Preparation of analogues of NAD+ as substrates for a sensitive fluorimetric assay of nucleotide pyrophosphatase.
1,N6-Etheno derivatives of pyridine analogues of NAD+ were synthesized, characterized and tested as substrates for a fluorimetric assay of nucleotide pyrophosphatase (EC 3.6.1.9). Upon cleavage of their pyrophosphate bond, the fluorescence of pyridine-1,N6-ethenoadenine dinucleotide (epsilon PdAD+) and of 4-hydrazinocarbonyl-pyridine-1,N6-ethenoadenine dinucleotide (epsilon hy4PdAD+) increased respectively 15-and 73-fold, at pH 7.4. This property allows a convenient steady-state assay of nucleotide pyrophosphatase by continuous monitoring of reaction progress. Both compounds were good substrates of this class of enzyme. The relative insensitivity of the fluorescence of epsilon PdAD+ and epsilon hy4PdAD+ to pH changes allowed assays under conditions preserving cellular integrity. epsilon PdAD+ is useful as a substrate for measuring nucleotide pyrophosphatase activity on the outside of mammalian cells because it is not a substrate for the external NAD+ glycohydrolase. epsilon Hy4PdAD+ proved useful when high sensitivity was needed.
D000227:Adenine Nucleotides / Q000138:chemical synthesis / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D005470:Fluorometry / Q000379:methods*; D006868:Hydrolysis; D009243:NAD / Q000031:analogs & derivatives* / Q000378:metabolism; D011755:Pyrophosphatases / Q000378:metabolism*; D013053:Spectrophotometry
D000227:Adenine Nucleotides; D009243:NAD; C043655:pyridine 1,N(6)-ethenoadenine dinucleotide; C043656:4-hydrazinocarbonylpyridine-1,N(6)-ethenoadenine dinucleotide; D011755:Pyrophosphatases; C021899:nucleotide pyrophosphatase