McCormick J I, Johnstone R M
Arch Biochem Biophys. 1986 Jul;248(1):379-89. doi: 10.1016/0003-9861(86)90434-0.
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.
对溶解的艾氏腹水癌细胞质膜和预先形成的与冻融循环偶联的大豆卵磷脂囊泡进行凝胶色谱分析，可重建3 - O - 甲基 - D - 葡萄糖转运。形成的脂质体的转运活性关键取决于重建过程中存在的阳离子。在钾离子存在下形成的脂质体显示出高水平的载体介导的3 - O - 甲基 - D - 葡萄糖摄取（495 pmol/分钟/毫克蛋白质），而在钠离子存在下形成的脂质体则不然（33 pmol/分钟/毫克蛋白质）。在钠离子存在下的无活性并非由于葡萄糖转运蛋白的掺入减少，也不是由于重建的载体分子与钾离子脂质体中的分子具有不同的取向。相反，钠离子脂质体中低葡萄糖转运水平与用钠离子形成的囊泡尺寸小有关。在钾离子存在下进行的第二次冻融循环导致钠离子脂质体的可用囊泡内体积增加两到三倍，并导致载体介导的3 - O - 甲基 - D - 葡萄糖摄取增加八倍。以相同方式处理的钾离子脂质体摄取仅增加两倍。通过[3H]细胞松弛素B的D - 葡萄糖抑制性光掺入，葡萄糖转运蛋白被鉴定为分子量范围为44.7至66.8 kDa的蛋白质。载体蛋白以非随机方式插入重建的囊泡中，至少80%的分子其胞质结构域朝向外部介质可及。相比之下，中性钠离子依赖性氨基酸转运系统似乎是随机重建的。
