An 125I-labeled N6-substituted azido analog of NAD+ for the photoaffinity labeling of NAD(+)-linked enzymes.
125I-N6-(N-[6-N-(5-iodo-4-azidosalicyl)-aminohexyl]- aminocarbamoylmethyl)-nicotinamide adenine dinucleotide (125I-N6-I-ASA-AH-NAD+) was synthesized by coupling N6-([6-aminohexyl]-carbamoylmethyl)-NAD+ with 4-azidosalicylic acid N-hydroxysuccinimide ester followed by radioiodination. The utility of 125I-N6-I-ASA-AH-NAD+ as an effective site-directed photoprobe was demonstrated by the photolabeling of both glutamate dehydrogenase and 15-hydroxyprostaglandin dehydrogenase. Both enzymes can be saturated with labeled probe with apparent dissociation constants comparable to those reported for NAD+. Photoincorporation of the probe into both enzymes was found to be protected specifically by NAD+. These results indicate that 125I-N6-I-ASA-AH-NAD+ can be a specific photoprobe for NAD(+)-linked enzymes.
Chen
P L
PL
Division of Medicinal Chemistry & Pharmaceutics, College of Pharmacy, University of Kentucky, Lexington 40536-0082.
Ensor
C M
CM
Tai
H H
HH
D000345:Affinity Labels / Q000737:chemistry*; D001386:Azides / Q000737:chemistry*; D005969:Glutamate Dehydrogenase / Q000737:chemistry; D006911:Hydroxyprostaglandin Dehydrogenases / Q000737:chemistry; D007457:Iodine Radioisotopes; D009243:NAD / Q000031:analogs & derivatives* / Q000737:chemistry; D010088:Oxidoreductases / Q000737:chemistry; D010777:Photochemistry
D000345:Affinity Labels; D001386:Azides; D007457:Iodine Radioisotopes; D009243:NAD; D010088:Oxidoreductases; D006911:Hydroxyprostaglandin Dehydrogenases; C035859:15-hydroxyprostaglandin dehydrogenase; D005969:Glutamate Dehydrogenase