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1.
《Journal of Molecular Catalysis .B, Enzymatic》2007,44(1):27-31
We evaluated the photochemical and enzymatic synthesis of methanol from formaldehyde with alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae and NAD+ photoreduction by the visible-light sensitization of zinc tetraphenylporphyrin tetrasulfonate (ZnTPPS) in the presence of methylviologen (MV2+), diaphorase, and triethanolamine (TEOA). When the sample solution containing ZnTPPS, MV2+, NAD+, diaphorase, and TEOA in potassium phosphate buffer solution was irradiated, the NADH produced increased with the irradiation time. After irradiation for 180 min, the conversion yield of NAD+ to NADH was about 60% under 0.1 mM NAD+ condition. The methanol production also depended on the conversion yield of NAD+ to NADH. After irradiation for 180 min, 0.38 μM of methanol was produced from formaldehyde (16 μM). The conversion ratio of formaldehyde to methanol was about 2.3%. This result indicates that a system for the photochemical synthesis of methanol from formaldehyde was developed with ADH and the NADH produced by the photosensitization of ZnTPPS in water media. 相似文献
2.
Ashok K. Srivastava Rafick-Pierre Sékaly Jean-Louis Chiasson 《Molecular and cellular biochemistry》1993,121(2):127-133
The effect of nicotinamide-adenine dinucleotides (NAD+ and NADP+) on Ca2+ transport in rat liver nuclei was investigated. Ca2+ uptake and release were determined with a Ca2+ electrode. Ca2+ uptake was dependent on adenosine triphosphate (ATP; 2mM). The presence of NAD+ (2mM) or NADP+ (1 and 2mM) caused a significant inhibition of Ca2+ uptake following addition of 2mM ATP. Ca2+, which accumulated in the nuclei during 6 min after ATP addition, was significantly released by the addition of NAD+ (0.5–2mM) or NADP+ (0.1–2mM). However, the effect of NADH (2mM) or NADPH (2mM) on Ca2+ uptake and release clearly weakened in comparison with the effects of NAD+ and NADP+. Meanwhile, ryanodine (10M), thapsigargin (10M) or oxalate (0.5mM) had no effect on Ca2+ uptake and release in rat liver nuclei. These reagents did not significantly alter the effects of 2mM NAD+ on Ca2+ uptake and release. Thus, NAD+ and NADP+ had a potent effect on Ca2+ transport in rat liver nuclei. The present findings suggest that the liver cytosolic NAD+ (NADP+) is a factor in the regulation of the nuclear Ca2+ concentration. (Mol Cell Biochem121: 127–133, 1993) 相似文献
3.
Fomes sclerodermeus produces manganese peroxidase (MnP) and laccase as part of its ligninolytic system. A Doehlert experimental design was applied in order to find the optimum conditions for MnP and laccase production. The factors studied were Cu2+, Mn2+ and asparagine. The present model and data analysis allowed us not only to define optimal media for production of both laccase and MnP, but also to show the combined effects between the factors. MnP was strongly influenced by Mn2+, which acts as an inducer. Under these conditions Cu2+ negatively affected MnP activity. At 13 days of growth 0.75 U ml–1 were produced in the optimized culture medium supplemented with 1 mM MnSO4 and 4 g l–1 asparagine. The laccase titer under optimized conditions reached maximum values at 16 days of growth: 13.5 U ml–1 in the presence of 0.2 mM CuSO4, 0.4 mM MnSO4 and 6 g l–1 asparagine. Mn2+ promoted production of both enzymes. There were important interactions among the nutrients evaluated, the most significant being those between Cu2+ and asparagine. 相似文献
4.
An NAD+-dependent xylitol dehydrogenase (XDH) from Fusarium oxysporum, a key enzyme in the conversion of xylose to ethanol, was purified to homogeneity and characterised. It was homodimeric with a subunit of M
r 48 000, and pI 3.6. It was optimally active at 45 °C and pH 9–10. It was fully stable at pH 6–7 for 24 h and 30 °C. K
m values for d-xylitol and NAD+ were 94 mM and 0.14 mM, respectively. Mn2+ at 10 mM increased XDH activity 2-fold and Cu2+ at 10 mM inhibited activity completely. 相似文献
5.
Ján Slezák Ludmila Okruhlicová Narcisa Tribulová Wolfgang Schulze Naranjan S. Dhalla 《Molecular and cellular biochemistry》1995,147(1-2):169-172
ATPases of cardiac cells are known to be among the most important enzymes to maintain the fluxes of vital cations by hydrolysis of the terminal high-energy phosphate of ATP. Biochemically the activities of Ca2+-pump ATPase, Ca2+/Mg2+-ecto ATPase, Na+,K+-ATPase and Mg2+-ATPase are determined in homogenates and isolated membranes as well as in myofibrillar and mitochondrial fractions of various purities. Such techniques permit estimation of enzyme activitiesin vitro under optimal conditions without precise enzyme topography. On the other hand, cytochemical methods demonstrate enzyme activityin situ, but not under optimal conditions. Until recently several cytochemical methods have been employed for each enzyme in order to protect its specific activity and precise localization but the results are difficult to interpret. To obtain more consistent data from biochemical and cytochemical point of view, we modified cytochemical methods in which unified conditions for each ATPase were used. The fixative solution (1% paraformaldehyde –0.2% glutaraldehyde in 0.1 M Tris Base buffer, pH 7.4), the same cationic concentrations of basic components in the incubation medium (0.1 M Tris Base, 2mM Pb(NO2)3, 5 mM MgSO4, 5 mM ATP) and selective stimulators or inhibitors were employed. The results reveal improved localization of Ca2+-pump ATPase, Na+–K+ ATPase and Ca2+/Mg2+-ecto ATPase in the cardiac membrane. 相似文献
6.
The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 catalyzes the H2-driven reduction of NAD+, as well as reverse electron transfer from NADH to H+, in the presence of O2. It comprises six subunits, HoxHYFUI2, and incorporates a [NiFe] H+/H2 cycling catalytic centre, two non-covalently bound flavin mononucleotide (FMN) groups and an iron-sulfur cluster relay for electron transfer. This study provides the first characterization of the diaphorase sub-complex made up of HoxF and HoxU. Sequence comparisons with the closely related peripheral subunits of Complex I in combination with UV/Vis spectroscopy and the quantification of the metal and FMN content revealed that HoxFU accommodates a [2Fe2S] cluster, FMN and a series of [4Fe4S] clusters. Protein film electrochemistry (PFE) experiments show clear electrocatalytic activity for both NAD+ reduction and NADH oxidation with minimal overpotential relative to the potential of the NAD+/NADH couple. Michaelis-Menten constants of 56 µM and 197 µM were determined for NADH and NAD+, respectively. Catalysis in both directions is product inhibited with K
I values of around 0.2 mM. In PFE experiments, the electrocatalytic current was unaffected by O2, however in aerobic solution assays, a moderate superoxide production rate of 54 nmol per mg of protein was observed, meaning that the formation of reactive oxygen species (ROS) observed for the native SH can be attributed mainly to HoxFU. The results are discussed in terms of their implications for aerobic functioning of the SH and possible control mechanism for the direction of catalysis. 相似文献
7.
The settling rates and intracellular levels of K+, Na+, Cl-, Mg2+ and Ca2+ were measured in Ditylum bright-welli (West) Grunow, grown axenically in an enriched seawater medium at 20 C at 4,000 lx on an 8:16 LD schedule. Cells at the end of the dark period have high Na+ (118 mM), low K+ (64 mM) and low Cl- (117 mM) relative to levels at the end of the light period when K+ (126 mM) and Cl- (154 mM) are high and Na+ (101 mM) is low. There is no significant change in Mg2+ (16–18 mM) or Ca2+ (3–4 mM) with time. The net result of the ion changes during the light period is to increase cell density by about 3.4 mg ml-1. This change can account for the increase in settling rate of ca. 0.3 day-1 during the same interval. The density of the cell contents, calculated from observed ion concentrations, is 15–18 mg.ml-1 less than that of the seawater medium. The ion and settling rate changes are light-dependent and do not persist in the dark or under constant light (ca. 850 lx), but cells do exhibit a free-running circadian rhythm in cell division under continuous dim illumination. The cell vacuole expands during the light period and contracts during the dark, apparently in response to the net ion fluxes. D. brightwelli appears to regulate its density by active ion selectivity accompanied by trans-vacuolar water movement. 相似文献
8.
《Journal of steroid biochemistry》1982,16(4):427-433
A partial characterization of human term placental 3ß-HSDH in mitochondria is reported. Apparent KM of pregnenolone: 70 nM. A dose-dependent stimulation of 3ß-HSDH by NAD+ or NADP+ was observed in the range from 10−6 to 10−3 M (KM value of NAD+: 20 μM). At equimolar concentrations NAD+ is more than 10-fold as effective a cofactor of the 3ß-HSDH than NADP+. pH optimum: 9.5 (glycine-NaOH buffer). Temperature optimum 40–45°C. A rapid loss of 3ß-HSDH activity was found after preincubation of the enzyme at 37°C after 30 min: less than 50% of initial enzyme activity is present. No inhibition was obtained by Mg2+, Ca2+ Sr2+ and Ba2+ (1–100 mM). A strong inhibition was achieved with 1 mM Zn2+, Cd2+, Cu2+ and 10 mM and 100 mM Fe2+, Mn2+, Co2+ and Ni2+. 相似文献
9.
Isolated, intact rat liver mitochondria, without extraneous substrates but loaded with Ca2+ (20 nmol/mg), can be observed to release Ca2+ when treated with ruthenium red. Such release can be inhibited by 0.33 mM dlisocitrate but not by 10 mM dl-β-hydroxybutyrate. Assays of NADP+, NADPH, NAD+, and NADH revealed that only the reduction of NADP+ can be linked with such inhibition of Ca2+ release, not that of NAD+. Since ruthenium redinsensitive Ca2+ release is a physiological (but normally masked) process, this experimental approach avoids some potential problems ascribed to strong pyridine nucleotide oxidation. It is suggested that specific NADP+:NADPH dependent reactions are part of a physiological mechanism regulating Ca2+ release/retention. 相似文献
10.
Nitrate reductase from Spinacea oleracea. Reversible inactivation by NAD(P)H and by thiols 总被引:5,自引:0,他引:5
E Palacián F De la Rosa F Castillo C Gómez-Moreno 《Archives of biochemistry and biophysics》1974,161(2):441-447
The enzymatic complex nitrate reductase from Spinacea oleracea is inactivated by NADH or NADPH and by simple thiols. The inactivation affects FNH2-nitrate reductase but not NADH-diaphorase. Reactivation can be achieved by addition of ferricyanide. The extent of inactivation by dithioerythritol is increased by NAD+, but not by NADP+. Nitrate protects against inactivation by NADH or NADPH, and abolishes the effect of NAD+ on the inactivation by dithioerythritol. The NAD(P)H-inactivation of nitrate reductase requires that the diaphorase moiety of the complex be functional. However, there is no proportionality between NADH-diaphorase or NADH-nitrate reductase activities and the susceptibility of the enzymatic preparation to NADH or NADPH. It seems likely that the nitrate reductase complex contains a specific regulatory site, different from the catalytic site, the reduction of which is accompanied by the production of an inactive form of the complex. 相似文献
11.
Michele Simonato Richard S. Jope Clementina Bianchi Lorenzo Beani 《Neurochemical research》1989,14(7):677-682
Ca2+ uptake was measured in purified rat cerebral cortex synaptosomes (P3 pellets) using45Ca2+ as a tracer. Ca2+ influx increased in time, and with an increase in external K+ concentration and temperature. The net (external K+-induced, depolarization-dependent) uptake follows a two-component course. The exponential term, due to the opening of voltage-operated calcium channels (VOC), has a rate constant which increases with an increase in the depolarization level (1.04 versus 0.54 nmol/s/mg protein for 50 mM—versus 15 mM [K+]-dependent net influx). The linear term, due to the Na+/Ca2+ exchange system, has a similar rate constant at all depolarization levels (0.16+/–0.05 and 0.11+/–0.02 nmol/s/mg protein). Excitatory amino acids (glutamate, kainate and n-methyl-d-aspartate-NMDA-) were tested on this preparation at doses ranging between 5×10–5 M and 5×10–3M and at multiple incubation times, under resting conditions and under two depolarizing conditions (partial depolarization: 15 mM external K+ and maximal depolarization: 50 mM external K+). NMDA was also tested in the absence of Mg2+. No effect was detectable under any of these experimental conditions. Hypotheses to interpret these data are discussed. Further studies on other preparations are needed in order to directly investigate the presynaptic effects of excitatory amino acids. 相似文献
12.
In the present study, we show that the extracellular addition of nicotinamide adenine dinucleotide (NAD+) induces a transient rise in [Ca2+]i in human monocytes caused by an influx of extracellular calcium. The NAD+-induced Ca2+ response was prevented by adenosine triphosphate (ATP), suggesting the involvement of ATP receptors. Of the two subtypes
of ATP receptors (P2X and P2Y), the P2X receptors were considered the most likely candidates. By the use of subtype preferential
agonists and antagonists, we identified P2X1, P2X4, and P2X7 receptors being engaged in the NAD+-induced rise in [Ca2+]i. Among the P2X receptor subtypes, the P2X7 receptor is unique in facilitating the induction of nonselective pores that allow entry of ethidium upon stimulation with
ATP. In monocytes, opening of P2X7 receptor-dependent pores strongly depends on specific ionic conditions. Measuring pore formation in response to NAD+, we found that NAD+ unlike ATP lacks the ability to induce this pore-forming response. Whereas as little as 100 μM ATP was sufficient to activate
the nonselective pore, NAD+ at concentrations up to 2 mM had no effect. Taken together, these data indicate that despite similarities in the action of
extracellular NAD+ and ATP there are nucleotide-specific variations. So far, common and distinct features of the two nucleotides are only beginning
to be understood. 相似文献
13.
Rafael Mejía-Alvarez Claudia Kettlun Eduardo Ríos Michael Stern Michael Fill 《The Journal of general physiology》1999,113(2):177-186
Single canine cardiac ryanodine receptor channels were incorporated into planar lipid bilayers. Single-channel currents were sampled at 1–5 kHz and filtered at 0.2–1.0 kHz. Channel incorporations were obtained in symmetrical solutions (20 mM HEPES-Tris, pH 7.4, and pCa 5). Unitary Ca2+ currents were monitored when 2–30 mM Ca2+ was added to the lumenal side of the channel. The relationship between the amplitude of unitary Ca2+ current (at 0 mV holding potential) and lumenal [Ca2+] was hyperbolic and saturated at ∼4 pA. This relationship was then defined in the presence of different symmetrical CsCH3SO3 concentrations (5, 50, and 150 mM). Under these conditions, unitary current amplitude was 1.2 ± 0.1, 0.65 ± 0.1, and 0.35 ± 0.1 pA in 2 mM lumenal Ca2+; and 3.3 ± 0.4, 2.4 ± 0.2, and 1.63 ± 0.2 pA in 10 mM lumenal Ca2+ (n > 6). Unitary Ca2+ current was also defined in the presence of symmetrical [Mg2+] (1 mM) and low [Cs+] (5 mM). Under these conditions, unitary Ca2+ current in 2 and 10 mM lumenal Ca2+ was 0.66 ± 0.1 and 1.52 ± 0.06 pA, respectively. In the presence of higher symmetrical [Cs+] (50 mM), Mg2+ (1 mM), and lumenal [Ca2+] (10 mM), unitary Ca2+ current exhibited an amplitude of 0.9 ± 0.2 pA (n = 3). This result indicates that the actions of Cs+ and Mg2+ on unitary Ca2+ current were additive. These data demonstrate that physiological levels of monovalent cation and Mg2+ effectively compete with Ca2+ as charge carrier in cardiac ryanodine receptor channels. If lumenal free Ca2+ is 2 mM, then our results indicate that unitary Ca2+ current under physiological conditions should be <0.6 pA. 相似文献
14.
Giovanni Testore Sebastiano Colombatto Francesca Silvagno Stefano Bedino 《The international journal of biochemistry & cell biology》1995,27(11):1201-1210
Oxidative deamination of putrescine, the precursor of polyamines, gives rise to γ-aminobutyraldehyde (ABAL). In this study an aldehyde dehydrogenase, active on ABAL, has been purified to electrophoretic homogeneity from rat liver cytoplasm and its kinetic behaviour investigated. The enzyme is a dimer with a subunit molecular weight of 51,000. It is NAD+-dependent, active only in the presence of sulphhydryl compounds and has a pH optimum in the range 7.3–8.4. Temperatures higher than 28°C promote slow activation and the process is favoured by the presence of at least one substrate. Km for aliphatic aldehydes decreases from 110 μM for ABAL and acetaldehyde to 2–3 μM for capronaldehyde. The highest relative V-values have been observed with ABAL (100) and isobutyraldehyde (64), and the lowest with acetaldehyde (14). Affinity for NAD+ is affected by the aldehyde present at the active site: Km for NAD+ is 70 μM with ABAL, 200 μM with isobutyraldehyde and capronaldehyde, and>800 μM with acetaldehyde. The kinetic behaviour at 37°C is quite complex; according to enzymatic models, NAD+ activates the enzyme (Kact 500 μM) while NADH competes for the regulatory site (Kin 70 μM). In the presence of high NAD+ concentrations (4 mM), ABAL promotes further activation by binding to a low-affinity regulatory site (Kact 10 mM). The data show that the enzyme is probably an E3 aldehyde dehydrogenase, and suggest that it can effectively metabolize aldehydes arising from biogenic amines. 相似文献
15.
Inclusion of an oligomeric enzyme, NAD+-dependent hydrogenase from the hydrogen-oxidizing bacterium Ralstonia eutropha, into a system of reverse micelles of different sizes resulted in its dissociation into catalytically active heterodimers and subunits, which were characterized in reactions with various substrates. It was found that: 1) the native tetrameric form of this enzyme catalyzes all types of studied reactions; 2) hydrogenase dimer, HoxHY, is a minimal structural unit catalyzing hydrogenase reaction with an artificial electron donor, reduced methyl viologen; 3) all structural fragments containing FMN and NAD+/NADH-binding sites exhibit catalytic activity in diaphorase reactions with one- and two-electron acceptors; 4) small subunits, HoxY and HoxU also exhibit activity in diaphorase reactions with artificial acceptors. These results can be considered as indirect evidence that the second FMN molecule may be associated with one of the small subunits (HoxY or HoxU) of the hydrogenase from R. eutropha.__________Translated from Biokhimiya, Vol. 70, No. 6, 2005, pp. 782–789.Original Russian Text Copyright © 2005 by Tikhonova, Kurkin, Klyachko, Popov. 相似文献
16.
We have previously reported the isolation by gel filtration and anionic exchange HPLC of two brain Na+, K+-ATPase inhibitors, II-A and II-E, and kinetics of enzyme interaction with the latter. In the present study we evaluated the kinetics of synaptosomal membrane Na+, K+-ATPase with II-A and found that inhibitory activity was independent of ATP (2–8 mM), Na+ (3.1–100 mM), or K+ (2.5–40 mM) concentration. Hanes-Woolf plots showed that II-A decreases Vmax in all cases; KM value decreased for ATP but remained unaltered for Na+ and K+, indicating respectively uncompetitive and noncompetitive interaction. However, II-A became a stimulator at 0.3 mM K+ concentration. It is postulated that brain endogenous factor II-A may behave as a sodium pump modulator at the synaptic region, an action which depends on K+ concentration. 相似文献
17.
Choong-Hyun Kim Jakob K. Kristjansson Michael M. White Thomas C. Hollocher 《Biochemical and biophysical research communications》1982,108(3):1126-1130
Measurements were made of the effect of dicationic (oxidized) and monocationic radical (reduced) forms of benzyl viologen (BV) and methyl viologen (MV) on the ion conductance across planar phospholipid bilayers under conditions of constant voltage. BV+ at 60 μM greatly increased ion conductance whereas BV2+, MV+ and MV2+ did not. Ion permeability ratios relative to nitrate were determined in the BV+ system. BV+ appears to be the first example of a perfectly anion-selective ionophore of the carrier type. BV+ probably functions both as an electron carrier and ionophore for nitrate while catalyzing the dithionite-nitrate reductase reaction in . 相似文献
18.
P. Rustin D. Chretien B. Parfait A. Rötig A. Munnich 《Molecular and cellular biochemistry》1997,174(1-2):115-119
Human cultured cells are widely used for the investigation of respiratory chain disorders. Oxidative properties are generally investigated by means of polarographic studies carried out on detergent-permeabilized cells. By studying the oxidative properties of Epstein-Barr virus-transformed B lymphocytes, we found that the respiration was significantly decreased after 3–4 days of cell culture. Simultaneously, we observed that NAD+-dependent oxidations (malate, glutamate, pyruvate) became dependent upon the addition of exogenous NAD+. The effect of NAD+ was shown to be related to an influx of catalytic amount of NAD+ into the mitochondrial matrix. A full ability to oxidize NAD+-dependent substrates was restored less than 2 h after a change of the culture medium.These observations suggested: (a) the occurrence of fluxes of catalytic amounts of NAD+ through the mitochondrial inner membrane in human cells; (b) an early control of mitochondrial metabolism by matrix NAD+ content in cells grown under limiting growth conditions; (c) the possible confusion between complex I deficiency and a decrease content of matrix NAD+ when using human cultured cells. (Mol Cell Biochem 115–119, 1997) 相似文献
19.
The role of monovalent cations in the photosynthesis of isolated intact spinach chloroplasts was investigated. When intact chloroplasts were assayed in a medium containing only low concentrations of mono- and divalent cations (about 3 mval l-1), CO2-fixation was strongly inhibited although the intactness of chloroplasts remained unchanged. Addition of K+, Rb+, or Na+ (50–100 mM) fully restored photosynthesis. Both the degree of inhibition and restoration varied with the plant material and the storage time of the chloroplasts in low-salt medium. In most experiments the various monovalent cations showed a different effectiveness in restoring photosynthesis of low-salt chloroplasts (K+>Rb+>Na+). Of the divalent cations tested, Mg2+ also restored photosynthesis, but to a lesser extent than the monovalent cations.In contrast to CO2-fixation, reduction of 3-phosphoglycerate was not ihibited under low-salt conditions. In the dark, CO2-fixation of lysed chloroplasts supplied with ATP, NADPH, and 3-phosphoglycerate strictly required the presence of Mg2+ but was independent of monovalent cations. This finding excludes a direct inactivation of Calvin cycle enzymes as a possible basis for the inhibition of photosynthesis under low-salt conditions.Light-induced alkalization of the stroma and an increase in the concentration of freely exchangeable Mg2+ in the stroma, which can be observed in normal chloroplasts, did not occur under low-salt conditions but were strongly enhanced after addition of monovalent cations (50–100 mM) or Mg2+ (20–50 mM).The relevance of a light-triggered K+/H+ exchange at the chloroplast envelope is discussed with regard to the light-induced increase in the pH and the Mg2+ concentration in the stroma, which are thought to be obligatory for light activation of Calvincycle enzymes. 相似文献
20.
R.Kenneth Burkhard 《Archives of biochemistry and biophysics》1982,218(1):207-212
Association constants were determined for the 1:1 interactions of calcium with NAD+, NADH, NADP+, and NADPH in aqueous systems (pH 7, 25 °C) by use of a calcium-sensitive electrode. The order of binding of calcium to these pyridine nucleotides appears to be NAD+ < NADH < NADP+ < NADPH with association constants of 0.2 × 102, 0.3 × 102, 0.9 × 102, and 2 × 102, respectively. Calorimetric experiments revealed that all of these interactions are endothermic with enthalpy changes of 1, 2, 2, and 3 kcal/mol, respectively. 相似文献