RESUMEN
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+ mobilizing second messenger which triggers Ca2+ release in both sea urchin egg homogenates and in mammalian cells. The NAADP binding protein has not been identified and the regulation of NAADP mediated Ca2+ release remains controversial. To address this issue, we have synthesized an NAADP analog in which 3-azido-5-azidomethylbenzoic acid is attached to the amino group of 5-(3-aminopropyl)-NAADP to produce an NAADP analog which is both a photoaffinity label and clickable. This 'all-in-one-clickable' NAADP (AIOC-NAADP) elicited Ca2+ release when microinjected into cultured human SKBR3 cells at low concentrations. In contrast, it displayed little activity in sea urchin egg homogenates where very high concentrations were required to elicit Ca2+ release. In mammalian cell homogenates, incubation with low concentrations of [32P]AIOC-NAADP followed by irradiation with UV light resulted in labeling 23â¯kDa protein(s). Competition between [32P]AIOC-NAADP and increasing concentrations of NAADP demonstrated that the labeling was selective. We show that this label recognizes and selectively photodervatizes the 23â¯kDa NAADP binding protein(s) in cultured human cells identified in previous studies using [32P]5-N3-NAADP.
Asunto(s)
Ácido Benzoico/síntesis química , Calcio/metabolismo , Química Clic/métodos , NADP/análogos & derivados , Etiquetas de Fotoafinidad/síntesis química , Animales , Sitios de Unión , Señalización del Calcio , Línea Celular Tumoral , Humanos , NADP/síntesis química , NADP/aislamiento & purificación , Etiquetas de Fotoafinidad/aislamiento & purificación , Unión Proteica , Erizos de MarRESUMEN
Nicotinamide adenine dinucleotides are critical redox-active substrates for countless catabolic and anabolic reactions. Ratios of NAD(+) to NADH and NADP(+) to NADPH are therefore considered key indicators of the overall intracellular redox potential and metabolic state. These ratios can be measured in bulk conditions using a highly sensitive enzyme cycling-based colorimetric assay (detection limit at or below 0.05 µM or 1 pmol) following a simple extraction procedure involving solutions of acid and base. Special considerations are necessary to avoid measurement artifacts caused by the presence of endogenous redox-active metabolites, such as phenazines made by diverse Pseudomonas species (see Chapter 25).
Asunto(s)
Bioquímica/métodos , NADP/análisis , NADP/aislamiento & purificación , Pseudomonas/metabolismo , Estándares de Referencia , Estadística como AsuntoRESUMEN
Clostridial glutamate dehydrogenase mutants, designed to accommodate the 2'-phosphate of disfavoured NADPH, showed the expected large specificity shifts with NAD(P)H. Puzzlingly, similar assays with oxidized cofactors initially revealed little improvement with NADP(+) , although rates with NAD(+) were markedly diminished. This article reveals that the enzyme's discrimination in favour of NAD(+) and against NADP(+) had been greatly underestimated and has indeed been abated by a factor of >â16,000 by the mutagenesis. Initially, stopped-flow studies of the wild-type enzyme showed a burst increase of A(340) with NADP(+) but not NAD(+), with amplitude depending on the concentration of the coenzyme, rather than enzyme. Amplitude also varied with the commercial source of the NADP(+). FPLC, HPLC and mass spectrometry identified NAD(+) contamination ranging from 0.04 to 0.37% in different commercial samples. It is now clear that apparent rates of NADP(+) utilization mainly reflected the reduction of contaminating NAD(+), creating an entirely false view of the initial coenzyme specificity and also of the effects of mutagenesis. Purification of the NADP(+) eliminated the burst. With freshly purified NADP(+), the NAD(+) : NADP(+) activity ratio under standard conditions, previously estimated as 300 : 1, is 11,000. The catalytic efficiency ratio is even higher at 80,000. Retested with pure cofactor, mutants showed marked specificity shifts in the expected direction, for example, 16 200 fold change in catalytic efficiency ratio for the mutant F238S/P262S, confirming that the key structural determinants of specificity have been successfully identified. Of wider significance, these results underline that, without purification, even the best commercial coenzyme preparations are inadequate for such studies.
Asunto(s)
Proteínas Bacterianas/metabolismo , Clostridium symbiosum/enzimología , Glutamato Deshidrogenasa/metabolismo , Proteínas Mutantes/metabolismo , NADP/metabolismo , NAD/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Clostridium symbiosum/genética , Contaminación de Medicamentos , Glutamato Deshidrogenasa/química , Glutamato Deshidrogenasa/genética , Concentración de Iones de Hidrógeno , Indicadores y Reactivos/química , Cinética , Proteínas Mutantes/química , NAD/análisis , NADP/análisis , NADP/aislamiento & purificación , Concentración Osmolar , Oxidación-Reducción , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Espectrometría de Fluorescencia , Espectrofotometría UltravioletaRESUMEN
Simultaneous extraction, separation and quantitation of reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) in Chinese Hamster Ovary (CHO) cells were investigated. The separation of flavins and nicotinamide cofactors was performed by capillary electrophoresis with laser-induced fluorescence detection at the excitation wavelength of 325 nm. The separation protocol was established by investigating the excitation wavelength, high voltage and effects of buffer nature, pH and concentration. All endogenous fluorophores riboflavin, FAD, FMN, NADH and NADPH show wide linear range of quantitation. The limits of detection for the five compounds ranged from 4.5 to 23 nM. Extraction conditions were optimized for high-efficiency recovery of all endogenous fluorophores from CHO cells. To account for the complex matrix of cell extracts, a standard addition method was used to quantify FAD, FMN, NADH and NADPH in CHO cells. The quantitative results should be useful to reveal the metabolic status of cells. The protocols for extraction, separation and quantitation are readily adaptable to normal and cancer cell lines for the analysis of endogenous fluorophores.
Asunto(s)
Electroforesis Capilar/métodos , Flavinas/aislamiento & purificación , Niacinamida/aislamiento & purificación , Animales , Células CHO , Cricetinae , Cricetulus , NAD/aislamiento & purificación , NADP/aislamiento & purificación , Reproducibilidad de los Resultados , Espectrometría de FluorescenciaRESUMEN
The measurement of changes in nicotinic acid adenine dinucleotide phosphate (NAADP) levels in cells has been, and remains, key to the investigation of the functions of NAADP as a Ca2+ -releasing second messenger. Here we provide details of how to isolate NAADP from cells by extraction with perchloric acid and then measure the NAADP using a radioreceptor assay. We demonstrate that NAADP is neither generated nor broken down during sample processing conditions and that radioreceptor assay is highly selective for the detection of NAADP under cell extract conditions. Furthermore, a number of improvements, such as solid-state detection of the radioactivity, are incorporated to enhance the safety of the procedure. Finally, we have developed a new method to prevent the endogenous metabolism of NAADP by chelating Ca2+ with bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), thereby reducing the difficulty of catching a small transient rise in NAADP levels. In summary, we have refined and improved a method for measuring NAADP levels and presented it in a manner accessible to a wide range of laboratories. It is expected that this will enhance research in the NAADP field.
Asunto(s)
Señalización del Calcio/fisiología , NADP/análogos & derivados , Animales , Calcio/metabolismo , Extractos Celulares/análisis , Quelantes/farmacología , Cromatografía Líquida de Alta Presión , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Embrión no Mamífero , Concentración de Iones de Hidrógeno , Masculino , Ratones , Ratones Endogámicos , Microinyecciones , Modelos Biológicos , NADP/análisis , NADP/aislamiento & purificación , NADP/metabolismo , Oocitos/metabolismo , Oxidantes/farmacología , Páncreas Exocrino/citología , Percloratos/farmacología , Radioisótopos de Fósforo/metabolismo , Unión Proteica , Ensayo de Unión Radioligante , Erizos de Mar/citología , Erizos de Mar/embriología , Sistemas de Mensajero Secundario , Espermatozoides/metabolismo , Factores de Tiempo , VolumetríaRESUMEN
Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to mobilize Ca(2+) from intracellular stores in a wide variety of organisms, ranging from plants to humans. We have developed a novel enzyme cycling assay for NAADP that involves coupled reactions catalyzed by four enzymes. In this system, NAADP is first converted into nicotinic acid adenine dinucleotide (NAAD) by alkaline phosphatase, after which the NAAD is converted to NAD, AMP, and PPi by NAD synthetase (NADS) in the presence of ATP and ammonia. The NAD is then amplified using an enzyme cycling system driven by glucose dehydrogenase and diaphorase. The resultant formation of formazan dye is measured spectrophotometrically based on the increase in absorbance at 450 nm. Using this method, NAADP (20-400 nM) was assayed, and a highly linear correlation was obtained between the NAADP concentration and the increase in absorbance at 450 nm. The cycling rate was approximately 95 cycles/min. In addition, the within-run coefficients of variation (CVs) for 25, 50, and 100 nM NAADP solutions were 9.33, 4.86, and 3.13%, respectively. Interference by NAD analogs (e.g., NAAD, NADP) in the sample was eliminated prior to running the assay by treating the sample with NADS and NAD nucleosidase (NADase). In sum, our findings indicate this enzyme cycling assay to be readily applicable for determination for NAADP in a variety of biological samples and to be particularly appropriate for use with an autoanalyzer.
Asunto(s)
Amida Sintasas/química , NADP/análogos & derivados , Fosfatasa Alcalina/química , Fosfatasa Alcalina/metabolismo , Amida Sintasas/metabolismo , Animales , Automatización , Bioensayo , Estudios de Evaluación como Asunto , Glucosa 1-Deshidrogenasa/química , Glucosa 1-Deshidrogenasa/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Modelos Químicos , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , NAD+ Nucleosidasa/química , NAD+ Nucleosidasa/metabolismo , NADP/química , NADP/aislamiento & purificación , NADP/metabolismo , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
In order to identify an enzyme capable of Fenton reaction in Synechocystis, we purified an enzyme catalyzing one-electron reduction of t-butyl hydroperoxide in the presence of FAD and Fe(III)-EDTA. The enzyme was a 26 kDa protein, and its N-terminal amino acid sequencing revealed it to be DrgA protein previously reported as quinone reductase [Matsuo M, Endo T and Asada K (1998) Plant Cell Physiol39, 751-755]. The DrgA protein exhibited potent quinone reductase activity and, furthermore, we newly found that it contained FMN and highly catalyzed nitroreductase, flavin reductase and ferric reductase activities. This is the first demonstration of nitroreductase activity of DrgA protein previously identified by a drgA mutant phenotype. DrgA protein strongly catalyzed the Fenton reaction in the presence of synthetic chelate compounds, but did so poorly in the presence of natural chelate compounds. Its ferric reductase activity was observed with both natural and synthetic chelate compounds with a better efficiency with the latter. In addition to small molecular-weight chemical chelators, an iron transporter protein, transferrin, and an iron storage protein, ferritin, turned out to be substrates of the DrgA protein, suggesting it might play a role in iron metabolism under physiological conditions and possibly catalyze the Fenton reaction under hyper-reductive conditions in this microorganism.
Asunto(s)
FMN Reductasa/metabolismo , Nitrorreductasas/metabolismo , Synechocystis/enzimología , terc-Butilhidroperóxido/metabolismo , Catálisis , Quelantes/farmacología , Ácido Edético/farmacología , FMN Reductasa/genética , Mononucleótido de Flavina/metabolismo , Hierro/metabolismo , Cinética , NAD/aislamiento & purificación , NAD/metabolismo , NADP/aislamiento & purificación , NADP/metabolismo , Nitrorreductasas/genética , Oxidación-Reducción , Especificidad por Sustrato , Synechocystis/genéticaRESUMEN
Nicotinamides are ubiquitous cofactors used by many biological systems as redox agents. Stereospecifically labeled cofactors are useful in many studies of nicotinamide-dependent enzymes. Enzyme-directed synthesis of these cofactors is rather common but their stability imposes significant challenges on yield, purity, and preservation. This paper presents the stereospecific synthesis of reduced R- and S-[4-3H] beta-nicotinamide adenine dinucleotide 2' phosphate (NADPH). The method of Valera et al. [Biochem. Biophys. Res. Commun. 148 (1987) 515] was modified to a synthetic procedure that produces both isotopic diastereomers within 2h with an improved yield of 75-90% after purification and lyophilization. In the synthesis, [4-3H]NADP+ was generated as an intermediate (which can be isolated if desired). The specific radioactivities reported here are 2.7 and 1.1 Ci/mmol for the S and R diastereomers, respectively. Specific radioactivities ranging from carrier-free to trace labeling can be achieved with a minor change to the procedure.
Asunto(s)
NADP/síntesis química , Alcohol Deshidrogenasa , Cromatografía Líquida de Alta Presión , Marcaje Isotópico , NADP/aislamiento & purificación , Oxidación-Reducción , Espectrofotometría Ultravioleta , Estereoisomerismo , Factores de Tiempo , TritioRESUMEN
Nicotinamide-containing cofactors are ubiquitous in biological systems. Consequently, numerous assays have been developed to study such systems that involve a variety of derivatives and isotopically labeled forms of these cofactors. Often the nicotinamide ring is labeled at the C-4 position which is directly involved in the hydride transfer chemistry catalyzed by nicotinamide-dependent enzymes. A label remote from the reactive center is often also required to follow the course of a reaction or the location of the cofactor. Since the necessary labeling pattern can be as unique as the designed experiment, these cofactors need to be synthesized, analyzed, and, most preferably, preserved. The micro-scale preservation of reduced nicotinamides has long been a challenge due to the inherent lability of the reduced cofactors. Furthermore, it has been found that the reduced 2'-phosphorylated cofactor is even less stable (i.e., reduced nicotinamide adenine dinucleotide phosphate (NADPH) is more labile than reduced nicotinamide adenine dinucleotide). Presented here are methods that were established to purify nicotinamide cofactors via reverse-phase high-performance liquid chromatography (HPLC) and, most importantly, to stabilize NADPH under optimal conditions for long-term storage. Additionally, an analytical HPLC method which achieves 7-min resolution between oxidized and reduced cofactors was developed. This method also results in over 4-min resolution of these nicotinamide cofactors from various derivatives of folic acid. This analysis affords a new analytical assay for the dihydrofolate reductase-catalyzed reaction and several dehydrogenases involved in folic acid metabolism.
Asunto(s)
NADP/aislamiento & purificación , Catálisis , Cromatografía Líquida de Alta Presión , Ácido Fólico/metabolismo , Oxidación-Reducción , Preservación Biológica , Espectrofotometría Ultravioleta , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
A synchronous enzyme-reaction system using water-soluble formazan and a non-enzymatic electron mediator was developed and applied to an enzyme immunoassay (EIA). The reaction system consists of four steps: (I) dephosphorylation of NADP(+) to produce NAD(+) by alkaline phosphatase (ALP), (II) reduction of NAD(+) to produce NADH with oxidation of ethanol to yield acetaldehyde by alcohol dehydrogenase (ADH), (III) reduction of water-soluble tetrazolium salt (WST-1) to produce formazan by NADH via 1-methoxy-5-methyl-phenazinium methyl sulfate (PMS), and (IV) re-reduction of NAD(+) to produce NADH by ADH. During each cycle, one molecule of tetrazolium is converted to one molecule of formazan. The concentration of formazan during the reaction was given by second-order polynomials of the reaction time. Kinetic studies strongly suggested that the synchronous enzyme-reaction system had the potential to detect an analyte at the attomole level in EIA. On the basis of the kinetic studies, optimal conditions for EIA incorporating the synchronous system were examined. NADP(+) was purified thoroughly to remove minor traces of NAD(+) in the preparation, and an ADH preparation contaminated with the lowest level of ALP activity was used. When the synchronous system was applied to a sandwich-type EIA for human C-reactive protein, the protein was detected with a sensitivity of 50 attomole per well of a micro-titer plate (0.1 ml) in a 1-h reaction. In addition, EIA with water-soluble formazan showed a more quantitative and sensitive result than that with insoluble formazan. These findings indicated that the (WST-1)-PMS system introduced in this study has a great potential for highly sensitive enzyme immunoassay.
Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Fosfatasa Alcalina/metabolismo , Técnicas para Inmunoenzimas , Metosulfato de Metilfenazonio/análogos & derivados , Anticuerpos Monoclonales , Proteína C-Reactiva/análisis , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Formazáns/análisis , Humanos , Cinética , Metosulfato de Metilfenazonio/farmacología , NADP/aislamiento & purificación , NADP/metabolismo , Oxidación-Reducción , Sensibilidad y Especificidad , Sales de TetrazolioRESUMEN
Our aims were to examine whether oxidative DNA damage was elevated in brain cells of male C57BL/6 mice after oxidative stress, and to determine whether neuronal nitric oxide synthase (nNOS) was involved in such damage. Oxidative stress was induced by occluding both common carotid arteries for 90 min, followed by reperfusion. Escherichia coli exonuclease III (Exo III) removes apyrimidinic or apurinic (AP) sites and 3'-phosphate termini in single-strand breaks, and converts these lesions to 3'OH termini. These ExoIII-sensitive sites (EXOSS) can then be postlabeled using digoxigenin-11-dUTP and Klenow DNA polymerase-I, and detected using fluorescein isothiocyanate-IgG against digoxigenin. Compared with the non-ischemia controls, the density of EXOSS-positive cells was elevated at least 20-fold (P < 0.01) at 15 min of reperfusion, and remained elevated for another 30 min. EXOSS mainly occurred in the cell nuclei of the astrocytes and neurons. Signs of cell death were detected at 24 h of reperfusion and occurred mostly in the neurons. Both DNA damage and cell death in the cerebral cortical neurons were abolished by treatment with 3-bromo-7-nitroindazole (30 mg/kg, intraperitoneal), which specifically inhibited nNOS. Our results suggest that nNOS, its activator (calcium), and peroxynitrite exacerbate oxidative DNA damage after brain ischemia.-Huang, D., Shenoy, A., Cui, J., Huang, W., Liu, P. In situ detection of AP sites and DNA strand breaks bearing 3'-phosphate termini in ischemic mouse brain.
Asunto(s)
Isquemia Encefálica/metabolismo , Daño del ADN , Histocitoquímica/métodos , Estrés Oxidativo/fisiología , Daño por Reperfusión/metabolismo , Animales , Núcleo Arqueado del Hipotálamo/química , Astrocitos/química , Muerte Celular , Corteza Cerebral/química , ADN Polimerasa I , Exodesoxirribonucleasas/metabolismo , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , NADP/aislamiento & purificación , Neuronas/química , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo I , Prosencéfalo/químicaRESUMEN
A method for the isolation of an intact, non-covalent complex formed by the interaction of aldose reductase, NADP(H) nucleotide, and inhibitor has been developed to aid in the discovery and development of novel aldose reductase inhibitors. In the complexes isolated, both the carboxylic acid-containing inhibitor tolrestat and the spirohydantoin-containing inhibitor AL1576 (2,7-difluorospirofluorene-9,5'-imidazolidine-2',4'-dione) tightly bound in a 1:1 ratio to aldose reductase complexed with either NADPH or NADP+. Inhibitor binding to either the enzyme-NADP+ or enzyme-NADPH complex appeared to be equal and pH-dependent, with maximum binding observed at a pH range of 7 to 8.5 where the inhibitors are ionized. These results indicated that the charge state of the cofactor (NADPH vs NADP+) is not critical for inhibitor binding to aldose reductase. Molecular modeling studies suggested that His110 plays a crucial role in directing charged inhibitors containing either a carboxylate or an ionizable hydantoin group to the active site of aldose reductase by providing charge interaction.
Asunto(s)
Aldehído Reductasa/antagonistas & inhibidores , Aldehído Reductasa/metabolismo , Inhibidores Enzimáticos/metabolismo , NADP/metabolismo , Aldehído Reductasa/aislamiento & purificación , Animales , Inhibidores Enzimáticos/aislamiento & purificación , Inhibidores Enzimáticos/farmacología , Fluorenos/metabolismo , Fluorenos/farmacología , Humanos , Hidantoínas/metabolismo , Hidantoínas/farmacología , Cristalino/enzimología , Modelos Moleculares , Músculos/enzimología , NADP/aislamiento & purificación , Naftalenos/metabolismo , Naftalenos/farmacología , Ftalazinas/metabolismo , Ftalazinas/farmacología , RatasRESUMEN
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a metabolite of NADP with Ca2+ mobilizing activity. The Ca2+ release mechanism activated by NAADP as well as the Ca2+ stores that it acts on are different from those activated by either cyclic ADP-ribose or inositol 1,4,5-trisphosphate (IP3) (Lee, H. C., and Aarhus, R. (1995) J. Biol. Chem. 270, 2152-2157). In order to demonstrate unambiguously that NAADP can mobilize Ca2+ stores in live cells, a caged analog was synthesized by reacting NAADP with 1-(2-nitrophenyl)diazoethane. Anion exchange high pressure liquid chromatography (HPLC) was used to purify one particular caged form from the mixture of products. Phosphate analyses following specific enzymatic cleavage indicate that the caging group is on the 2'-phosphate. This is confirmed by 31P NMR spectroscopy, showing that the 2'-phosphate of the caged compound exhibits an altered chemical shift of -2.6 ppm as compared with 2.3 ppm determined for the 2'-phosphate of NAADP. Caged NAADP had no Ca2+ releasing activity at a concentration as high as 1 micro;M when tested on sea urchin egg microsomes. After photolysis, it released Ca2+, was effective in nanomolar range, and was indistinguishable from authentic NAADP. The regeneration of NAADP after photolysis was also confirmed by HPLC analyses. The analog is particularly susceptible to UV and can be efficiently photolyzed using a spectrofluorimeter. To demonstrate its utility in live cells, caged NAADP was microinjected into sea urchin eggs. Photolysis effectively regenerated NAADP and activated Ca2+ oscillations in the eggs. Removal of external Ca2+ did not prevent the Ca2+ oscillations but only delayed the second Ca2+ peak by about 45 s, indicating that the oscillations are due to release from internal stores and not caused by Ca2+ influx. A mechanism based on sensitization of the Ca2+ release by Ca2+ loading is proposed to account for the Ca2+ oscillation observed.
Asunto(s)
NADP/análogos & derivados , Animales , Calcio/metabolismo , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Microsomas/efectos de los fármacos , Microsomas/metabolismo , NADP/síntesis química , NADP/aislamiento & purificación , NADP/farmacología , Óvulo/efectos de los fármacos , Óvulo/metabolismo , Erizos de MarRESUMEN
Two main stable chromium complexes were detected in the aqueous extract of Cr-rich yeast using high-performance liquid chromatographic (HPLC) separation on a reversed-phase RP-8 column and atomic absorption spectrometric detection of chromium. However, the complexes could not be separated and purified sufficiently to allow their reliable identification. Both complexes behaved like ionic or very polar substances with a weak affinity to the reverse-phase column. Yeast extract was then spiked with some synthetic Cr complexes which were prepared considering known data (UV spectra, molecular mass, etc.) on candidates glucose tolerance factor (GTF) compounds. Among the complexes tested, attention was paid especially to the complex of Crm with beta-nicotinamide adenine dinucleotide phosphate (NADP), which accompanies one of two Cr complexes in yeast extract during the HPLC separation. The complex with a Cr:NADP stoichiometric ratio 1:2 has not previously been reported. The complex was tested for its glycaemic activity using glucose tolerance test on rabbits. Significant hyperglycaemia was caused in the animals. Considering the NADP is generally present in tissues of nearly all living organisms, its complexes with transient metals could play a very important role in oxidation-reduction processes of metabolic pathways.
Asunto(s)
Glucemia/metabolismo , Candida/química , Cromo/farmacología , NADP/farmacología , Animales , Cromatografía Líquida de Alta Presión , Cromo/química , Cromo/aislamiento & purificación , NADP/química , NADP/aislamiento & purificación , ConejosRESUMEN
A novel type of pyridine nucleotide, containing two adenosine triphosphate ribose residues rather than one, was isolated from Azotobacter vinelandii strain O. The nucleotide was shown to be 2"- or 3"-(2'-phosphoadenosine-5'-diphosphoribosyl)nicotinamide adenine dinucleotide phosphate, in which 2'-phospho-5'-diphosphoadenosylribose was glycosidically linked to the NADP at position 2' or 3' of the nicotinamide mononucleotide moiety. The ATPribosylNADP did not show coenzyme activity for yeast glucose 6-phosphate dehydrogenase, nor was it cleaved by Neurospora crassa NAD(P) glycohydrolase, indicating that the biological properties conferred on the beta-NADP molecule were largely modified by the attachment of the ATP-ribose group.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Azotobacter vinelandii/química , NADP/análogos & derivados , Adenosina Trifosfato/aislamiento & purificación , Fosfatasa Alcalina , Conformación de Carbohidratos , Hidrólisis , Estructura Molecular , NADP/aislamiento & purificación , Fosfodiesterasa I , Hidrolasas Diéster Fosfóricas , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Especificidad por SustratoRESUMEN
The levels of creatine phosphate, purine, and pyridine nucleotides in tissues provide important information on energetic and oxidative cellular states. Nevertheless, technical, theoretical, and methodological difficulties in extraction and quantification procedures have so far limited our understanding of the exact role that these substances play in metabolic processes which take place in cells. The objective of our study was to find an easy and rapid method for extracting, separating, and quantifying creatine phosphate, purine, and pyridine nucleotides in solid tissues. We adapted the classic acid-extraction procedure with HClO4 for purine and oxidized pyridine nucleotides and then developed a new alkaline extraction with phenol in a phosphate buffer solution (pH 7.8) for reduced pyridine nucleotides. Biopsies of myocardial tissue were frozen and ground at -180 degrees C using the appropriate extraction procedure. The separation and quantification of the metabolites were performed using a reversed-phase 3-microns Supelchem C18 column, with the addition of tetrabutylammonium as an ion-pair agent to the buffer solution, by ultraviolet detection. The recovery of the external and internal standards always exceeded 90%. The autooxidation or interconversion processes were almost insignificant for each reduced form. This technique allowed us to avoid complex enzymatic procedures and difficulties in the selective assay of pyridine nucleotides with chemiluminescence and surface spectroscopy.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Miocardio/química , NADP/aislamiento & purificación , NAD/aislamiento & purificación , Fosfocreatina/aislamiento & purificación , Nucleótidos de Purina/aislamiento & purificaciónRESUMEN
(4R)-Deuterated-reduced nicotinamide adenine dinucleotide phosphate, (4R)-[2H]NADPH, was prepared by reduction of NADP+ using an NADP(+)-dependent alcohol dehydrogenase (EC 1.1.1.2) from Thermoanaerobium brockii and isopropanol-d8 as substrate at 43 degrees C, pH 9. More than 80% of the product was identified as reduced cofactor by reverse-phase (ODS) HPLC, and a 1H NMR study showed that all of the reduced cofactor was (4R)-deuterated. Less than 10% of the product was oxidized cofactor, the remainder being impurities from the breakdown of the dinucleotide compound. Subsequent purification carried out by semipreparative reverse-phase HPLC with 0.1 M NaCl at pH 8.5 gave a compound of more than 96% purity. Separated (4R)-[2H]NADPH fractions were freeze-dried and the white solid was stored at 5 degrees C with desiccant.
Asunto(s)
Deuterio , NADP/síntesis química , 1-Propanol , Alcohol Deshidrogenasa , Cromatografía Líquida de Alta Presión/métodos , Marcaje Isotópico/métodos , Espectroscopía de Resonancia Magnética/métodos , NADP/química , NADP/aislamiento & purificación , Oxidación-ReducciónRESUMEN
The nucleotide concentrations in acid and alkaline erythrocyte extracts have been measured by RP-HPLC in healthy controls and in patients bearing different inherited disorders, with altered erythrocyte NAD(P) levels. The objective was the simultaneous determination of the nucleotide profile and of the oxidative state of pyridine coenzymes by the most suitable extraction method. Both alkaline and acid extractions were necessary to obtain the complete pattern, due to defective recovery of the oxidized or reduced coenzymes, respectively, during the extraction procedures. Purine nucleotide quantification seemed to be reliable by all methods. High NADP+ levels were confirmed in two glucose-6-phosphate dehydrogenase deficient patients, coupled with raised NAD levels, lowered NADPH/NADP+ ratio and increased NADH/NAD+ ratio. Higher NAD+ and normal or lower NADH/NAD+ ratios were found in two hypoxanthine-phosphoribosyltransferase deficient patients, while a patient with superactive phosphoribosylpyrophosphate synthetase showed a decreased NADH level in addition to the low NAD+ level previously found.
Asunto(s)
Eritrocitos/química , NADP/sangre , NAD/sangre , Cromatografía Líquida de Alta Presión , Enfermedad del Almacenamiento de Glucógeno Tipo I/sangre , Humanos , Síndrome de Lesch-Nyhan/sangre , NAD/aislamiento & purificación , NADP/aislamiento & purificación , Oxidación-Reducción , Purina-Nucleósido Fosforilasa/deficiencia , Ribosa-Fosfato Pirofosfoquinasa/metabolismoRESUMEN
Analysis by HPLC of the protein-free supernatant obtained after denaturation of aldose reductase shows that the native form of the enzyme (ARb) contains a tightly bound NADP+, which is absent in the oxidatively modified form (ARa). The absorption, fluorescence, and circular dichroism spectra of ARb and ARa are consistent with the presence of the cofactor only in the native form of aldose reductase. On the other hand, the modified enzyme, in appropriate thiol reducing conditions, can tightly bind NADP+. This indicates a potential reversibility of the modification of aldose reductase, at least in terms of retention of the cofactor.