AJP - Endocrinology and Metabolism, Vol 249, Issue 4 398-E408, Copyright © 1985 by American Physiological Society

ARTICLES

Insulin binding and glucose transport in rat skeletal muscle sarcolemmal vesicles

G. K. Grimditch, R. J. Barnard, S. A. Kaplan and E. Sternlicht

A new method is described for isolation of sarcolemma (SL) from skeletal muscle of rats that produces vesicles of high purity and yield. There was a mean 59-fold purification (n = 22) of the SL marker enzyme K+-p-nitrophenylphosphatase. Specific activities of marker enzymes for sarcoplasmic reticulum and mitochondria were low, indicating minimal contamination. Despite the high purity and low contamination, a relatively high protein yield was achieved (0.43 +/- 0.03 mg/g wet wt, n = 25). Electron microscopy showed that the membranes were primarily vesicles. Specific 125I-insulin binding association constants derived from the high- and low-affinity portion of the Scatchard plots were 0.764 +/- 0.154 and 0.0096 +/- 0.0012 X 10(9) M-1, whereas the apparent number of receptors were 15.0 +/- 4.1 and 925 +/- 80 X 10(9) per mg of SL protein. Equilibrium exchange glucose transport studies at 37 degrees C indicated that the SL vesicles exhibited specific D-glucose transport which was responsive to in vivo insulin stimulation. We conclude that this isolation procedure, especially in light of the high purity and yield, provides a good and practical experimental model for studying insulin binding and glucose transport in skeletal muscle.

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