QUANTITATION OF NA+ K+-ATPASE AND GLUCOSE-TRANSPORTER ISOFORMS IN RATADIPOCYTE PLASMA-MEMBRANE BY IMMUNOGOLD LABELING/

We have quantitated and studied the topology of isoforms of the Na+/K-ATPase and of the glucose transporter in rat adipocyte plasma membran es. Adipocytes were incubated with or without insulin for 15 min. Shee ts of native plasma membrane, with the cytoplasmic face exposed, were prepared by adsorption to EM grids. Grids were incubated in parallel w ith monoclonal antibodies against the K+-ATPase isoforms alpha1 and al pha2, and the glucose transporter isoforms GLUT1 and GLUT4, followed b y immunogold labeling, negative staining and quantitation by counting of the gold particles in electron micrographs. In addition, the distri bution of glucose transporters and Na+/K+-ATPase isoforms in subcellul ar membrane fractions prepared by an established fractionation procedu re 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 va st majority of the Na+/K+-ATPase molecules in the adipocyte plasma mem brane were of the alpha2 isoform; GLUT4 was the dominating glucose tra nsporter isoform. The total number of Na+/K+-ATPase molecules labeled in the plasma membrane was 3.5 x 10(5) per cell, independent of insuli n stimulation. Concomitantly, insulin increased GLUT4 labeling sevenfo ld to a value of 3.5 x 10(5) per cell.