ISSN:
1432-1424
Keywords:
Insulin
;
Immuno-electron microscopy
;
Caveolae
;
Na+/K+-ATPase
;
Glucose transporters
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
Abstract 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 α1 and α2, 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 α2 isoform; GLUT4 was the dominating glucose transporter isoform. The total number of Na+/K+-ATPase molecules labeled in the plasma membrane was 3.5×105 per cell, independent of insulin stimulation. Concomitantly, insulin increased GLUT4 labeling sevenfold to a value of 3.5×105 per cell.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00241490
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