Identification And Characterization Of The Hexose Transporters In Rat L6 Myoblasts
Transport kinetic studies have demonstrated that two hexose transport systems are present in undifferentiated rat L6 myoblasts. These hexose transport systems were identified and characterized in this study using a number of different approaches. Cytochalasin B (CB), a potent inhibitor of hexose transport, was first used as a probe for these systems. CB binding studies with whole cells and membrane preparations revealed the presence of two distinct CB binding sites, CB{dollar}sb{lcub}rm H{rcub}{dollar} and CB{dollar}sb{lcub}rm L{rcub}{dollar}. They differ not only in their binding affinities, but also in their response to various biochemical, physiological and genetic manipulations. Based on the correlation between transport activity and the level of CB binding sites, CB{dollar}sb{lcub}rm L{rcub}{dollar} and CB{dollar}sb{lcub}rm H{rcub}{dollar} are thought to be associated with the high and low affinity hexose transport system (HAHT and LAHT), respectively. These CB binding components were further identified and characterized by CB photoaffinity labelling. Both mutant and glucose analogue inhibition studies suggested that the CB photolabelled components (40-60 kDa) were associated with both HAHT and LAHT. Proteolytic digestion studies indicated that the external surface of the hexose transporter was resistant to trypsin cleavage, and that the CB binding sites might be located on the cytoplasmic side of the hexose transporter. Immunoblotting studies of membrane preparations with specific antisera suggested that the 63 and 60 kDa polypeptides might be associated with HAHT and LAHT, respectively. Hexose transport mutants have proven to be indispensable in the identification and characterization of CB binding sites, CB photolabelled components and components recognized by specific antisera.;The effects of growth conditions and myogenic differentiation on hexose transport activity were also investigated. These studies revealed that the hexose transport systems were very sensitive to the growth conditions, and that myogenesis might result in the degradation or modification of HAHT.
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