NADP System Research Articles

The determination of the redox states and phosphorylation potential in living tissues and their relationship to metabolic control of disease phenotypes

This paper reviews the development in the 1950s of methods to determine the redox states of the free [NAD(+) ]/[NADH] in cytoplasm of yeast by Helmut Holzer and Feodore Lynen and in rat liver by Theodore Bucher and Martin Klingenberg. This work was extended in the 1960s in the laboratory of Hans Krebs, where the use of basic thermodynamic and kinetic principles allowed the extension of this approach to the determination of the free mitochondrial [NAD(+) ]/NADH] in mitochondria and the redox state of the free NADP system in cytoplasm and mitochondria. This work also outlined the linkage between the redox states in the various couples to the phosphorylation state or the free [ATP]/[ADP][P(i) ] ratio, the central energy parameter of living cells. This work has since been extended to include other energy-linked systems including the gradients of inorganic ions between extra and intracellular phases of the cell and the redox state of the co-enzyme Q couple of mitochondria. This system of linked near-equilibrium redox and phosphorylation potentials constitutes a framework of primitive metabolic control that is altered in a number of disease phenotypes. The alteration of such disease phenotypes by substrate availability is discussed, as well as the importance of a thorough grounding in basic kinetics and thermodynamics in designing new therapies to normalize the metabolic abnormalities that are the proximate cause of many common and some rare diseases states.

Highly sensitive spectrofluorimetric determination of trace amounts NADP using Europium ion–doxycycline complex probe

A new spectrofluorimetric method was developed for determination of trace amount of Coenzyme II (NADP). Using europium ion-doxycycline (DC) as a fluorescent probe, in the buffer solution of pH 8.44, NADP can remarkably enhance the fluorescence intensity of the Eu 3+–DC complex at λ = 612 nm and the enhanced fluorescence intensity is in proportion to the concentration of NADP. Optimum conditions for the determination of NADP were also investigated. The dynamic range for the determination of NADP is 3.3 × 10 −7 to 6.1 × 10 −6 mol l −1 with detection limit of 6.8 × 10 −8 mol l −1. This method is simple, practical and relatively free interference from coexisting substances and can be successfully applied to determination of NADP in synthetic water samples and in serum samples. Moreover, the enhancement mechanisms of the fluorescence intensity in the Eu 3+–DC system and the Eu 3+–DC–NADP system have been also discussed.

Photosystem I-dependent cyclic electron flow in intact spinach chloroplasts: Occurrence, dependence on redox conditions and electron acceptors and inhibition by antimycin A

Photosystem I-dependent cyclic electron transport is shown to operate in intact spinach chloroplasts with oxaloacetate, but not with nitrite or methylviologen as electron acceptors. It is regulated by the redox state of the chloroplast NADP system. Inhibition of cyclic electron transport by antimycin A occurs immediately on addition of this antibiotic in the light. It is unrelated to a different function of antimycin A, inhibition of nonphotochemical quenching of chlorophyll fluorescence, which requires prior dissipation of the transthylakoid proton gradient before antimycin A can become effective.

Pyridine nucleotide levels and activities of dehydrogenases in cambial derivatives ofRobinia pseudoacacia L.

Despite the importance of the vascular cambial differentiation, little is known about its regulation. In order to address this problem we attempted to biochemically characterize differentiating xylem and phloem elements during the early stages of development. By applying techniques of quantitative histochemistry we show that the total pool size of pyridine nucleotides is similar in the phloem (PD) and xylem (XD) oriented derivatives of the cambial zone of trees ofRobinia pseudoacacia L. Within the PD zone, the amount of NAD + NADH exceeded that of NADP + NADPH [around 600 versus 200 pmol (mg dry weight)-1], possibly indicative of a preponderance of catabolic pathways (ratio of NADH∶NAD about 1). In contrast, the NADP(H) system dominated in the XD zone. This coincided with a high activity of NAD kinase. In addition, the extractable activities of the key enzymes of the oxidative pentose phosphate pathway, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, were greatly increased. At a ratio of NADPH∶NADP of approximately 1, this could be indicative of increased rates of reductive biosyntheses, and could thus well be involved in early steps of the formation of phenols and lignin monomers. Taken together, this first approach clearly shows that phloem-oriented and xylem-oriented cambial descendents exhibit distinct differences in their biochemical patterns even in early stages of differentiation.

Adenine Nucleotide Levels, the Redox State of the NADP System, and Assimilatory Force in Nonaqueously Purified Mesophyll Chloroplasts from Maize Leaves under Different Light Intensities

Recently, a nonaqueous fractionation method of obtaining highly purified mesophyll chloroplasts from maize leaves was established. This method is now used to determine adenine nucleotide levels, the redox states of the NADP system, Pi levels and dihydroxyacetone phosphate/3-phosphoglycerate ratios in mesophyll chloroplasts of Zea mays L. leaves under different light intensities. The sum of the ATP, ADP, and AMP levels was estimated to be 1.4 millimolar and the ATP/ADP ratio was 1 in the dark and 2.5 to 4 in the light. The adenine nucleotides were equilibrated by adenylate kinase. The total concentration of NADP(H) in the chloroplasts was 0.3 millimolar in the dark and 0.48 millimolar in the light. The ratio of NADPH/NADP was 0.1 to 0.18 in the dark and 0.23 to 0.48 in the light. The Pi level was estimated to be 20 millimolar in the dark and 10 to 17 millimolar in the light. The 3-phosphoglycerate reducing system was under thermodynamic equilibrium in the light. The calculated assimilatory forces were 8 per molar and 40 to 170 per molar in the dark and the light, respectively. There was no relationship between the degree of activation of pyruvate, Pi dikinase, and adenylate energy charge, or ATP/ADP ratio or ADP level under various light intensities. Only a weak relationship was found between the degree of activation of NADP-malate dehydrogenase and the NADPH/NADP ratio or NADP(H) level with increasing light intensity. A possible regulatory mechanism which is responsible for the regulation of activation of pyruvate,Pi dikinase and NADP-malate dehydrogenase is discussed.

Capacity of the malate/oxaloacetate shuttle for transfer of reducing equivalents across the envelope of leaf chloroplasts

A steady-state model is used to estimate the capacity of the malate/oxaloacetate shuttle for indirect transfer of reducing equivalents between cytoplasm and chloroplast stroma of leaf cells. The dependence of transenvelope exchange on the malate and oxaloacetate gradients is described by a kinetic model of the dicarboxylate translocator. This model accounts for competition of transferable compounds, for counterexchange, and for saturation kinetics. Kinetic parameters are derived from known data, except the K m value for oxaloacetate, which is estimated from oxaloacetate-dependent oxygen evolution of isolated intact chloroplasts. Transfer capacity of the translocator is estimated not to exceed 10 μmol/mg chlorophyll per hour under physiological conditions. Using known data on stromal pyridine nucleotide concentrations it is shown that the stromal NADP-linked malate dehydrogenase cannot contribute to oxidation of malate in the stroma. Stromal malate can be oxidized by NAD-linked malate dehydrogenase, as the NAD system is more oxidized than the NADP system. However, according to this calculation, rates of steady-state transport do not exceed 3–4 μmol/mg chlorophyll per hour. Likewise, rates of export of reducing equivalents to the cytosol are shown to be physiologically insignificant. It is concluded that the malate/oxaloacetate shuttle cannot contribute significantly to the communication of cytosolic and stromal reducing equivalents.

The redox state of the NADP system in illuminated chloroplasts

Pyridine nucleotide levels were measured in intact spinach chloroplasts. The NADPH/NADP ratio was close to unity in darkened chloroplasts. On illumination, chloroplast NADP levels decreased rapidly. The decrease was more prominent at low than at high light intensities. In the presence of bicarbonate, NADP subsequently increased to reach a steady-state level. The kinetics of the increase were related in general, but not in detail, to the lag phase of photosynthesis. In the steady state, chloroplast NADP was sometimes, particularly during photosynthesis at high light intensities, less reduced in the light than in the dark. In the dark-light transition, phosphoglycerate reduction is driven by increases in the ratios NADPH/NADP and ATP/ADP. When photosynthesis accelerates after the initial lag phase, the NADPH/NADP ratio decreases and a high ratio of phosphoglycerate to triose phosphate becomes an important factor in driving carbon reduction. Under photosynthetic flux conditions, the redox state of the chloroplast NADP system appeared to be governed largely by the chloroplast ratio of phosphoglycerate to dihydroxyacetone phosphate and by the phosphorylation potential [ATP]/[ADP] [P i]. The inhibitor of cyclic electron transport, antimycin A, increased reduction of the chloroplast NADP system. Even when reduction was almost complete in the presence of 5 μM antimycin A, photosynthesis was still significant at low light intensities. Electrons appeared to be effectively distributed between the cyclic electron-transport pathway and the noncyclic route to NADP at NADPH/NADP ratios as low as about 1. When bicarbonate was absent, the NADP system remained largely reduced in the light. The energy-transfer inhibitor, Dio-9, and uncouplers and agents which interfered with pH regulation of the Calvin cycle increased reduction of the NADP system while decreasing photosynthesis.

Pyridine nucleotide distributions and enzyme mass action ratios in hepatocytes from fed and starved rats

Hepatocytes isolated from fed or starved rats were rapidly lysed using the recently described technique of turbulent flow (M. E. Tischler, P. Hecht, and J. R. Williamson, 1977, Arch. Biochem. Biophys., 181, 278–292) . Pyridine nucleotide and metabolite contents were measured in the particulate fraction of both whole and disrupted cells after centrifugation through silicone oil. Lactate/pyruvate, β-hydroxybutyrate/acetoacetate, isocitrate/α-ketoglutarate, and malate/pyruvate ratios were determined for calculation of the free NADH NAD + and NADPH NADP + ratios in the cytosol and mitochondria. Lactate/pyruvate ratios measured in the extracellular and cytosolic compartments were in good agreement. Ratios of β-hydroxybutyrate/acetoacetate measured in the extracellular, cytosolic, and mitochondrial compartments also agreed well. Addition of ammonia to fed or starved rat liver cells incubated with lactate, pyruvate, β-hydroxybutyrate, and acetoacetate caused an oxidation of both the NAD and NADP redox states in the mitochondria and cytosol, although the NADP system was oxidized to a greater extent. Calculation of the free NADH and NAD concentrations in the cytosol provided values of about 1 and 400 to 500 μ m, respectively, under control conditions. The concentrations of free NADH and NAD in the mitochondria were considerably higher, being 300 to 400 μ m and 4 to 6 m m, respectively. The free andm bound NAD systems in both the cytosol and mitochondria were more oxidized in the presence of ammonia. NAD and NADP redox potential differences across the mitochondrial membrane (Δ E h) were not significantly affected by ammonia addition and were generally similar in cells from both fed and starved rats: −52 and −56 mV for the NAD system and −19 to −29 mV for the NADP system. For the NAD system the cytosolic potential was −260 mV in the absence of ammonia and −250 mV in its presence, the mitochondrial values being −315 and −303 mV, respectively. The average cytosolic NADP potential, on the other hand, was −400 mV in the absence and −384 mV in the presence of ammonia. The mitochondrial fractions yielded NADP potentials of −420 mV in the absence of ammonia with both fed and starved rats. Ammonia decreased the mitochondrial NADP potential to −404 mV in fed rats and to −415 mV in starved rats. The calculated free NADH NAD + and NADPH NADP + ratios as well as metabolite concentrations were used to evaluate the mass action ratios of both cytosolic and mitochondrial enzymes. Cytosolic alanine aminotransferase remained near equilibrium in the absence and presence of ammonia, while cytosolic and mitochondrial aspartate aminotransferase reactions deviated up to fivefold. The glutamate dehydrogenase reaction was in near equilibrium with the NAD system, but deviated by three to four orders of magnitude from equilibrium with the NADP system in the direction favoring glutamate synthesis rather than deaminatión. Cytosolic malate dehydrogenase deviated from equilibrium by about one order of magnitude, while mitochondrial malate dehydrogenase and citrate synthase deviated by two to six orders of magnitude. These data emphasize the importance of regulation of the citric acid cycle at the citrate synthase step.

Properties of photoreductions by photosystem II in isolated chloroplasts. III. The effect of uncouplers on phenylenediamine shuttles across the membrane in the presence of dibromothymoquinone

In the presence of the plastoquinone antagonist dibromothymoquinone the photoreduction of ferricyanide by isolated chloroplast membranes is attributed to Photosystem II. The reaction is stimulated by the addition of phenylenediamine or C-substituted phenylenediamines (which may form a diimine on oxidation) but not of N-substituted phenylenediamines (which form a stable radical on oxidation). Phenylenediamines also restore NADP reduction (and O 2 evolution) in 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone (DBMIB)-treated chloroplasts. In this bypassing of the inhibition site, N-substituted phenylenediamines are very effective, whereas p-phenylenediamine and C-substituted phenylenediamines are inefficient. Uncouplers exhibit a surprising effect on these systems. Even under coupling conditions uncouplers inhibit electron flow to ferricyanide mediated by phenylenediamine in the pH range 7.3–8.0, whereas the restoration of the NADP system is stimulated. For the interpretation of the results the side of the membrane involved is considered. It is proposed that in ferricyanide reduction by Photosystem II, a phenylenediimine/diamine shuttle operates which moves reducing equivalents from the inside to the outside across the membrane. This shuttle requires a pH gradient across the membrane because of different optimal ratios of diimine/diamine inside and outside. This pH difference is abolished by the uncoupler, accounting for the observed inhibition. The restoration of electron flow from water to NADP in DBMIB-treated chloroplasts is assumed to be a bypass of the inhibition site inside the membrane via a phenylenediamine. Because the imine/amine ratio brought about by the pH gradient is not favorable for the inside oxidation an uncoupler stimulates NADP reduction even under coupling conditions. Also in photoreductions by Photosystem I, for example NADP reduction at the expense of P-phenylenediamine/ascorbate, a shuttle of reducing equivalents across the membrane occurs but this time from outside to inside.

Oxidation in the NADP system and release of GSSG from hemoglobin-free perfused rat liver during peroxidatic oxidation of glutathione by hydroperoxides

Both of these enzyme activities have been studied extensively in recent years in rat liver homogenates and subcellular fractions [l-6]. In addition to Hz 02, several organic hydroperoxides were reported as active substrates for rat liver glutathione peroxidase [2]; some of these, e.g. t-butyl hydroperoxide or cumene hydroperoxide, do not react with catalase, and the corresponding alcohols do not react with alcohol dehydrogenase. Therefore, a selective transition in the 2GSH/GSSG redox system is effected in intact liver cells by these substrates.

Quantitative Histochemistry Of Nicotinamide Adenine Nucleotides In Human Skin

The NAD and NADP system was measured in freeze-dried samples (0.3 μg) of epidermis, hair follicle, sebaceous gland, sweat gland, and dermis by means of enzyme cycling methods. The absolute concentration of nucleotides ranged from 10‒15 to 10‒12 moles in an assay with 1: 40,000 tissue dilution at the enzyme cycling step. The total NAD was distributed similarly in the cutaneous organs, and its concentration was approximately 1 mμ mole per mg dry weight. The degree of oxidation of NAD was lowest in sebaceous gland. Less than 40 per cent of the total NAD was in the reduced state in the skin. The concentration of total NADP ranged from 100 to 300 μμ moles per mg dry weight, 50–70 per cent of which was in the reduced form. The concentration of both total NADP and NADPH was highest in sebaceous gland; the ratio of NAD to NADP was lowest in sebaceous gland. The higher concentration and higher degree of reduction of the NADP system in sebaceous gland coincides with the fact that the activities of NADP-dependent enzymes are higher in sebaceous gland and that this gland is the most active site of lipogenesis in skin. The concentration of nicotinamide adenine nucleotides and the degree of their reduction increased in proliferating and migrating epithelium during wound healing.

Positive increase of redox potential of the extramitochondrial NADP(H) system by mixed function oxidations in hemoglobin-free perfused rat liver

FEBS LettersVolume 9, Issue 4 p. 205-208 Full-length articleFree Access Positive increase of redox potential of the extramitochondrial NADP(H) system by mixed function oxidations in hemoglobin-free perfused rat liver Helmut Sies, Helmut Sies Institut für Physiologische Chemie und Physikalische Biochemie der Universität München, GermanySearch for more papers by this authorMartina Kandel, Martina Kandel Institut für Physiologische Chemie und Physikalische Biochemie der Universität München, GermanySearch for more papers by this author Helmut Sies, Helmut Sies Institut für Physiologische Chemie und Physikalische Biochemie der Universität München, GermanySearch for more papers by this authorMartina Kandel, Martina Kandel Institut für Physiologische Chemie und Physikalische Biochemie der Universität München, GermanySearch for more papers by this author First published: August 17, 1970 https://doi.org/10.1016/0014-5793(70)80356-8Citations: 25AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 Th. Bücher, H. Sund Pyridine Nucleotide Dependent Dehydrogenases (1970), Springer Berlin, Heidelberg, New York 439– 2 H.A. Krebs, R.L. Veech, S. Papa J.M. Tager E. Quagliariello E.C. Slater The Energy Level and Metabolic Control in Mitochondria (1969), Adriatica Editrice Bari 329– 3 H. Sies and B. Brauser, European J. Biochem., in press. 4 B. Brauser, H. Versmold, Th. Bücher, Z. Physiol. Chem., 349, (1968), 1589– 5 B. Brauser, H. Sies, Th. Bücher, FEBS Letters, 2, (1969), 170– 6 W.J. Marshall, A.E.M. McLean, Biochem. Pharmacol., 18, (1969), 153– 7 B. Chance, F. Jöbsis, Nature, 184, (1959), 195– 8 M. Klingenberg, in: Methoden der enzymatischen Analyse, ed. H.U. Bergmeyer, 2nd edition (Verlag Chemie, Weinheim) in press. 9 J.R. Williamson, B.E. Corkey, J.M. Lowenstein Methods in Enzymology 13, (1969), Academic Press New York, London 434– 10 H.J. Hohorst, F.H. Kreutz, Th. Bücher, Biochem. Z., 332, (1959), 18– 11 Th. Bücher, M. Klingenberg, Angew. Chem., 70, (1958), 552– 12 M. Klingenberg, 11 Coll. Ges. Physiol. Chem. (1961), Springer Berlin, Heidelberg, New York 82– 13 J.R. Williamson, S. Papa J.M. Tager E. Quagliariello E.C. Slater The Energy Level and Metabolic Control in Mitochondria (1969), Adriatica Editrice Bari 385– 14 R.G. Thurman, R. Scholz, Z. Physiol. Chem., 351, (1970), 294– 15 R. Scholz, R.G. Thurman, Federation Proc., 29, (1970), 925– Abstr. 3873 16 M.J. Waterman, H.S. Mason, Biochem. Biophys. Res. Commun., 39, (1970), 450– Citing Literature Volume9, Issue4August 17, 1970Pages 205-208 ReferencesRelatedInformation