Trad C H, Chavan A J, Clemens J, Haley B E
Physics Department, American University of Beirut, Lebanon.
Arch Biochem Biophys. 1993 Jul;304(1):58-64. doi: 10.1006/abbi.1993.1321.
Photoaffinity labeling of ovine prolactin with the NAD+ photoaffinity analog [alpha-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 microM probe with an apparent Kd of approximately 25 microM. Protection of photoinsertion was observed with NAD+ and NADH. The photoinsertion was decreased by 75% and greater than 95%, respectively, upon addition of 200 microM 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.
利用NAD⁺光亲和类似物[α-³²P]烟酰胺-2-叠氮腺嘌呤二核苷酸对绵羊催乳素进行光亲和标记，以鉴定NADH/NADPH结合位点。通过在生理相关浓度下的饱和及标记保护实验，证明了核苷酸相互作用的特异性。在约100μM探针处观察到光插入饱和，表观解离常数约为25μM。NAD⁺和NADH可观察到对光插入的保护作用。加入200μM上述化合物后，光插入分别减少了75%和超过95%。NADP⁺和NADPH获得的保护作用分别处于相同水平。通过胰蛋白酶和糜蛋白酶消化光标记的催乳素，并通过硼酸亲和色谱和固定化Fe³⁺亲和色谱纯化光标记的肽段，鉴定了NADH/NADPH结合位点的腺嘌呤环结合结构域。该肽段被鉴定为Ala²²-Tyr²⁸。这些研究表明，催乳素含有一个NADH/NADPH结合位点，这可能在该激素的作用机制中具有重要意义。
