Chavan A J, Ensor C M, Wu P, Haley B E, Tai H H
Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University of Kentucky, Lexington 40536-0082.
J Biol Chem. 1993 Aug 5;268(22):16437-42.
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.
许多前列腺素在C-15位的氧化会导致15-酮代谢物的形成，这些代谢物的生物活性降低。该反应由NAD(+)依赖性15-羟基前列腺素脱氢酶催化。使用NAD+的光亲和类似物[α-32P]烟酰胺-2-叠氮腺嘌呤二核苷酸，我们在15-羟基前列腺素脱氢酶的NAD+结合位点的腺嘌呤环结合结构域中鉴定出一种肽。通过饱和和保护实验证明了光标记的特异性。在约45-50 microM处观察到光标记的饱和，表观Kd为8-10 microM。当在10 microM探针存在下对蛋白质进行光解时，约90%的光标记可被200 microM NAD+保护。用金黄色葡萄球菌V8或胰凝乳蛋白酶消化光标记的蛋白质，然后通过硼酸酯亲和色谱或Fe+3螯合色谱，随后进行反相HPLC纯化光标记的肽。光标记的肽区域被鉴定为Val32-Glu40。
