Photoaffinity labeling of human placental NAD(+)-linked 15-hydroxyprostaglandin dehydrogenase with [alpha-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+, [alpha-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 microM with an apparent Kd of 8-10 microM. Approximately 90% of photolabeling could be protected by 200 microM NAD+ when the protein was photolyzed in the presence of 10 microM 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.
Chavan
A J
AJ
Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University of Kentucky, Lexington 40536-0082.
Ensor
C M
CM
Wu
P
P
Haley
B E
BE
Tai
H H
HH
D000225:Adenine / Q000032:analysis; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001386:Azides; D002846:Chromatography, Affinity; D006801:Humans; D006911:Hydroxyprostaglandin Dehydrogenases / Q000737:chemistry*; D006913:Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009243:NAD / Q000031:analogs & derivatives / Q000737:chemistry*; D010084:Oxidation-Reduction; D010455:Peptides / Q000032:analysis; D010777:Photochemistry; D010920:Placenta / Q000201:enzymology*
D000345:Affinity Labels; D000596:Amino Acids; D001386:Azides; D010455:Peptides; D009243:NAD; C062879:nicotinamide 2-azidoadenine dinucleotide; D006913:Hydroxysteroid Dehydrogenases; D006911:Hydroxyprostaglandin Dehydrogenases; C035859:15-hydroxyprostaglandin dehydrogenase; D000225:Adenine