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1.
Mitochondria from the parasitic helminth, Hymenolepis diminuta, catalyzed both NADPH:NAD+ and NADH:NADP+ transhydrogenase reactions which were demonstrable employing the appropriate acetylpyridine nucleotide derivative as the hydride ion acceptor. Thionicotinamide NAD+ would not serve as the oxidant in the former reaction. Under the assay conditions employed, neither reaction was energy linked, and the NADPH:NAD+ system was approximately five times more active than the NADH:NADP+ system. The NADH:NADP+ reaction was inhibited by phosphate and imidazole buffers, EDTA, and adenyl nucleotides, while the NADPH:NAD+ reaction was inhibited only slightly by imidazole and unaffected by EDTA and adenyl nucleotides. Enzyme coupling techniques revealed that both transhydrogenase systems functioned when the appropriate physiological pyridine nucleotide was the hydride ion acceptor. An NADH:NAD+ transhydrogenase system, which was unaffected by EDTA, or adenyl nucleotides, also was demonstrable in the mitochondria of H. diminuta. Saturation kinetics indicated that the NADH:NAD+ reaction was the product of an independent enzyme system. Mitochondria derived from another parasitic helminth, Ascaris lumbricoides, catalyzed only a single transhydrogenase reaction, i.e., the NADH:NAD+ activity. Transhydrogenase systems from both parasites were essentially membrane bound and localized on the inner mitochondrial membrane. Physiologically, the NADPH:NAD+ transhydrogenase of H. diminuta may serve to couple the intramitochondrial metabolism of malate (via an NADP linked “malic” enzyme) to the anaerobic NADH-dependent ATP-generating fumarate reductase system. In A. lumbricoides, where the intramitochondrial metabolism of malate depends on an NAD-linked “malic” enzyme which is localized primarily in the intermembrane space, the NADH:NAD+ transhydrogenase activity may serve physiologically in the translocation of hydride ions across the inner membrane to the anaerobic energy-generating fumarate reductase system. 相似文献
2.
Glutamate dehydrogenase, GDH (l-glutamate: NAD+ oxidoreductase (deaminating) EC 1.4.1.2) was purified from the plant fraction of lupin nodules and the purity of the preparation established by gel electrophoresis and electrofocusing. The purified enzyme existed as 4 charge isozymes with a MW of 270000. The subunit MW, as determined by dodecyl sulphate electrophoresis, was 45 000. On the basis of the results of the MW determinations a hexameric structure is proposed for lupin-nodule GDH. The pH optima for the enzyme were pH 8.2 for the amination reaction and pH 8.8 for the deamination reaction. GDH from lupin nodules showed a marked preference for NADH over NADPH in the amination reaction and used only NAD+ for the deamination reaction. Pyridoxal-5′-P and EDTA inhibited activity. The enzyme displayed Michaelis-Menten kinetics with respect to all substrates except NAD+. When NAD+ was the varied substrate, there was a deviation from Michaelis-Menten behaviour towards higher activity at high concentrations of NAD+. 相似文献
3.
New enzyme cycling method for determination of oxidized and reduced nicotinamide adenine dinucleotide 总被引:2,自引:0,他引:2
A highly sensitive enzyme cycling method for the measurement of NAD+(H) concentration in tissue extracts has been described. The assay measured propanediol formed during the coupled oxidation-reduction reactions between ethanol and lactaldehyde as catalyzed by liver alcohol dehydrogenase in the presence of catalytic concentrations of NAD+(H). The assay can be used to determine NAD+(H) concentrations in the range of 0.4 to 4 pmoles of coenzyme in a 0.1 ml reaction volume. 相似文献
4.
PIERRETTE BALDY 《Physiologia plantarum》1977,40(2):91-97
Metabolism of γ-Aminobutyrate in Agaricus bisporus. III. The Succinate-Semialdehyde: NAD (P)+ Oxidoreductase. The succinate-semialdehyde:NAD(P)+ oxidoreductase (E.C. 1.2.1.16) is responsible for the second step in the catabolism of γ-aminobutyrate: the irreversible enzymatic conversion of succinic semialdehyde (SSA) to succinate. Succinate semialdehyde dehydrogenase was extracted from mitochondrial fraction of fruit-bodies of Agaricus bisporus Lge. The mitochondrial pellet was sonicated and centrifuged at 110,000 g; the supernatant obtained was designated the “crude extract”. The enzyme was extremely unstable on storage, unless 1 mM EDTA and 20% glycerol were added. Kinetic studies were carried out at 30°C, and the formation of NADH or NADPH was followed by measuring increase of absorbance at 340 nm with a spectrophotometer. The dehydrogenase was completely inactive when the reaction was run in the absence of thiol and was more active with NAD+ than with NADP+. In the “crude extract” the activity with NADP+ had a pH optimum between 8.6 and 9.1 and the Km values for SSA and NADP+ were 2.0 × 10?4M and 1.4 × 10?4M respectively. The pH optimum with NAD+ was found between 8.6 and 8.8 and the Km value for SSA is 4.8 × 10?4M and for NAD+ 2.0 × 10?3M. With NAD+, the kinetic values (pH, Km) of the “crude extract” chromatographed on hydroxylapatite were unchanged. Inhibition by thiamine pyrophosphate (TPP) was uncompetitive with respect to NAD+, those by malate, ATP, ADP and NADPH non-competitive and that by NADH competitive. These results and the fact that activity with NAD+ was lost more slowly than with NADP+ indicate the possibility of at least two mitochondrial succinate-semialdehyde dehydrogenases, even though the activities of this enzyme assayed with NAD+ and NADP+ respectively were not able to be separated from each other by hydroxylapatite column chromatography. Some speculations on the metabolic regulation of this dehydrogenase and considerations on the significance of these results in the physiology of respiration in Agaricus bisporus Lge are given. 相似文献
5.
Michael A. Moxley Daniel A. Beard Jason N. Bazil 《The Journal of biological chemistry》2016,291(6):2712-2730
Mammalian E3 is an essential mitochondrial enzyme responsible for catalyzing the terminal reaction in the oxidative catabolism of several metabolites. E3 is a key regulator of metabolic fuel selection as a component of the pyruvate dehydrogenase complex (PDHc). E3 regulates PDHc activity by altering the affinity of pyruvate dehydrogenase kinase, an inhibitor of the enzyme complex, through changes in reduction and acetylation state of lipoamide moieties set by the NAD+/NADH ratio. Thus, an accurate kinetic model of E3 is needed to predict overall mammalian PDHc activity. Here, we have combined numerous literature data sets and new equilibrium spectroscopic experiments with a multitude of independently collected forward and reverse steady-state kinetic assays using pig heart E3. The latter kinetic assays demonstrate a pH-dependent transition of NAD+ activation to inhibition, shown here, to our knowledge, for the first time in a single consistent data set. Experimental data were analyzed to yield a thermodynamically constrained four-redox-state model of E3 that simulates pH-dependent activation/inhibition and active site redox states for various conditions. The developed model was used to determine substrate/product conditions that give maximal E3 rates and show that, due to non-Michaelis-Menten behavior, the maximal flux is different compared with the classically defined kcat. 相似文献
6.
7.
《DNA Repair》2014
Nicotinamide adenine dinucleotide, NAD+, is a small metabolite coenzyme that is essential for the progress of crucial cellular pathways including glycolysis, the tricarboxylic acid cycle (TCA) and mitochondrial respiration. These processes consume and produce both oxidative and reduced forms of NAD (NAD+ and NADH). NAD+ is also important for ADP(ribosyl)ation reactions mediated by the ADP-ribosyltransferase enzymes (ARTDs) or deacetylation reactions catalyzed by the sirtuins (SIRTs) which use NAD+ as a substrate. In this review, we highlight the significance of NAD+ catabolism in DNA repair and cell death through its utilization by ARTDs and SIRTs. We summarize the current findings on the involvement of ARTD1 activity in DNA repair and most specifically its involvement in the trigger of cell death mediated by ARTD1 activation and energy depletion. By sharing the same substrate, the activities of ARTDs and SIRTs are tightly linked, are dependent on each other and are thereby involved in the same cellular processes that play an important role in cancer biology, inflammatory diseases and ischaemia/reperfusion. 相似文献
8.
M I Horowitz 《Archives of biochemistry and biophysics》1991,288(2):317-323
Browning reactions of amino sugars were observed in a variety of sterile pH buffers at 25-37 degrees C. These reactions were signaled by an increase in absorbance at 273 nm, followed by an increase in absorbance at 320-360 nm. The reactions were maximal at pH 7.0 in phosphate buffer. Acidic solutions (pH less than 2.2) of 50 mM D-glucosamine hydrochloride gave only a negligible reaction and 2-acetamido-2-deoxy-D-glucose was unreactive. Half of the D-glucosamine in a 100 mM solution in sterile 0.2 M sodium phosphate buffer, pH 7.4, at 37 degrees C decomposed or was transformed in 27 h. A comparison of reactivity in generating A273 and A340 chromophores showed D-mannosamine greater than D-galactosamine greater than D-glucosamine. Permanganate oxidation of incubated glucosamine solutions afforded a compound which chromatographed like 2,5-pyrazinedicarboxylic acid and gave the same ultraviolet absorption spectrum. This, together with fractionating and thin-layer chromatography of the products of glucosamine incubation, suggests that 2,5-bis(tetrahydroxybutyl)pyrazine is formed as one of the products of autocondensation of D-glucosamine in accord with the report of Candiano et al. (1988, Carbohydr. Res. 184, 67-75) on products formed in glucosamine-lysine incubation mixtures. Formation of products absorbing at 325-360 nm was inhibited by the chelator diethylene-triaminepentaacetic acid. This suggests that the later reactions may be mediated by a metal-stimulated free radical mechanism. After 4 days incubation high molecular weight products with absorbance maxima at 273 nm and 325-360 nm were detected. Some of these were retained by dialysis membranes of molecular weight cut-off greater than 3500 and greater than 12,000. 相似文献
9.
The assay of the bromelains using N alpha-CBZ-L-lysine p-nitrophenyl ester and N-CBZ-glycine p-nitrophenyl ester as substrates 总被引:1,自引:0,他引:1
R M Silverstein 《Analytical biochemistry》1974,62(2):478-484
Two new esterolytic assays of the pineapple stem bromelains are described. They use as substrates the p-nitrophenyl esters of Nα-CBZ-l-lysine (CLN) and N-CBZ-glycine (CGN). The activity is monitored by the direct spectrophotometric measurement of the enzyme-catalyzed hydrolysis of these esters at 340 nm. The bromelains are rapidly activated with 1 mm l-cysteine at pH 4.6 for the CLN assay and pH 6.1 for the CGN assay. EDTA has no measurable effect. The sensitivities of the assays approach 10 μg/ml in a reaction time of 3 min. 相似文献
10.
An assay for the determination of NAD has been developed utilizing the coupled oxidoreductase activity of liver alcohol dehydrogenase. The coupled reaction between ethanol and lactaldehyde is driven by the removal of one of the products, acetaldehyde, into a semicarbazide solution. Under the stated conditions, a linear relationship exists between the absorbance of acetaldehyde semicarbazone and NAD concentration in the reaction mixture. The principal advantages of this method are speed and simplicity. NAD+ and NADH are assayed by the same procedure, which is also used to measure NADP+ and NADPH after these nucleotides have been converted to NAD+ and NADH, respectively. 相似文献
11.
The NAD+ analogue, 3-aldoxime pyridine adenine dinucleotide, is prepared by transglycosidation. Contrary to the published data, this analogue shows no activity as coenzyme with alcohol dehydrogenase from horse liver or from yeast. This is demonstrated by three methods: no increase of absorption at 331 nm by the enzymic oxidation of ethanol; no increase at 290 nm with cinnamic alcohol; and no exchange reaction. The inhibition by this analogue of the oxidation of ethanol by NAD+ is competitive at pH 7.6 and 9.5 with yeast alcohol dehydrogenase; with liver alcohol dehydrogenase, it is of the mixed type at pH 7.6 and non-competitive at pH 9.5. The lack of activity of the analogue and inhibition of the competitive or mixed type may be explained by the fact that the binary complex does not bind the substrate or that in the ternary complex the hydride shift does not occur. The non-competitive inhibition at pH 9.5 with the horse liver alcohol dehydrogenase may be explained by the existence of binding sites specific for this analogue. 相似文献
12.
Chicken liver lactate dehydrogenase (l-lactate: NAD+ oxidoreductase, EC 1.1.1.27) catalyses the reversible reduction reaction of hydroxypyruvate to l-glycerate. It also catalyses the oxidation reaction of the hydrated form of glyoxylate to oxalate and the reduction of the non-hydrated form to glycolate. At pH 8, these latter two reactions are coupled. The coupled system equilibrium is attained when the NAD+/NADH ratio is greater than unity.Hydroxypyruvate binds to the enzyme at the same site as the pyruvate. When there are substances with greater affinity to this site in the reaction medium and their concentration is very high, hydroxypyruvate binds to the enzyme at the l-lactate site. In vitro and with purified preparation of lactate dehydrogenase, hydroxypyruvate stimulates the production of oxalate from glyoxylate-hydrated form and from NAD; the effect is due to the fact that hydroxypyruvate prevents the binding of non-hydrated form of glyoxylate to the lactate dehydrogenase in the pyruvate binding site. At pH 8, the l-glycerate stimulates the production of glycolate from glyoxylate-non-hydrated form and NADH since hydroxypyruvate prevents the binding of glyoxylate-hydrated form to the enzyme. 相似文献
13.
The inhibition of urocanase from Pseudomonas putida by O-methylhydroxylamine has been characterized as being due to the formation of an adduct between CH3ONH2 and NAD+, the latter of which has been recently shown to be a tightly bound coenzyme for this urocanase. Inhibition is maximal at pH 8.5 and is blocked by the presence of the substrate analog imidazole propionate. Loss of catalytic activity corresponds directly with the binding of 1 mol of 14CH3ONH2 per mole of enzyme, and partial reversibility of the modification, achieved by dialysis at pH 7.5, is accompanied by concomitant restoration of enzymatic activity. No incorporation of 14CH3ONH2 into urocanase is seen when enzyme-bound NAD+ is first converted to NADH or when NAD+ is removed by prior treatment of urocanase with 8 m urea. Stability and spectral properties of the CH3ONH · NAD adduct are consistent with previous data reported for the product of the hydroxylamine reaction with NAD+. It is concluded that other urocanases which exhibit inhibition by hydroxylamine may likewise contain NAD+ as an essential coenzyme and that the use of 14CH3ONH2 as a reversible modification reagent for NAD+ should prove helpful for studies on the role of NAD+ in the urocanase catalytic process. 相似文献
14.
Yuta Mutaguchi Taketo Ohmori Haruhiko Sakuraba Kazunari Yoneda Katsumi Doi Toshihisa Ohshima 《Analytical biochemistry》2011,(1):1
Methods with which to simply and rapidly assay l-aspartate (l-Asp) and d-aspartate (d-Asp) would be highly useful for physiological research and for nutritional and clinical analyses. Levels of l- and d-Asp in food and cell extracts are currently determined using high-performance liquid chromatography. However, this method is time-consuming and expensive. Here we describe a simple and specific method for using an l-aspartate dehydrogenase (l-AspDH) system to colorimetrically assay l-Asp and a system of three hyperthermophilic enzymes—aspartate racemase (AspR), l-AspDH, and l-aspartate oxidase (l-AO)—to assay d-Asp. In the former, the reaction rate of nicotinamide adenine dinucleotide (NAD+)-dependent l-AspDH was measured based on increases in the absorbance at 438 nm, reflecting formation of formazan from water-soluble tetrazolium-1 (WST-1), using 1-methoxy-5-methylphenazinum methyl sulfate (mPMS) as a redox mediator. In the latter, d-Asp was measured after first removing l-Asp in the sample solution with l-AO. The remaining d-Asp was then changed to l-Asp using racemase, and the newly formed l-Asp was assayed calorimetrically using NAD+-dependent aspartate dehydrogenase as described above. This method enables simple and rapid spectrophotometric determination of 1 to 100 μM l- and d-Asp in the assay systems. In addition, methods were applicable to the l- and d-Asp determinations in some living cells and foods. 相似文献
15.
The reaction of 4,4'-bis-dimethylaminodiphenylcarbinol with the sulfhydryl group. A new reagent for sulfhydryl analysis 总被引:3,自引:0,他引:3
M S Rohrbach B A Humphries F J Yost W G Rhodes S Boatman R G Hiskey J H Harrison 《Analytical biochemistry》1973,52(1):127-142
4,4′-bis-Dimethylaminodiphenylcarbinol (BDC-OH) dissociates in aqueous buffers at pH values below neutrality to form a resonance-stabilized carbonium-immonium ion (BDC+) which exhibits an absorbance maximum at 606 nm. In the presence of 4.0 M guanidine hydrochloride, BDC+ has an apparent molar absorption coefficient of 70,800 M?1cm?1 and an absorbance maximum of 612 nm. Sulfhydryl groups react with the cation to form S-(4,4′-bis-dimethylaminodiphenylmethyl-) derivatives with a concomitant quantitative loss of the 612-nm absorbance. This quantitative interaction has been exploited in the development of a new and convenient technique for the quantitative determination of sulfhydryl groups in proteins. Results of sulfhydryl determinations on simple thiols and five proteins are presented, along with comparison data obtained via other sulfhydryl techniques. 相似文献
16.
Summary A quantitative cytochemical assay for NAD+ kinase-like activity in the guinea-pig thyroid gland is described. The NADP+ produced by the activity of the kinase was used to drive the NADP+-dependent enzyme glucose-6-phosphate dehydrogenase which is endogenous to the tissue. The activity of glucose-6-phosphate dehydrogenase is greatly in excess of that of the kinase and was unaffected by the constituents of the kinase incubation medium (ATP, Mg2+ and NAD+) either alone or in combination. Kinase activity was dependent both on ATP and Mg2+, with maximal activity seen when the Mg-ATP ratio was between 1:1 and 4:1. Free ATP inhibited the activity of the enzyme. Enzyme activity was exhibited over a broad pH range (7–9) with a peak at pH 8.2. The sulphhydryl-blocking agents,p-chloromercuribenzoate, iodoacetate and iodoacetamide (at 1 mM), completely abolished kinase activity but were without effect on glucose-6-phosphate dehydrogenase activity.N-ethylmaleimide and citrate (both at 1 mM) had no effect on either kinase or glucose-6-phosphate dehydrogenase activities. 相似文献
17.
By means of a Sephadex G-50 column and anionic exchange HPLC a cerebral cortex soluble fraction (II-E) which highly inhibits neuronal Na+-K+-ATPase activity has been previously obtained. Herein, II-E properties are compared with those of the cardenolide ouabain, the selective and specific Na+, K+-ATPase inhibitor. It was observed that alkali treatment destroyed II-E but not ouabain inhibitory activity. II-E presented a maximal absorbance at 265 nm both at pH 7 and pH 2 which diminished at pH 10. Ouabain showed a maximum at 220 nm which was not altered by alkalinization. II-E was not retained in a C-18 column, indicating its hydrophilic nature, whereas ouabain presented a 26-min retention time in reverse phase HPLC. Therefore, it is concluded that the inhibitory factor present in II-E is structurally different to ouabain. 相似文献
18.
An H-ATPase Assay: Proton Pumping and ATPase Activity Determined Simultaneously in the Same Sample 总被引:8,自引:4,他引:4
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Palmgren MG 《Plant physiology》1990,94(3):882-886
A continuous spectrophotometric assay of H+-ATPase activity was developed by combining two well-known methods for measuring proton pumping and ATPase activity. Proton uptake into plasma membrane vesicles from Avena sativa L. (cv Rhiannon) was monitored as the absorbance decrease at 495 nm of the ΔpH probe acridine orange. Simultaneously, ATPase activity was measured by following the absorbance decrease at 340 nanometers by coupling ATP hydrolysis enzymatically to the oxidation of NADH. This H+-ATPase assay is convenient for determining the relative relationship between ATP hydrolysis and proton pumping. 相似文献
19.
Electrochemically generated dimers of N′-methylnicotinamide and NAD+ both undergo photooxidation on irradiation at 254 nm in aqueous medium to yield the respective parent monomers. For the NAD dimer the quantum yield for photodissociation was about 0.01, whether irradiated at 254 nm or at wavelengths to the red of 320 nm. Irradiation at the latter wavelengths, where the NAD+ monomer itself does not absorb and is not photosensitive, led to quantitative regeneration of coenzyme activity in the alcohol dehydrogenase system. The photodissociation reaction exhibited no oxygen effect. The photochemically generated dimer of N′-methylnicotinamide was also photooxidized to the parent monomer by irradiation at 254 nm at pH 9.5. The foregoing process of electrochemical (or photochemical) reduction and photochemical oxidation, comprising a closed cycle of electron and proton transport, is similar to that previously observed for a number of pyrimidine analogs. Furthermore, the NAD dimer is a substrate of snake venom nucleotide pyrophosphatase and is hydrolyzed to release NMN dimer which, on irradiation at 254 nm, also undergoes photooxidation to the parent NMN+ monomer. A mechanism for the photooxidation reaction is formulated and relevant biological implications of the foregoing are presented. 相似文献
20.
Barbara Czochralska Wiesław Kawczynski Grzegorz Bartosz David Shugar 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,801(3):403-409
It has been shown that direct excitation of NADH (or NADPH) in aqueous medium at 254 nm, or at wavelengths longer than 320 nm (where only the reduced nicotinamide moiety absorbs), leads to generation of NAD+ (or NADP+). The reaction proceeds both in the presence and absence of oxygen. Under aerobic conditions the reaction is accompanied by formation of H2O2 at a level equimolar with that of the NADH present in solution. On irradiation at wavelengths longer than 320 nm, conversion of NADH to enzymatically active NAD+ is about 75%. Under analogous irradiation conditions, the dimers (NAD)2 and (NADP)2 undergo disproportionation to NAD+ and NADP+, respectively, to the extent of 90%. Both physicochemical and enzymatic criteria were employed to formulate mechanisms for the photooxidation of NADH and the photodisproportionation of the dimer (NAD)2. 相似文献