Stereospecific hexose transport by membrane vesicles from mouse fibroblasts: membrane vesicles retain increased hexose transport associated with viral transformation

Abstract

Membrane vesicles isolated from nontransformed BALB/c 3T3 mouse fibroblasts (3T3) and from those cells transformed by simian virus 40 (SV3T3) displayed carrier-mediated and stereospecific uptake of hexose as measured by the difference between D-[(14)C]glucose or its analogues and L-[(3)H]glucose uptake. Stereospecific uptake appeared to be linear for 5 sec and reached a maximum at 5-10 min. Stereospecific D-[(14)C]glucose uptake, osmotically sensitive and temperature dependent, was inhibited by unlabeled D-glucose or its analogues and was stimulated by the countertransport of accumulated unlabeled D-glucose. As with whole cells, the initial rate of stereospecific uptake by SV3T3 membrane vesicles was approximately 2.5-fold greater than that by 3T3 vesicles. Efflux of preloaded D-[(14)C]glucose was also faster from SV3T3 than from 3T3 membrane vesicles. The K(m) value was 5 mM for both the 3T3 and the SV3T3 membrane vesicles, but the V(max) values were 36 and 86 nmol/mg of protein per min, respectively, suggesting an increase in the number or availability of hexose carriers in transformed cell membranes. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicles. The binding of cytochalasin B to the SV3T3 membrane vesicles was significantly greater than that to 3T3 vesicles. Thus, the membrane vesicles retained many of the features of the altered hexose transport observed in whole cells in association with viral transformation.