Asymmetric reconstitution of the glucose transporter from Ehrlich ascites cell plasma membrane: role of alkali cations.
Gel chromatography of solubilized Ehrlich cell plasma membranes and preformed asolectin vesicles coupled to a freeze-thaw cycle results in the reconstitution of 3-O-methyl-D-glucose transport. The transport activity of the liposomes formed is critically dependent on the cation present during reconstitution. Liposomes formed in K+ show high levels of carrier-mediated 3-O-methyl-D-glucose uptake (495 pmol/min/mg protein) while those formed in Na+ do not (33 pmol/min/mg protein). The inactivity in Na+ is not due to a diminished incorporation of glucose transporter nor is it due to carrier molecules reconstituted with a different orientation from those in K+ liposomes. Instead, the low glucose transport level in Na+ liposomes is related to the small size of vesicles formed with Na+. A second freeze-thaw cycle in K+ causes a two- to threefold increase in the available intravesicular volume of Na+ liposomes and results in an eightfold increase in carrier-mediated 3-O-methyl-D-glucose uptake. K+ liposomes, treated in an identical manner, show only a twofold increase in uptake. The glucose transporter was identified as a protein with a molecular mass range of 44.7 to 66.8 kDa, by the D-glucose-inhibitable photoincorporation of [3H]cytochalasin B. The carrier protein is inserted in reconstituted vesicles in a nonrandom manner with at least 80% of the molecules oriented with the cytoplasmic domain accessible to the external medium. In contrast, the neutral Na+-dependent amino acid transport system appears to be randomly reconstituted.
D019325:3-O-Methylglucose; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism*; D002414:Cations, Monovalent; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D003571:Cytochalasin B / Q000378:metabolism; D005615:Freezing; D008081:Liposomes / Q000378:metabolism*; D008757:Methylglucosides / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology*; D012964:Sodium / Q000494:pharmacology*; D014357:Trypsin / Q000494:pharmacology
D000596:Amino Acids; D002414:Cations, Monovalent; D008081:Liposomes; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D019325:3-O-Methylglucose; D003571:Cytochalasin B; D012964:Sodium; D014357:Trypsin; D011188:Potassium