Photoaffinity labeling of human placental NAD⁺-linked 15-hydroxyprostaglandin dehydrogenase with [α-³²P]2N₃NAD⁺: Identification of a peptide in the adenine ring binding domain

Oxidation of many prostaglandins at C-15 results in the formation of 15-keto metabolites, which have reduced biological activity. This reaction is catalyzed by NAD⁺-dependent 15-hydroxyprostaglandin dehydrogenase. Using the photoaffinity analog of NAD⁺, [α-³²P]nicotinamide-2-azidoadenine dinucleotide, we have identified a peptide in the adenine ring binding domain of the NAD⁺ binding site of 15-hydroxyprostaglandin dehydrogenase. The specificity of photolabeling was demonstrated by saturation and protection experiments. Saturation of photolabeling was observed at approximately 45-50 μM with an apparent Kd of 8-10 μM. Approximately 90% of photolabeling could be protected by 200 μM NAD⁺ when the protein was photolyzed in the presence of 10 μM probe. The photolabeled protein was digested with Staphylococcus aureus V8 or chymotrypsin, and the photolabeled peptides were purified by either boronate affinity chromatography or Fe⁺³ chelate chromatography followed by reverse phase HPLC. The photolabeled peptide region was identified to be Val³²-Glu⁴⁰.