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Synthesis of 8‐N 3 ‐[2'‐ 32 P]NADP(H), a new photoaffinity label for NADP(H)‐specific enzymes
Author(s) -
Hartog A.F.,
Berden J.A.
Publication year - 1990
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(90)80661-2
Subject(s) - nad+ kinase , enzyme , photoaffinity labeling , chemistry , reagent , affinity label , stereochemistry , nucleotide , phosphorylation , biochemistry , binding site , organic chemistry , gene
The synthesis of the photoaffinity label 8‐N 3 ‐[2'‐ 32 P]NADP + is described. The synthesis consists of the following steps: first 8‐N 3 ‐AMP is synthesized from AMP or ATP and then the 8‐N 3 ‐AMP is coupled with NMN, using DCCD as coupling reagent. Finally, the formed 8‐N 3 ‐NAD + is phosphorylated with [γ‐ 32 P]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 + .