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 microL/mg protein, whereas fraction B was composed of membrane fragments and much smaller vesicles, with an intravesicular volume of 7 microL/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 microM 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.
A method for the selection and isolation of hexose transport mutants in undifferentiated rat myoblast L6 cells is reported; 2-deoxy-D-glucose (2-DOG)-and 2-deoxy-2-fluoro-D-glucose (2FG)-resistant mutants were selected after mutagenization of L6 cells with ethyl methanesulfonate. Of these, D18 and D23 (selected with 0.1 mM 2-DOG) and F72 and F76 (selected with 0.1 mM 2FG) exhibited the lowest hexose transport activity. Uptake of 0.06 mM 2-DOG, 2FG, or 3-O-methyl-D-glucose (3-OMG) by mutants grown in fructose medium supplemented with 0.05 mM 2FG was about four- to five-fold lower than the parental L6 cells. These mutants contain normal levels of ATP and glycolytic enzyme activities. They also exhibit normal transport activities for alpha-aminoisobutyric acid and fructose. Furthermore, hexose transport was observed to be decreased in plasma membrane vesicles prepared from these mutants. Kinetic analysis of 2-DOG and 3-OMG transport in mutant F72 demonstrated that the Vmax for 2-DOG uptake was significantly reduced, whereas the Vmax for 3-OMG transport was not affected. In all cases, the affinity for these hexose analogues was unaffected. In addition mutant F72 was found to be only slightly affected by treatment with various energy inhibitors and sulfhydryl reagents. The results suggest that this mutant is defective in, or has low levels of, a plasma membrane component(s) involved in the high-affinity hexose transport system.
Treatment of glucose-grown L6 rat myoblasts with rabbit or sheep anti-(L6-rat myoblast) antibody for 35 min or glucose starvation for at least 8 h results in a 2-fold increase in the Vmax. of 2-deoxy-D-glucose (dGlc) and 3-O-methyl-D-glucose uptake. In both cases, apparent transport affinities were not affected. Furthermore, once stimulation has occurred, further increases in hexose uptake could not be produced. Assays of antibody binding to whole cells suggested that the antibody is not internalized but remains bound on the cell surface. To elucidate the site and mechanism of antibody action, plasma-membrane vesicles from L6 cells were prepared. Anti-L6 antibody was found to cause a time- and dosage-dependent stimulation of dGlc transport in these vesicles. Maximum activation was achieved after 30 min exposure. This antibody-mediated activation could be inhibited by treatment of vesicles with various proteinase inhibitors. Treatment of vesicles with trypsin was also found to activate dGlc transport to levels observed with antibody. These results are virtually identical with those obtained with whole cells and suggest that antibody-mediated activation of hexose transport results from interaction of antibody with a specific membrane component(s).
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