An enzymatic cycling assay for nicotinic acid adenine dinucleotide phosphate using NAD synthetase.
Anal Biochem
; 364(2): 97-103, 2007 May 15.
Article
en En
| MEDLINE
| ID: mdl-17395143
Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to mobilize Ca(2+) from intracellular stores in a wide variety of organisms, ranging from plants to humans. We have developed a novel enzyme cycling assay for NAADP that involves coupled reactions catalyzed by four enzymes. In this system, NAADP is first converted into nicotinic acid adenine dinucleotide (NAAD) by alkaline phosphatase, after which the NAAD is converted to NAD, AMP, and PPi by NAD synthetase (NADS) in the presence of ATP and ammonia. The NAD is then amplified using an enzyme cycling system driven by glucose dehydrogenase and diaphorase. The resultant formation of formazan dye is measured spectrophotometrically based on the increase in absorbance at 450 nm. Using this method, NAADP (20-400 nM) was assayed, and a highly linear correlation was obtained between the NAADP concentration and the increase in absorbance at 450 nm. The cycling rate was approximately 95 cycles/min. In addition, the within-run coefficients of variation (CVs) for 25, 50, and 100 nM NAADP solutions were 9.33, 4.86, and 3.13%, respectively. Interference by NAD analogs (e.g., NAAD, NADP) in the sample was eliminated prior to running the assay by treating the sample with NADS and NAD nucleosidase (NADase). In sum, our findings indicate this enzyme cycling assay to be readily applicable for determination for NAADP in a variety of biological samples and to be particularly appropriate for use with an autoanalyzer.
Buscar en Google
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Amida Sintasas
/
NADP
Tipo de estudio:
Diagnostic_studies
/
Evaluation_studies
/
Prognostic_studies
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Anal Biochem
Año:
2007
Tipo del documento:
Article
País de afiliación:
Japón
Pais de publicación:
Estados Unidos