An improved cycling assay for nicotinamide adenine dinucleotide

“…the values for lA, lB, 2A, 2B, 2C and 2D are 1.83 m M -' s-', 3.33 mM-' s-', 0.75 mM-' s-', 1.42 mM-' s-', 1.68 mM-' s -' and 2.03 mM-' s -I , respectively. The rate constants, k, and k3, for the reactions of the reduced form of 2B with oxygen and with 3-(4,5'-dimethyl~hiazole-2-yl)-2,5-diphenyltetrazolium ion, respectively, were k2 = 1.21 mM-' sC1 and k3 = 91 mM-' s-'.…”

mentioning

confidence: 88%

Synthesis and characterization of 1‐substituted 5‐alkylphenazine derivatives carrying functional groups

Yomo

Sawai

Urabe

et al. 1989

European Journal of Biochemistry

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The following 1-substituted derivatives of Smethylphenazine and 5-ethylphenazine were synthesized: 1-(3-carboxypropyloxy)-5-methylphenazine (lB), l-(3-carboxypropyloxy)-5-ethylphenazine (2B), 1-(3-ethoxycarbonylpropyloxy)-5-ethylphenazine (2C) and l-[N-(2-aminoethyl)carbamoylpropyloxy]-5-~thylphenazine (2D); their spectra, stability and reactivity as electron mediators were investigated, together with those of 5-methylphenazine (1A) and 5-ethylphenazine (2A). The 1-substituted derivatives are all insensitive to light and the derivatives of 5-ethylphenazine are more stable than those of 5-methylphenazine under neutral and alkaline conditions; 2 8 is the most stable of all the derivatives. The spectral properties of the decomposed compounds showed that photodecomposition of 1A and 2A is associated with hydroxylation at position 1, alkali decomposition of 1A and 1 B with elimination of the 5-methyl group and alkali decomposition of 2A, 2B, and 2D with a ring-opening reaction. The second-order rate constant kl for the reaction of the phenazine derivatives with NADH was measured under steady-state conditions. The k l values vary depending on the substituents at positions 1 and 5 :the values for lA, lB, 2A, 2B, 2C and 2D are 1.83 m M -' s-', 3.33 mM-' s-', 0.75 mM-' s-', 1.42 mM-' s-', 1.68 mM-' s -' and 2.03 mM-' s -I , respectively. The rate constants, k, and k3, for the reactions of the reduced form of 2B with oxygen and with 3-(4,5'-dimethyl~hiazole-2-yl)-2,5-diphenyltetrazolium ion, respectively, were k2 = 1.21 mM-' sC1 and k3 = 91 mM-' s-'. These phenazine derivatives have potential applications in the biochemical field. (Fig. 1, 1A) is a unique electron mediator as it can accept electrons from NAD(P)H and is widely used in various redox reactions. However, its application is still limited, mainly because of its instability to light and to alkaline solution. 5-Ethylphenazine (Fig. 1, 2A) has been recommended as a more stable electron mediator in alkaline solutions [l, 21 and light-insensitive derivatives have also been prepared and used in the assay of dehydrogenases [3 -61, but the mechanisms of these instabilities are still unclear. 5-MethylphenazineWe have studied co-enzyme-recycling systems [7 -91 and now we intend to use phenazine derivatives as NAD(P)+ regenerators. For this purpose, it is necessary to prepare stable phenazine derivatives with a functional group suitable for binding them to other molecules, such as water-soluble polymers, solid supports and enzymes. These derivatives are also useful for investigating the mechanisms of the electron-transfer reactions from NAD(P)H. Here, we describe the preparation of new phenazine derivatives carrying a carboxyl group (Fig. I , 1 B and 2B) and an amino group (Fig. 1, 2D) and also their stability and electron-transfer activity. The kinetic constants obtained in this work are necessary for understanding the coupled-reaction system of NAD(P)+ regeneration.

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“…The supernatant was used for assaying NAD(P)(H) levels. The assay was based on the methods of Burch et al (1967) and Bernofsky & Swan (1973). The oxidized forms are stable in 0-01 M-H2SO4/0-1 M -N~~S O~, whereas the reduced forms are destroyed by heating for 30min at 60°C.…”

Section: Growth Of Plants Soybean Plants [Glycine Maxmentioning

confidence: 99%

Factors contributing to the accumulation of glutamate in Bradyrhizobium japonicum bacteroids under microaerobic conditions

Salminen

Streeter

1990

Journal of General Microbiology

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Previous studies with labelled N and C have indicated synthesis and accumulation of glutamate in Brudyrhizobium japonicum bacteroids under microaerobic conditions similar to those found in soybean nodules. Low 2-oxoglutarate dehydrogenase (OGDH) activity might have accounted for this observation, but similar levels of enzyme activity were found in bacteroids isolated anaerobically or aerobically and in cultured bacteria. However, OGDH from B.japonicum bacteroids was strongly inhibited by NADH, and the degree of inhibition depended on the NADH: NAD ratio. Determination of endogenous levels of NAD and NADH gave NADH: NAD ratios of 0.19 and 0.83 in bacteroids isolated under aerobic and anaerobic conditions, respectively. A ratio of 0.83 resulted in more than 50% inhibition of OGDH in uitro, and this would be consistent with channelling of 2-oxoglutarate to glutamate. [ 14C)Glutamate supplied to bacteroids was metabolized to CO, slowly relative to the respiration of malate, and essentially no labelling of products of glutamate metabolism such as arginine, proline, glutamine and 4aminobutyrate (GAB) was found. Attempts to trap 14C in GAB by supplying unlabelled GAB or transaminase inhibitors with [ 14C]glutamate were unsuccessful. The finding that glutamate decarboxylase was essentially absent in six different strains of B. japonicum was consistent with the labelling results and indicated that conversion of glutamate to succinate via GAB is slow or nil. The inhibition of OGDH by a high NADH:NAD ratio and the absence of the GAB shunt are complementary mechanisms which probably account for the accumulation of glut ama te .

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An improved cycling assay for nicotinamide adenine dinucleotide

Cited by 355 publications

References 3 publications

Effects of the overexpression of fructose-1,6-bisphosphate aldolase on fermentation pattern and transcription of the genes encoding lactate dehydrogenase and pyruvate formate-lyase in a ruminal bacterium, Streptococcus bovis

Effects of the overexpression of fructose-1,6-bisphosphate aldolase on fermentation pattern and transcription of the genes encoding lactate dehydrogenase and pyruvate formate-lyase in a ruminal bacterium, Streptococcus bovis

Synthesis and characterization of 1‐substituted 5‐alkylphenazine derivatives carrying functional groups

Factors contributing to the accumulation of glutamate in Bradyrhizobium japonicum bacteroids under microaerobic conditions

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