Synthesis of 8‐N3‐[2'‐32P]NADP(H), a new photoaffinity label for NADP(H)‐specific enzymes

FEBS Letters

Hartog, A.F.¹  Berden, J.A.¹ 

[1] E.G. Slater Institute for Biochemical Research, University of Amsterdam, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands

关键词: NADP-specific enzyme;    Photoaffinity probe;    NADP analog;    [32P]NADP+;    8-azido;    NAD-kinase;    8-N3-AMP;    8-azido-adenosine-5'-monophosphate;    8-N3-ATP;    8-azido-adenosine-5'-triphosphate;    8-N3-NAD(H);    nicotinamide-8-azido-adenine dinucleotide;    8-N3-NADP(H);    nicotinamide-8-azido-adenine dinucleotide phosphate;    TEA · HCO3;    triethylamine-bicarbonate;   

DOI  :  10.1016/0014-5793(90)80661-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The synthesis of the photoaffinity label 8-N₃-[2'-³²P]NADP⁺ is described. The synthesis consists of the following steps: first 8-N₃-AMP is synthesized from AMP or ATP and then the 8-N₃-AMP is coupled with NMN, using DCCD as coupling reagent. Finally, the formed 8-N₃-NAD⁺ is phosphorylated with [γ-³²P]ATP, using NAD-kinase. The formed product is purified and analysed and is shown to react well with NADP-specific enzymes and not to react with NAD-specific enzymes. Additionally, it is proposed that in the NAD-kinase reaction 2 mol ATP are used for the formation of 1 mol NADP: one molecule of ATP is hydrolyzed to ADP and phosphate while the other delivers the phosphate for the phosphorylation of NAD⁺.

【 授权许可】

Unknown