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. 1984 Nov 1;223(3):715–721. doi: 10.1042/bj2230715

Preparation of analogues of NAD+ as substrates for a sensitive fluorimetric assay of nucleotide pyrophosphatase.

C D Muller, C Tarnus, F Schuber
PMCID: PMC1144355  PMID: 6150704

Abstract

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.

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Selected References

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