Zala C A, Perdue J F
Biochim Biophys Acta. 1980 Jul 16;600(1):157-72. doi: 10.1016/0005-2736(80)90421-6.
Mixed membrane vesicles prepared from cultured chick embryo fibroblasts possess a stereospecific D-glucose transport system, the properties of which are identical to those of the system in intact cells. Uptake of D-glucose proceeds without chemical alteration. The rate of stereospecific uptake of D-glucose into the mixed vesicles is 70% greater than that of the homogenate and uptake is directly proportional to membrane protein concentration. Stereospecific D-glucose uptake appears linear for 0.3 min, reaches a maximum at 2--5 min, and declines to zero by 5 h as L-glucose enters the vesicles. Uptake is osmotically sensitive and inhibited by cytochalasin B (Ki = 0.13 microM) and the structural analogues of D-glucose : D-mannose, 2-deoxy-D-glucose, 3-O-methyl-D-glucose, D-galactose and maltose, but not by sucrose of L-glucose. Uphill counterflow can be demonstrated and the apparent activation energy displays a transition from 47.7 kcal/mol below 11 degrees C to 18.1 kcal/mol above 11 degrees C. Stereospecific uptake rates of mixed vesicles prepared from Rous sarcoma virus-transformed cells are increased 30% over control values, and are increased 66% in vesicles derived from cells incubated for 24 h in glucose-free medium. Plasma membrane vesicles prepared from these cells by a dextran cushion centrifugation procedure display a 9-fold increase in the specific activity of stereospecific D-glucose uptake relative to the homogenate. Extraction of these membranes with dimethylmaleic anhydride (5 mg/mg protein) results in substantial or complete removal of major polypeptides of molecular weight 40 000, 55 000, 75 000, 78 000 and 200 000 with no loss in total uptake activity. Following extraction, major polypeptides of molecular weight 28 000, 33 000 and 68 000 remain in the membrane residue.
从培养的鸡胚成纤维细胞制备的混合膜泡具有立体特异性D - 葡萄糖转运系统，其性质与完整细胞中的系统相同。D - 葡萄糖的摄取过程中没有化学变化。D - 葡萄糖立体特异性摄取到混合膜泡中的速率比匀浆高70%，摄取与膜蛋白浓度成正比。立体特异性D - 葡萄糖摄取在0.3分钟内呈线性，在2 - 5分钟达到最大值，随着L - 葡萄糖进入膜泡，在5小时时降至零。摄取对渗透压敏感，并受到细胞松弛素B（Ki = 0.13 microM）以及D - 葡萄糖的结构类似物：D - 甘露糖、2 - 脱氧 - D - 葡萄糖、3 - O - 甲基 - D - 葡萄糖、D - 半乳糖和麦芽糖的抑制，但不受蔗糖或L - 葡萄糖的抑制。可以证明存在上坡逆流，表观活化能在11℃以下从47.7千卡/摩尔转变为11℃以上的18.1千卡/摩尔。从劳氏肉瘤病毒转化细胞制备的混合膜泡的立体特异性摄取速率比对照值增加30%，在无葡萄糖培养基中孵育24小时的细胞衍生的膜泡中增加66%。通过葡聚糖垫层离心程序从这些细胞制备的质膜膜泡相对于匀浆，立体特异性D - 葡萄糖摄取的比活性增加了9倍。用二甲基马来酸酐（5毫克/毫克蛋白质）提取这些膜会导致分子量为40000、55000、75000、78000和200000的主要多肽大量或完全去除，而总摄取活性没有损失。提取后，分子量为28000、33000和68000的主要多肽保留在膜残渣中。
