Identification and Characterization of a Nucleotide Binding Site of Ovine Prolactin with 2-Azido-NAD

Photoaffinity labeling of ovine prolactin with the NAD+ photoaffinity analog [α-32P]nicotinamide-2-azidoadenine dinucleotide has been used to identify an NADH/NADPH binding site. Specificity of nucleotide interaction was demonstrated by saturation and protection of labeling at physiologically relevant concentrations. Saturation of photoinsertion was observed at approximately 100 μM probe with an apparent Kd of approximately 25 μM. Protection of photoinsertion was observed with NAD+ and NADH. The photoinsertion was decreased by 75% and greater than 95%, respectively, upon addition of 200 μM of the above-mentioned compounds. The protection obtained with NADP+ and NADPH was of the same order, respectively. The adenine ring binding domain of NADH/NADPH binding site was identified by trypsin and chymotrypsin digestion of the photolabeled prolactin and purification of the photolabeled peptide by boronate affinity chromatography and immobilized Fe3+ affinity chromatography. The peptide was identified to be Ala22-Tyr28. These studies demonstrate that prolactin contains an NADH/NADPH binding site which may be significant in the mechanism of action of this hormone.