A NEW PROCEDURE FOR THE ISOLATION OF PLASMA-MEMBRANES, T-TUBULES, ANDINTERNAL MEMBRANES FROM SKELETAL-MUSCLE

A new subcellular fractionation procedure for the simultaneous isolati on of plasma membranes and transverse (T) tubule membranes from rat sk eletal muscle was developed. This new technique allows the isolation a nd separation of plasma membranes and T tubules in distinct subcellula r fractions, as revealed by the membrane distribution of enzymatic and immunologic markers of both cell surface compartments. The procedure also yields a novel membrane fraction that is devoid of markers of bot h surface domains but is markedly enriched with GLUT-4 glucose transpo rters, thus strongly suggesting that it represents an intracellular po ol of GLUT-4. Using this new procedure, we found that acute in vivo in sulin administration (30 min) increased GLUT-4 protein content in the plasma membrane and a T tubule fraction (by similar to 80%), whereas a smaller elevation (35%) was observed in another fraction enriched wit h T tubules. Insulin induced a concomitant reduction (similar to 40%) in GLUT-4 abundance in the intracellular fraction. These results furth er support the hypothesis that T tubules are involved in the regulatio n of glucose transport in skeletal muscle. This novel fractionation me thod will be useful in investigating the regulation of muscle GLUT-4 t ransporters in other physiological and disease states such as diabetes , where defective translocation of the transporter protein to either o ne or both cell surface domains is suspected to occur.