Hexose transport in plasma membrane vesicles of rat myoblast L6

Abstract

To determine the molecular mechanism of hexose transport in rat myoblasts, transport studies were carried out with purified plasma membrane vesicles. Rat myoblasts were homogenized and fractionated by differential and sucrose gradient centrifugation. Six different fractions were obtained. Studies with marker enzymes revealed that two fractions (A and B) were composed of only plasma membrane. These two fractions differed considerably in their physical properties. Fraction A was composed of large multilaminated vesicles, with an intravesicular volume of 50 μL/mg protein, whereas fraction B was composed of membrane fragments and much smaller vesicles, with an intravesicular volume of 7 μL/mg protein. Based on the response of the ouabain-sensitive Na+,K+-ATPase activity to sodium dodecyl sulfate and ionophore treatments, it seemed likely that fraction A was composed of a significant amount of sealed right-side-out vesicles, whereas fraction B was composed of mainly membrane sheets or leaky vesicles. The initial rate of hexose influx into the membrane vesicles was determined by the flow dialysis technique. The optimal conditions for 2-deoxyglucose (2-DG) uptake into the plasma membrane vesicles were either 50 mM phosphate buffer or 10 mM 2-(N-2-hydroxyethylpiperazin-N′-yl)ethanesulfonic acid buffer at pH 7.0. In the presence of 500 μM 2-DG, the initial rates of 2-DG influx were 295 and 49 nmol/min per milligram protein for fractions A and B, respectively. In other words, after 1 min of incubation, the intravesicular concentration of 2-DG was around 6 mM, about 10 times the extravesicular concentration. D-Glucose was taken up to a similar extent (333 nmol/min per milligram protein), whereas L-glucose only equilibrated across the plasma membrane. Analysis of the fate of 2-DG revealed that the substrate was not phosphorylated upon incubation with the vesicles. Transport activity can be abolished either by disruption of the membrane vesicles or by reduction of the electrical potential across the membrane.