Hamilton R T, Nilsen-Hamilton M
J Biol Chem. 1978 Nov 25;253(22):8247-56.
Membrane vesicles were prepared from mouse fibroblasts transformed by SV40 virus (SV3T3). Following disruption of the cells by nitrogen cavitation, the membrane vesicles were obtained by differential centrifugation. As measured by enzyme markers, they consist mainly of membrane from the plasma membrane and smooth and rough endoplasmic reticulum. The vesicles transport Pi by two separate, mediated systems: one is independent of Na+, and the other is secondary active transport driven by a Na+ gradient. Electrical and chemical energy can be provided by a Na+ gradient to drive the concentrative uptake of Pi by the vesicles, one or both forces being used to energize transport. Evidence is provided that both the electrical and chemical potentials produced by the asymmetric distribution of Na+ across the membrane of SV3T3 membrane vesicles are utilized to concentrate phosphate in the vesicles. Phosphate transport by the vesicles cannot be accounted for by a small contamination of this fraction with mitochondria (1 to 4%). The Pi transport properties of the membrane vesicles differ from those of the fraction enriched in mitochondria in the following respects: their kinetic properties, and their responses to a Na+ gradient, N-ethylmaleimide, mersalyl, and succinate/acetate. However, the membrane vesicles share some properties of Pi transport with mitochondria. Cyanide, azide, oligomycin, 2,4-dinitrophenol, and carbonyl cyanide m-cholophenylhydrazone, inhibitors of Pi transport by mitochondria, also inhibit membrane vesicle, Pi transport. The vesicles retain all the features of Pi transport by SV3T3 cells that have been examined. They provide a simplified system for a determination of the details of the mechanism of Pi transport under conditions where transport is dissociated from intracellular reactions and in the presence of a defined electrochemical driving force.
膜泡是从经SV40病毒转化的小鼠成纤维细胞（SV3T3）制备而来。通过氮空化作用破坏细胞后，经差速离心获得膜泡。用酶标记物测定，它们主要由质膜、光滑内质网和粗糙内质网的膜组成。这些膜泡通过两个独立的介导系统转运无机磷酸盐（Pi）：一个不依赖于钠离子（Na+），另一个是由Na+梯度驱动的继发性主动转运。Na+梯度可提供电能和化学能，以驱动膜泡对Pi的浓缩摄取，其中一种或两种力用于为转运供能。有证据表明，SV3T3膜泡膜上Na+的不对称分布所产生的电势和化学势都被用于在膜泡中浓缩磷酸盐。膜泡对Pi的转运不能用该组分中少量的线粒体污染（1%至4%）来解释。膜泡的Pi转运特性在以下方面与富含线粒体的组分不同：它们的动力学特性，以及它们对Na+梯度、N - 乙基马来酰亚胺、汞撒利和琥珀酸盐/醋酸盐的反应。然而，膜泡在Pi转运方面与线粒体有一些共同特性。氰化物、叠氮化物、寡霉素、2,4 - 二硝基苯酚和羰基氰化物间氯苯腙，这些线粒体Pi转运的抑制剂，也抑制膜泡的Pi转运。这些膜泡保留了已检测的SV3T3细胞Pi转运的所有特征。它们提供了一个简化的系统，用于在转运与细胞内反应分离且存在明确的电化学驱动力的条件下，确定Pi转运机制的细节。
