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AJP - Cell Physiology, Vol 265, Issue 6 C1716-C1722, Copyright © 1993 by American Physiological Society
ARTICLES |
A. Marette, J. Krischer, L. Lavoie, C. Ackerley, J. L. Carpentier and A. Klip
Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.
The cellular localization of the alpha 2-subunit of the Na(+)-K(+)-ATPase was defined by immunoelectron microscopy, and the effect of insulin on the amount of alpha 2-immunoreactive subunits on the cell surface was quantitated. Two protocols were used for tissue fixation and immunolocalization. Protocol 1 was characterized by fixation with 2% paraformaldehyde, use of a monoclonal antibody, and detection with 3-nm-diameter gold-labeled Fab fragments or 10-nm gold-labeled immunoglobulin G. Protocol 2 was characterized by fixation with 4% paraformaldehyde plus 0.1% glutaraldehyde, use of a polyclonal antibody, and detection with 10-nm gold-labeled protein A. In control muscle, the alpha 2-subunit of the Na(+)-K(+)-ATPase was present at the plasma membrane and in intracellular tubular and vesicular structures located in subsarcolemmal and triadic regions. Acute insulin stimulation increased the number of immunolabeled alpha 2-subunits in the plasma membrane after both fixation protocols. The gain in the plasma membrane ranged from 1.5- to 3.7-fold and was significant at the level of P < 0.005. These results provide morphological quantitative evidence that the alpha 2-subunit of the Na(+)-K(+)-ATPase is present both at the plasma membrane and intracellularly in mammalian skeletal muscle and that insulin acutely increases its abundance in the muscle surface.
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