D'Souza C, Clarke J T, Cook H W, Spence M W
Biochim Biophys Acta. 1983 Mar 23;729(1):1-8. doi: 10.1016/0005-2736(83)90448-0.
Murine neuroblastoma cells (strain N1E-115) were incubated with 2-[fatty acyl-14 C]acylphosphatidylcholine/sphingomyelin or phosphatidylcholine/[choline-3H]sphingomyelin liposomes (1:1, mol/mol; 1.2 mumol total lipid/mg cell protein) in the presence of partially purified rat liver phospholipid transfer protein (2.5 mg/ml), cytochalasin B (50 microM) and 2-deoxyglucose (50 mM) for 10 min. Washed cells were chased for periods of up to 45 min at 37 degrees C with medium containing transfer protein and unlabeled liposomes. Total transfer protein-dependent incorporation of [14C]phosphatidylcholine ([14C] PC) and [3H]sphingomyelin was 136.7 +/- 26.5(n = 5) and 23.7 +/- 5.4(n = 6) nmol/mg protein per 10 min incubation, respectively, (mean +/- S.D.). Incorporation of [14C]PC into the mitochondrial membrane fraction was 128-fold greater (nmol/mg protein) than incorporation of [3H]sphingomyelin. In contrast, incorporation of [3H]sphingomyelin into a fraction enriched in plasma membrane and into microsomes was 1.4- and 2.6-fold greater, respectively, than incorporation of [14C]PC. During the chase periods, the specific activities of total cellular phospholipids decreased as intact [14C]PC and [3H]sphingomyelin accumulated in the culture medium. In the case of cells labeled with [14C]PC, the effect was due primarily to a decrease in the amount of labeled phospholipid in the mitochondrial fraction; in the case of cells labeled with [3H]sphingomyelin, the decrease in activity was greatest in microsomal and plasma membrane phospholipids. The rate and extent of non-endocytotic incorporation of exogenous phosphatidylcholine into the cell membrane of cultured neuroblastoma cells, and its subsequent subcellular disposition, is different from that of exogenous sphingomyelin. Whereas PC is evidently incorporated into and turned over most rapidly in fraction enriched in mitochondrial membranes, sphingomyelin appears to be preferentially incorporated into microsomal and plasma membrane.
将鼠神经母细胞瘤细胞（N1E - 115株）与2 - [脂肪酰基 - 14C]酰基磷脂酰胆碱/鞘磷脂或磷脂酰胆碱/[胆碱 - 3H]鞘磷脂脂质体（1:1，摩尔/摩尔；1.2微摩尔总脂质/毫克细胞蛋白）在部分纯化的大鼠肝磷脂转移蛋白（2.5毫克/毫升）、细胞松弛素B（50微摩尔）和2 - 脱氧葡萄糖（50毫摩尔）存在的情况下孵育10分钟。用含有转移蛋白和未标记脂质体的培养基在37℃下对洗涤后的细胞进行长达45分钟的追踪。每10分钟孵育，[14C]磷脂酰胆碱（[14C]PC）和[3H]鞘磷脂依赖于转移蛋白的总掺入量分别为136.7±26.5（n = 5）和23.7±5.4（n = 6）纳摩尔/毫克蛋白，（平均值±标准差）。[14C]PC掺入线粒体膜部分的量（纳摩尔/毫克蛋白）比[3H]鞘磷脂高128倍。相反，[3H]鞘磷脂掺入富含质膜的部分和微粒体中的量分别比[14C]PC高1.4倍和2.6倍。在追踪期间，随着完整的[14C]PC和[3H]鞘磷脂在培养基中积累，总细胞磷脂的比活性下降。在用[14C]PC标记的细胞中，这种影响主要是由于线粒体部分中标记磷脂的量减少；在用[3H]鞘磷脂标记的细胞中，微粒体和质膜磷脂的活性下降最大。外源性磷脂酰胆碱非内吞性掺入培养的神经母细胞瘤细胞膜的速率和程度及其随后的亚细胞分布与外源性鞘磷脂不同。虽然PC显然最迅速地掺入富含线粒体膜的部分并在其中周转，但鞘磷脂似乎优先掺入微粒体和质膜。
