Voldstedlund M, Tranum-Jensen J, Vinten J
Department of Medical Physiology, Panum Institute, University of Copenhagen, Denmark.
J Membr Biol. 1993 Oct;136(1):63-73. doi: 10.1007/BF00241490.
We have quantitated and studied the topology of isoforms of the Na+/K(+)-ATPase and of the glucose transporter in rat adipocyte plasma membranes. Adipocytes were incubated with or without insulin for 15 min. Sheets of native plasma membrane, with the cytoplasmic face exposed, were prepared by adsorption to EM grids. Grids were incubated in parallel with monoclonal antibodies against the Na+/K(+)-ATPase isoforms alpha 1 and alpha 2, and the glucose transporter isoforms GLUT1 and GLUT4, followed by immunogold labeling, negative staining and quantitation by counting of the gold particles in electron micrographs. In addition, the distribution of glucose transporters and Na+/K(+)-ATPase isoforms in subcellular membrane fractions prepared by an established fractionation procedure was monitored by Western blotting. We found that the Na+/K(+)-ATPases and the glucose transporters were confined to the planar part of the plasma membrane, without association to caveolar invaginations. The vast majority of the Na+/K(+)-ATPase molecules in the adipocyte plasma membrane were of the alpha 2 isoform; GLUT4 was the dominating glucose transporter isoform. The total number of Na+/K(+)-ATPase molecules labeled in the plasma membrane was 3.5 x 10(5) per cell, independent of insulin stimulation. Concomitantly, insulin increased GLUT4 labeling sevenfold to a value of 3.5 x 10(5) per cell.
我们已对大鼠脂肪细胞质膜中钠钾ATP酶异构体和葡萄糖转运蛋白的拓扑结构进行了定量研究。脂肪细胞在有或无胰岛素的情况下孵育15分钟。通过吸附到电子显微镜网格上制备出细胞质面外露的天然质膜片。网格与针对钠钾ATP酶异构体α1和α2以及葡萄糖转运蛋白异构体GLUT1和GLUT4的单克隆抗体平行孵育，随后进行免疫金标记、负染色，并通过对电子显微镜照片中的金颗粒计数进行定量分析。此外，通过蛋白质印迹法监测了通过既定分级分离程序制备的亚细胞膜组分中葡萄糖转运蛋白和钠钾ATP酶异构体的分布。我们发现，钠钾ATP酶和葡萄糖转运蛋白局限于质膜的平面部分，与小窝内陷无关。脂肪细胞质膜中绝大多数钠钾ATP酶分子为α2异构体；GLUT4是主要的葡萄糖转运蛋白异构体。质膜中标记的钠钾ATP酶分子总数为每个细胞3.5×10⁵个，与胰岛素刺激无关。与此同时，胰岛素使GLUT4标记增加了七倍，达到每个细胞3.5×10⁵个的水平。
