Qian Y, Tiffany-Castiglioni E, Harris E D
Department of Biochemistry, Texas A&M University, College Station 77843, USA.
Am J Physiol. 1995 Oct;269(4 Pt 1):C892-8. doi: 10.1152/ajpcell.1995.269.4.C892.
C6 rat glioma cells accumulate and efflux 67Cu. Both processes showed saturation kinetics with increasing 67Cu concentration. The Michaelis constant (Km) for uptake was 0.63 +/- 0.14 microM; maximum velocity (Vmax) was 3.29 +/- 0.57 pmol Cu.mg protein-1.min-1. The Km for efflux was 0.15 +/- 0.06 microM; Vmax was 1.08 +/- 0.71 pmol Cu.mg protein-1.min-1. p-Chloromercuribenzoate (p-CMB) totally blocked 67Cu efflux but had no effect on Km or Vmax of uptake. Total 67Cu in the cell after 50 min was partitioned equally between particulate and soluble fractions. p-CMB-treated cells accumulated more 67Cu, but < 10% was bound to the particulate (membrane) fraction. Pb also increased 67Cu accumulation without affecting Km and Vmax of 67Cu uptake. These data suggest that carriers for import and export of Cu in C6 cells are distinct or operate in two different cellular environments. Efflux is a sulfhydryl-dependent process subject to inhibition by Pb. The data are consistent with a P-type ATPase in the efflux of Cu from cells and the potential for Pb to inhibit the efflux mechanism.
C6大鼠胶质瘤细胞能积累和外排67Cu。随着67Cu浓度增加，这两个过程均呈现出饱和动力学。摄取的米氏常数（Km）为0.63±0.14微摩尔；最大速度（Vmax）为3.29±0.57皮摩尔铜·毫克蛋白-1·分钟-1。外排的Km为0.15±0.06微摩尔；Vmax为1.08±0.71皮摩尔铜·毫克蛋白-1·分钟-1。对氯汞苯甲酸（p-CMB）完全阻断了67Cu外排，但对摄取的Km或Vmax没有影响。50分钟后细胞内的总67Cu在颗粒部分和可溶部分中平均分配。经p-CMB处理的细胞积累了更多的67Cu，但<10%与颗粒（膜）部分结合。铅也增加了67Cu的积累，而不影响67Cu摄取的Km和Vmax。这些数据表明，C6细胞中铜的导入和输出载体是不同的，或者在两种不同的细胞环境中起作用。外排是一个依赖巯基的过程，易受铅的抑制。这些数据与细胞中铜外排的P型ATP酶以及铅抑制外排机制的可能性一致。
