adrenodoxin reductase provides the thermodynamic driving force for formation of a fully reduced flavoprotein form under conditions wherein incomplete reduction would otherwise be expected. Stopped flow studies demonstrate that reduction of adrenodoxin reductase by equimolar NADPH to form the ARH2-NADP+ complex is first order (k=28 s-1). When a large excess of NADPH is used, a second apparently first order process is observed (k=4.25 s-1), which is interpreted as replacement of NADPH for NADP+ in the ARH2-NADP+ complex. Comparison of these rate constants to catalytic flavin turnover numbers for reduction of various oxidants by NADPH, suggests an ordered sequential mechanism in which reduction of oxidant is accomplished by the ARH2-NADP+ complex, followed by dissociation of NADP+. The absolute dependence of NADPH-cytochrome c reduction on both adrenodoxin reductase and adrenodoxin is confirmed...     

NADPH regulates human NAD kinase, a NADP+-biosynthetic enzyme

NAD kinase (NADK, EC 2.7.1.23) is the sole NADP(+)-biosynthetic enzyme that catalyzes phosphorylation of NAD(+) to yield NADP(+) using ATP as a phosphoryl donor, and thus, plays a vital role in the cell and represents a potentially powerful antimicrobial drug target. Although methods for expression and purification of human NADK have been previously established (Lerner et al. Biochem Biophys Res Commun 288:69-74, 2001), the purification procedure could be significantly improved. In this study, we improved the method for expression and purification of human NADK in Escherichia coli and obtained a purified homogeneous enzyme only through heat treatment and single column chromatography. Using the purified human NADK, we revealed a sigmoidal kinetic behavior toward ATP and the inhibitory effects of NADPH and NADH, but not of NADP(+), on the catalytic activity of the enzyme. These inhibitory effects provide insight into the regulation of intracellular NADPH synthesis. Furthermore, these attributes may provide a clue to design a novel drug against Mycobacterium tuberculosis in which this bacterial NADK is potently inhibited by NADP(+).     

Aldose reductase and sorbitol dehydrogenase distribution in rat kidney.

The sorbitol pathway catalyzes the conversion of glucose to fructose via the intermediate sorbitol. It consists of aldose reductase (AR) and sorbitol dehydrogenase (SDH). In adult (44 day) kidney zones, AR was highest in the outer medulla. In substructures AR was highest in distal convoluted tubule. The AR was greatest in newborn and 8-day zones of developing rat kidney. Acute alloxan diabetes was associated with decreased AR in small arteries, but not glomeruli. The SDH was lowest in outer medulla. It was most active in glomeruli and distal convoluted tubules. The diabetic state leads to no change of SDH in arteries but an increase in glomeruli. SDH increased with development. This study demonstrates AR and SDH in substructures of the kidney. The pathway is present in developing kidney. In diabetes the enzymatic changes would tend to decrease accumulation of sorbitol.     

NADPH/NADP+ ratios in photosynthesizing reconstituted chloroplasts.

Levels of reduced and oxidized triphosphopyridine nucleotides have been determined in reconstituted spinach chloroplasts and compared with levels in whole isolated chloroplasts during photosynthesis and darkness. The ratio of NADPH/NADP+ reaches values slightly above 1.0 at the beginning of photosynthesis, less than half the ratio attained with whole chloroplasts. Nonetheless these lower ratios are sufficient to maintain high rates of photosynthetic carbon dioxide fixation and reduction, which are comparable in the reconstituted chloroplasts to the rates found with whole chloroplasts. As with whole chloroplasts there is a decline in the ration of NADPH/NADP+ as a function of time of photosynthesis. The effect of addition of bicarbonate (6 mM) in causing a transient drop in the ratio of NADPH/NADP/ is described and discussed in terms of the reversibility of the reduction of 3-phosphoglycerate to triose phosphate. The ratio NADPH/NADP+ can be improved by the addition of more lamellae either before or during the course of photosynthesis, and this improvement in ratio is accompanied by an improved rate of CO2 fixation or a more sustained rate of CO2 fixation with time of photosynthesis. The importance of NADPH/NADP+ ratio not only to the reduction of 3-phosphoglycerate to triose phosphate but also to the activation of the ribulose-1,5-diphosphate carboxylasemediated step is discussed.     

Higher plant NADP+-dependent isocitrate dehydrogenases,ammonium assimilation and NADPH production

The assimilation of ammonium into glutamate is mainly achieved by the GS/GOGAT pathway and requires carbon skeletons in the form of 2-oxoglutarate. To date, the exact enzymatic origin of this organic acid for plant ammonium assimilation is unknown. NADP⁺-dependent isocitrate dehydrogenases can carry out this function and the recent efforts concentrated on evaluating the involvement of different isoforms, distinguished by their subcellular localisation, are analysed. Furthermore, a possible role for these enzymes in the production of NADPH for redox-regulated cell metabolism, such as the recycling of glutathione required in response to oxidative stress will be discussed.     

NADPH Production from NADP+ by a Formate-utilizing Methanogenic Bacterium

Resting cells of the methanogen strain HU, a formate-utilizing methanogenic bacterium, was able to utilize formate or hydrogen as electron donor for the production of NADPH from NADP⁺ under suitable conditions. In the presence of 0.2% Triton X-100 and 0.3 m potassium phosphate, pH 9.0 at 30°C, the resting cells could convert ca. 60% of the exogenous NADP⁺ into NADPH yielding ca. 6 g NADPH/liter. Phosphate ions greatly enhanced the NADPH production.     

Reduction of trioses by NADPH-dependent aldo-keto reductases. Aldose reductase, methylglyoxal, and diabetic complications.

The substrate specificities of human aldose reductase and aldehyde reductase toward trioses, triose phosphates, and related three-carbon aldehydes and ketones were evaluated. Both enzymes are able to catalyze the NADPH-dependent reduction of all of the substrates used. Aldose reductase shows more discrimination among substrates than does aldehyde reductase and is generally the more efficient catalyst. The best substrate for aldose reductase is methylglyoxal (kcat = 142 min-1, kcat/Km = 1.8 x 10(7) M-1 min-1), a toxic 2-oxo-aldehyde that is produced nonenzymatically from triose phosphates and enzymatically from acetone/acetol metabolism. D- and L-glyceraldehyde and D- and L-lactaldehyde are also good substrates for aldose reductase. The aldose reductase-catalyzed reduction of methylglyoxal produces 95% acetol, 5% D-lactaldehyde. Further reduction of acetol produces only L-1,2-propanediol. Acetol and propanediol are two products that accumulate in uncontrolled diabetes. Both acetol and methylglyoxal were compared with glucose for their abilities to produce covalent modification of albumin. All three of these carbonyl compounds reacted with albumin to produce modified proteins with new absorption and emission bands that are spectrally similar. Both methylglyoxal and acetol are much more reactive than glucose. A new integrative model of diabetic complications is proposed that combines the aldose reductase/polyol pathway theory and the nonenzymatic glycation theory except that emphasis is placed both on methylglyoxal/acetol metabolism and on glucose metabolism.     

The mitochondrial external NADPH dehydrogenase modulates the leaf NADPH/NADP+ ratio in transgenic Nicotiana sylvestris

Plant mitochondria contain alternative external NAD(P)H dehydrogenases,which oxidize cytosolic NADH or NADPH and reduce ubiquinonewithout inherent linkage to proton pumping and ATP production.In potato, St-NDB1 is an external Ca²⁺-dependent NADPH dehydrogenase.The physiological function of this enzyme was investigated inhomozygous Nicotiana sylvestris lines overexpressing St-ndb1and co-suppressing St-ndb1 and an N. sylvestris ndb1. In leafmitochondria isolated from the overexpressor lines, higher activityof alternative oxidase (AOX) was detected. However, the AOXinduction was substantially weaker than in the complex I-deficientCMSII mutant, previously shown to contain elevated amounts ofNAD(P)H dehydrogenases and AOX. An aox1b and an aox2 gene wereup-regulated in CMSII, but only aox1b showed a response, albeitsmaller, in the transgenic lines, indicating differences inAOX activation between the genotypes. As in CMSII, the increaseof AOX in the overexpressing lines was not due to a generaloxidative stress. The lines overexpressing St-ndb1 had consistentlylowered leaf NADPH/NADP⁺ ratios in the light and variably decreasedlevels in darkness, but unchanged NADH/NAD⁺ ratios. CMSII insteadhad similar NADPH/NADP⁺ and lower NADH/NAD⁺ ratios than thewild type. These results demonstrate that St-NDB1 is able tomodulate the cellular balance of NADPH and NADP⁺ at least inthe day and that reduction of NADP(H) and NAD(H) is independentlycontrolled. Similar growth rates, chloroplast malate dehydrogenaseactivation and xanthophyll ratios indicate that the change inreduction does not communicate to the chloroplast, and thatthe cell tolerates significant changes in NADP(H) reductionwithout deleterious effects.     

Complex-forming properties of butanedione-modified ferredoxin-NADP+ reductase with NADP+ and ferredoxin.

The plastidic ferredoxin-NADP⁺ reductase from the xanthophycean alga Bumilleriopsis forms a stoichiometric 1:1 complex with ferredoxin and NADP⁺ which is demonstrated by difference spectra of both complexes. Butanedione modification of the flavoprotein results in loss of its enzymatic activities (transhydrogenase and diaphorase) concurrently with its capability to form a complex with NADP⁺, whereas the ferredoxin-binding site is practically not influenced by the modifying reagent and complex formation is still possible. It is assumed, therefore, that butanedione specifically reacts with the arginine residue of the protein involved in binding of pyridine nucleotides at the active site. Further, the data presented strongly support the previous proposal of different binding sites for ferredoxin and pyridine nucleotides at the reductase.     

NADP+ and NADPH in glucose-6-phosphate dehydrogenase-deficient erythrocytes under oxidative stimulation.

A mild oxidative stimulation of the hexose monophosphate pathway of human glucose-6-phosphate dehydrogenase (EC 1.1.1.49)-deficient erythrocytes (Mediterranean variant) causes a significant drop in NADPH. These results, other than to confirm that glucose-6-phosphate dehydrogenase deficiency is a product deficiency disorder, demonstrate that under oxidative stimulation glutathione reductase may become functionally impaired and GSSG cannot be reduced at a sufficient rate.     

Crystal structures of adrenodoxin reductase in complex with NADP+ and NADPH suggesting a mechanism for the electron transfer of an enzyme family

Adrenodoxin reductase is a flavoenzyme that shuffles electrons for the biosynthesis of steroids. Its chain topology belongs to the glutathione reductase family of disulfide oxidoreductases, all of which bind FAD at equivalent positions. The three reported structures of adrenodoxin reductase were ligated with reduced and oxidized NADP and have now confirmed this equivalence also for the NADP-binding site. Remarkably, the conformations and relative positions of the prosthetic group FAD and the cofactor NADP have been conserved during protein evolution despite very substantial changes in the polypeptide. The ligated enzymes showed small changes in the domain positions. When compared with the structure of the NADP-free enzyme, these positions correspond to several states of the domain motion during NADP binding. On the basis of the observed structures, we suggest an enzymatic mechanism for the subdivision of the received two-electron package into the two single electrons transferred to the carrier protein adrenodoxin. The data banks contain 10 sequences that are closely related to bovine adrenodoxin reductase. Most of them code for gene products with unknown functions. Within this family, the crucial residues of adrenodoxin reductase are strictly conserved. Moreover, the putative docking site of the carrier is rather well conserved. Five of the family members were assigned names related to ferredoxin:NADP(+) reductase, presumably because adrenodoxin reductase was considered a member of this functionally similar family. Since this is not the case, the data bank entries should be corrected.     

NADPH/NADP+ ratio: regulatory implications in yeast glyoxylic acid cycle

Summary The utilization by yeast of two carbon sources is carried out through the operation of the glyoxylic acid cycle. Kinetic data from the isocitrate transforming enzymes suggest that the flow of isocitrate through the glyoxylic acid cycle depends upon the inhibition of the isocitrate decarboxylating enzymes. Both isocitrate dehydrogenases are inhibited by a mixture of glyoxylate + oxaloacetate, but for the reasons described in the text we consider that this inhibition is of no physiological significance. On the other hand, we have found that NADPH is a competitive inhibitor of NADP-isocitrate dehydrogenase with respect to NADP⁺, with a K_I similar to its K_M. It also produces an additive effect on the NADH-produced inhibition of NAD-isocitrate dehydrogenase. We propose NADPH as the compound that channels the utilization of isocitrate into the glyoxylic acid cycle. This is supported by the finding of an increased NADPH/NADP⁺ ratio in acetate grown yeast with respect to glucose grown cells.     

Aldose reductase inhibition counteracts nitrosative stress and poly(ADP-ribose) polymerase activation in diabetic rat kidney and high-glucose-exposed human mesangial cells

Both increased aldose reductase (AR) activity and oxidative/nitrosative stress have been implicated in the pathogenesis of diabetic nephropathy, but the relation between the two factors remains a subject of debate. This study evaluated the effects of AR inhibition on nitrosative stress and poly(ADP-ribose) polymerase (PARP) activation in diabetic rat kidney and high-glucose-exposed human mesangial cells. In animal experiments, control (C) and streptozotocin-diabetic (D) rats were treated with/without the AR inhibitor fidarestat (F, 16 mg kg(-1) day(-1)) for 6 weeks starting from induction of diabetes. Glucose, sorbitol, and fructose concentrations were significantly increased in the renal cortex of D vs C (p < 0.01 for all three comparisons), and sorbitol pathway intermediate, but not glucose, accumulation, was completely prevented in D + F. F at least partially prevented diabetes-induced increase in kidney weight as well as nitrotyrosine (NT, a marker of peroxynitrite-induced injury and nitrosative stress), and poly(ADP-ribose) (a marker of PARP activation) accumulation, assessed by both immunohistochemistry and Western blot analysis, in glomerular and tubular compartments of the renal cortex. In vitro studies revealed the presence of both AR and PARP-1 in human mesangial cells, and none of these two variables were affected by high glucose or F treatment. Nitrosylated and poly(ADP-ribosyl)ated proteins (Western blot analysis) accumulated in cells cultured in 30 mM D-glucose (vs 5.55 mM glucose, p < 0.01), but not in cells cultured in 30 mM L-glucose or 30 mM D-glucose plus 10 microM F. AR inhibition counteracts nitrosative stress and PARP activation in the diabetic renal cortex and high-glucose-exposed human mesangial cells. These findings reveal new beneficial properties of the AR inhibitor F and provide the rationale for detailed studies of F on diabetic nephropathy.