High channel-mediated water permeability in rabbit erythrocytes: characterization in native cells and expression in Xenopus oocytes.
High channel-mediated water permeability in rabbit erythrocytes: characterization in native cells and expression in Xenopus oocytes.
复制标题
兔红细胞中高通道介导的水渗透性：天然细胞的表征和爪蟾卵母细胞的表达。
DOI：
10.1021/bi00222a013
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Verkman,AS
中科院分区：
文献类型：
--
作者：
Tsai,ST;Zhang,RB;Verkman,AS
材料和方法细胞和幽灵制剂。从兔或人供体(A.S.Verkman)的新鲜红细胞通过静脉穿刺法获得，并用肝素抗凝。细胞洗涤两次，红细胞颗粒悬浮在140 mM氯化钠-5 mM磷酸二氢钾，pH 7.4(PBS)中，用于停流测量。通过对Steck和Kant(1974)的方法进行改进，从洗过的红细胞中制备了红细胞幽灵。细胞裂解，在5 mM磷酸二氢钠(pH 8.0)中洗三次，悬浮在PBS中，在37℃下孵育60分钟封闭。在英国威尔特郡的高科技SF-51仪器上，用停流90光散射法(510 Nm)测定渗透水的渗透性。将红细胞悬浮液(-0.25%的红细胞压积)或封闭的鬼膜(-0.5 mg蛋白质/毫升)与等体积的含蔗糖的高渗PBS混合，引起渗透水外流和光散射增加。如前面所述，根据光散射和红细胞表面积体积比的时间进程计算P(Dix等人，1985年)。在一些实验中，在停流测量之前，水转运抑制剂与红细胞孵育特定的时间。汞化合物pCMBS、pCMB和HgCl2在使用前立即制备，并在孵育期间屏蔽光线。从乙醇的原液中加入阿米洛利和菲洛维汀。卵母细胞表达研究。如前所述，从兔网织红细胞中制备mRNA(Zhang等，
Materials and MethodsCells and Ghost Preparation. Fresh erythrocytes from rabbits or a human donor (A. S. Verkman) were obtained by venipuncture and anticoagulated with heparin. Cells were washed twice, and the erythrocyte pellet was suspended in 140 mM NaCl-5 mM potassium phosphate, pH 7.4 (PBS), for stopped-flow measurements. Red cell ghosts were prepared from washed erythrocytes by a modification of the procedure of Steck and Kant (1974). Cells were lysed and washed three times in 5 mM sodium phosphate (pH 8.0), suspended in PBS, and sealed by a 60-min incubation at 37 C. Stopped-Flow Measurements. Osmotic water permeability was measured by stopped-flow 90 light scattering (510 nm) in a Hi-Tech SF-51 apparatus (Wiltshire, England). A sus-pension of erythrocytes (—0.25% hematocrit) or sealed ghost membranes (^—0.5 mg of protein/mL) was mixed with an equal volume of hyperosmotic PBS containing sucrose to cause osmotic water efflux and increased light scattering. P (was calculated from the time course of light scattering and erythrocyte surface-to-volume ratio as described previously (Dix et al., 1985). In some experiments water transport inhibitors were incubated with erythrocytes for specified times prior to stopped-flow measurements. The mercurials pCMBS, pCMB, and HgCl2 were prepared immediately before use and shielded from light duringincubations. Amiloride and phlo-retin were added from stock solutions in ethanol. Oocyte Expression Studies. mRNA was prepared from rabbit reticulocytes as described previously (Zhang et al.,