Shailubhai K, Illeperuma C, Tayal M, Vijay I K
Department of Animal Sciences, University of Maryland, College Park 20742.
J Biol Chem. 1990 Aug 25;265(24):14105-8.
UDP-Glc:dolichol phosphate glucosyltransferase from lactating rat mammary gland has been partially purified by a combination of (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography on DEAE-TSK, and affinity chromatography. The partially purified enzyme exhibited several protein bands when examined by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions; among these, a 35-kDa polypeptide was quite prominent and appeared to be enriched during purification. Photoaffinity labeling of the partially purified enzyme preparation with 5-azido-[beta-32P]UDP-Glc identified a 35-kDa polypeptide. Labeling of a solubilized enzyme preparation from crude and stripped microsomes also revealed a 35-kDa band on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Photoinsertion of the probe in this polypeptide is enhanced by the presence of dolichol phosphate and Mg2+. Competition studies with UDP-Glc, UDP-glucuronic acid, other sugar nucleotides, and Glc-1-phosphate provide evidence to validate the specificity of photoaffinity labeling. These studies indicate that this 35-kDa polypeptide is involved in the synthesis of dolichol-P-Glc in rat mammary tissue. The possibility that this polypeptide may represent glucosyltransferase has been discussed.
通过硫酸铵分级分离、凝胶过滤、DEAE - TSK离子交换色谱和亲和色谱相结合的方法，对哺乳期大鼠乳腺中的UDP - 葡萄糖：磷酸多萜醇葡萄糖基转移酶进行了部分纯化。在还原条件下，用10%十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳检测时，部分纯化的酶呈现出几条蛋白带；其中，一条35 kDa的多肽非常突出，并且在纯化过程中似乎有所富集。用5 - 叠氮基 - [β - 32P]UDP - 葡萄糖对部分纯化的酶制剂进行光亲和标记，鉴定出一条35 kDa的多肽。对粗微粒体和脱脂微粒体的溶解酶制剂进行标记，在10%十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上也显示出一条35 kDa的条带。磷酸多萜醇和Mg2 + 的存在增强了探针在该多肽中的光插入。用UDP - 葡萄糖、UDP - 葡萄糖醛酸、其他糖核苷酸和葡萄糖 - 1 - 磷酸进行的竞争研究为验证光亲和标记的特异性提供了证据。这些研究表明，这条35 kDa的多肽参与大鼠乳腺组织中多萜醇 - P - 葡萄糖的合成。已经讨论了这条多肽可能代表葡萄糖基转移酶的可能性。
