Kandror K, Pilch P F
Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118.
J Biol Chem. 1994 Jan 7;269(1):138-42.
It has previously been shown that in fat cells, the intracellular reservoir of the glucose transporter isoform GLUT4 consists of a membrane vesicle population highly enriched in this transporter (approximately 15% of the protein content) that has a relatively simple protein pattern as revealed by SDS-polyacrylamide gel electrophoresis and silver staining (Zorzano, A., Wilkinson, W., Kotliar, N., Thoidis, G., Wadzinski, B. E., Ruoho, A. E., and Pilch, P. F. (1989) J. Biol. Chem. 264, 12358-12363). Upon exposure of adipocytes to insulin, the cell-surface (plasma membrane) content of GLUT4 is dramatically enhanced, and this transporter appears to continually cycle from intracellular vesicles to the plasma membrane. To identify other proteins that may recycle in a similar fashion to GLUT4 and that may participate in the insulin-dependent alteration in the cellular location of GLUT4 and other membrane proteins, we performed immunoadsorption of GLUT4-enriched vesicles together with biotinylation of membranes using a cell-impermeant analog of biotin. We find that immunoadsorbed GLUT4-containing vesicles can be fractionated into Triton X-100-soluble and -insoluble fractions. The first includes three major glycoprotein components with molecular masses of 110, 160, and 230 kDa and a few minor polypeptides with lower molecular masses. A Triton X-100-resistant fraction consists of GLUT4 and an approximately 25-kDa protein. All three major Triton-soluble proteins (110, 160, and 230 kDa) isolated from the immunoimmobilized vesicles on wheat germ agglutinin-agarose are strongly biotinylated in an insulin-dependent fashion, i.e. they cycle to and from the cell surface in an apparently identical manner to GLUT4. Sequence analysis of two tryptic fragments from p160 reveals that it is a novel protein containing sequence with no homology to known proteins.
先前的研究表明，在脂肪细胞中，葡萄糖转运异构体GLUT4的细胞内储存库由高度富集该转运蛋白的膜泡群体组成（约占蛋白质含量的15%），通过SDS-聚丙烯酰胺凝胶电泳和银染显示，其蛋白质模式相对简单（佐尔扎诺，A.，威尔金森，W.，科蒂亚尔，N.，索伊迪斯，G.，瓦津斯基，B.E.，鲁霍，A.E.，和皮尔希，P.F.（1989年）《生物化学杂志》264卷，12358 - 12363页）。脂肪细胞暴露于胰岛素后，GLUT4在细胞表面（质膜）的含量显著增加，并且该转运蛋白似乎不断地从细胞内囊泡循环至质膜。为了鉴定可能以与GLUT4类似的方式循环且可能参与GLUT4和其他膜蛋白细胞定位的胰岛素依赖性改变的其他蛋白质，我们使用生物素的细胞不渗透类似物对富含GLUT4的囊泡进行免疫吸附并对膜进行生物素化。我们发现，免疫吸附的含GLUT4囊泡可分为Triton X - 100可溶性和不溶性部分。第一部分包括三种主要糖蛋白成分，分子量分别为110、160和230 kDa，以及一些分子量较低的次要多肽。Triton X - 100抗性部分由GLUT4和一种约25 kDa的蛋白质组成。从麦胚凝集素 - 琼脂糖上免疫固定的囊泡中分离出的所有三种主要Triton可溶性蛋白（110、160和230 kDa）均以胰岛素依赖性方式被强烈生物素化，即它们以与GLUT4明显相同的方式在细胞表面循环进出。对p160的两个胰蛋白酶片段的序列分析表明，它是一种新型蛋白质，其序列与已知蛋白质无同源性。
