Baldwin J M, Gorga J C, Lienhard G E
J Biol Chem. 1981 Apr 25;256(8):3685-9.
The transport function of the purified monosaccharide transporter from human erythrocytes has been investigated. By a cycle of cholate solubilization and removal, the purified transporter was incorporated into phospholipid vesicles of about 300 A diameter, at a density of about one per vesicle. This distribution permitted an all-or-none assay for transport activity, in which the fraction of the intravesicular volume that rapidly equilibrated with D-glucose in the medium yielded an estimate of the moles of protein functional in transport. It was found that almost every transporter molecule capable of binding cytochalasin B was also capable of transport. The rate of transporter-catalyzed exchange of D-glucose between the medium and the vesicles at equilibrium was also measured. In this assay, the transport activity of the purified protein, expressed per mol of cytochalasin B binding of site, was about 5% of that of the transporter in the intact erythrocyte under similar conditions. These results show that the reduced transport activity of the purified transporter found in various rate assays is due to most of the molecules functioning at a lower rate, rather than a few molecules functioning at the in vivo rate.
对从人红细胞中纯化得到的单糖转运蛋白的转运功能进行了研究。通过胆酸盐增溶和去除的循环过程，将纯化的转运蛋白整合到直径约300埃的磷脂囊泡中，密度约为每个囊泡一个。这种分布使得能够对转运活性进行全或无测定，其中囊泡内体积中与培养基中的D-葡萄糖快速平衡的部分给出了参与转运的蛋白质摩尔数的估计值。发现几乎每个能够结合细胞松弛素B的转运蛋白分子也都具有转运能力。还测量了在平衡状态下转运蛋白催化的培养基与囊泡之间D-葡萄糖交换的速率。在该测定中，在类似条件下，以每摩尔细胞松弛素B结合位点表示的纯化蛋白的转运活性约为完整红细胞中转运蛋白活性的5%。这些结果表明，在各种速率测定中发现的纯化转运蛋白转运活性降低是由于大多数分子以较低速率发挥作用，而不是少数分子以体内速率发挥作用。
