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Nicotinamide-adenine dinucleotide pyrophosphatase in the growing and aging mosquito.

ABSTRACT: 1. The disappearance of pyridine nucleotides during incubation with mosquito homogenates proceeds through the hydrolysis of the pyrophosphate linkage of these compounds as demonstrated by the formation of NMN and AMP from NAD(+). This reaction was also demonstrated by the loss in the coenzyme functioning property of NAD(+) (yeast alcohol dehydrogenase reaction) without a concomitant loss in reactivity towards cyanide. Transglycosidase activity was not observed in the mosquito homogenates, and low concentrations of nicotinamide did not inhibit the NAD(+) splitting activity of these homogenates. These observations are all in accord with the presence in these homogenates of a NAD(+) pyrophosphatase rather than a NADase. 2. The NAD(+) pyrophosphatase is destroyed by boiling, is not heat-activated, and has a pH optimum at pH8.75. In addition to NAD(+), other dinucleotides such as NADP(+), the 3-acetylpyridine and thionicotinamide analogues of NAD(+) and the thionicotinamide analogue of NADP(+), function as substrates in the hydrolysis catalysed by the pyrophosphatase. 3. A decrease in the specific activity of NAD(+) pyrophosphatase was observed during larval development, and a barely detectable activity was found in the pupa and adult. 4. Enzyme activity per organism increased in the larva but decreased to a very low value in the pupa and adult. These results indicate that the decrease in specific activity was due to a decrease in enzyme concentration rather than an increase in amounts of protein.

SUBMITTER: Anderson BM

PROVIDER: S-EPMC1270119 | biostudies-other | 1966 Nov

REPOSITORIES: biostudies-other

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Project description:AimsAccurate analysis of dinucleotide redox cofactors nicotinamide adenine dinucleotide phosphate reduced (NADPH), nicotinamide adenine dinucleotide phosphate (NADP+), nicotinamide adenine dinucleotide reduced (NADH), and nicotinamide adenine dinucleotide (NAD+) from biological samples is important to understanding cellular redox homeostasis. In this study, we aimed to develop a simple protocol for quenching metabolism and extracting NADPH that avoids interconversion among the reduced forms and the oxidized forms.ResultsWe compared seven different solvents for quenching and extraction of cultured mammalian cells and mouse tissues: a cold aqueous buffer commonly used in enzyme assays with and without detergent, hot aqueous buffer, and cold organic mixtures (80% methanol, buffered 75% acetonitrile, and acidic 40:40:20 acetonitrile:methanol:water with either 0.02 M or 0.1 M formic acid). Extracts were analyzed by liquid chromatography-mass spectrometry (LC-MS). To monitor the metabolite interconversion, cells were grown in 13C6-glucose medium, and unlabeled standards were spiked into the extraction solvents. Interconversion between the oxidized and reduced forms was substantial except for the enzyme assay buffer with detergent, 80% methanol and 40:40:20 acetonitrile:methanol:water, with the 0.1 M formic acid mix giving the least interconversion and best recoveries. Absolute NAD+, NADH, NADP+, and NADPH concentrations in cells and mouse tissues were measured with this approach.InnovationWe found that the interconversion between the reduced and oxidized forms during extraction is a major barrier to accurately measuring NADPH/NADP+ and NADH/NAD+ ratios. Such interconversion can be monitored by isotope labeling cells and spiking NAD(P)(H) standards.ConclusionExtraction with 40:40:20 acetonitrile:methanol:water with 0.1 M formic acid decreases interconversion and, therefore, is suitable for measurement of redox cofactor ratios using LC-MS. This solvent is also useful for general metabolomics. Samples should be neutralized immediately after extraction to avoid acid-catalyzed degradation. When LC-MS is not available and enzyme assays are accordingly used, inclusion of detergent in the aqueous extraction buffer reduces interconversion. Antioxid. Redox Signal. 28, 167-179.

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Nicotinamide-adenine dinucleotide pyrophosphatase in the growing and aging mosquito.

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