Muller C D, Tarnus C, Schuber F
Biochem J. 1984 Nov 1;223(3):715-21. doi: 10.1042/bj2230715.
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.
合成了NAD⁺吡啶类似物的1,N⁶-乙烯基衍生物，对其进行了表征，并作为核苷酸焦磷酸酶（EC 3.6.1.9）荧光测定的底物进行了测试。在其焦磷酸键断裂时，吡啶-1,N⁶-乙烯基腺嘌呤二核苷酸（εPdAD⁺）和4-肼羰基吡啶-1,N⁶-乙烯基腺嘌呤二核苷酸（εhy4PdAD⁺）的荧光在pH 7.4时分别增加了15倍和73倍。这一特性使得通过连续监测反应进程来方便地进行核苷酸焦磷酸酶的稳态测定成为可能。这两种化合物都是这类酶的良好底物。εPdAD⁺和εhy4PdAD⁺的荧光对pH变化相对不敏感，这使得在保持细胞完整性的条件下进行测定成为可能。εPdAD⁺可用作测量哺乳动物细胞外核苷酸焦磷酸酶活性的底物，因为它不是细胞外NAD⁺糖水解酶的底物。当需要高灵敏度时，εhy4PdAD⁺被证明是有用的。
