Jakubowski J, Jakob A
Department of Biochemistry, University of Basel, Switzerland.
Eur J Biochem. 1990 Oct 24;193(2):541-9. doi: 10.1111/j.1432-1033.1990.tb19370.x.
Uptake of 22Na+ by liver plasma membrane vesicles, reflecting Na+ transport by (Na+, K+)ATPase or Na+/H+ exchange was studied. Membrane vesicles were isolated from rat liver homogenates or from freshly prepared rat hepatocytes incubated in the presence of [Arg8]vasopressin or pervanadate and insulin. The ATP dependence of (Na+, K+)ATPase-mediated transport was determined from initial velocities of vanadate-sensitive uptake of 22Na+, the Na(+)-dependence of Na+/H+ exchange from initial velocities of amiloride-sensitive uptake. By studying vanadate-sensitive Na+ transport, high-affinity binding sites for ATP with an apparent Km(ATP) of 15 +/- 1 microM were observed at low concentrations of Na+ (1 mM) and K+ (1mM). At 90 mM Na+ and 60 mM K+ the apparent Km(ATP) was 103 +/- 25 microM. Vesiculation of membranes and loading of the vesicles prepared from liver homogenates in the presence of vasopressin increased the maximal velocities of vanadate-sensitive transport by 3.8-fold and 1.9-fold in the presence of low and high concentrations of Na+ and K+, respectively. The apparent Km(ATP) was shifted to 62 +/- 7 microM and 76 +/- 10 microM by vasopressin at low and high ion concentrations, respectively, indicating that the hormone reduced the influence of Na+ and K+ on ATP binding. In vesicles isolated from hepatocytes preincubated with 10 nM vasopression the hormone effect was conserved. Initial velocities of Na+ uptake (at high ion concentrations and 1 mM ATP) were increased 1.6-1.7-fold above control, after incubation of the cells with vasopressin or by affinity labelling of the cells with a photoreactive analogue of the hormone. The velocity of amiloride-sensitive Na+ transport was enhanced by incubating hepatocytes in the presence of 10 nM insulin (1.6-fold) or 0.3 mM pervanadate generated by mixing vanadate plus H2O2 (13-fold). The apparent Km(Na+) of Na+/H+ exchange was increased by pervanadate from 5.9 mM to 17.2 mM. Vesiculation and incubation of isolated membranes in the presence of pervanadate had no effect on the velocity of amiloride-sensitive Na+ transport. The results show that hormone receptor-mediated effects on (Na+, K+)ATPase and Na+/H+ exchange are conserved during the isolation of liver plasma membrane vesicles. Stable modifications of the transport systems or their membrane environment rather than ionic or metabolic responses requiring cell integrity appear to be involved in this regulation.
研究了肝质膜囊泡对22Na+的摄取，该摄取反映了(Na+,K+)ATP酶或Na+/H+交换介导的Na+转运。膜囊泡从大鼠肝脏匀浆或在[精氨酸8]加压素、过氧钒酸盐和胰岛素存在下孵育的新鲜制备的大鼠肝细胞中分离得到。(Na+,K+)ATP酶介导的转运对ATP的依赖性通过22Na+钒酸盐敏感性摄取的初始速度来确定，Na+/H+交换对Na+的依赖性通过氨氯地平敏感性摄取的初始速度来确定。通过研究钒酸盐敏感性Na+转运，在低浓度Na+(1 mM)和K+(1 mM)下观察到ATP的高亲和力结合位点，其表观Km(ATP)为15±1 microM。在90 mM Na+和60 mM K+时，表观Km(ATP)为103±25 microM。在加压素存在下，从肝脏匀浆制备的膜囊泡的囊泡化和装载分别在低浓度和高浓度的Na+和K+存在下使钒酸盐敏感性转运的最大速度增加了3.8倍和1.9倍。在低离子浓度和高离子浓度下，加压素分别使表观Km(ATP)变为62±7 microM和76±10 microM，表明该激素降低了Na+和K+对ATP结合的影响。在用10 nM加压素预孵育的肝细胞中分离得到的囊泡中，该激素的作用得以保留。在用加压素孵育细胞或用该激素的光反应类似物对细胞进行亲和标记后，Na+摄取的初始速度(在高离子浓度和1 mM ATP下)比对照增加了1.6 - 1.7倍。在10 nM胰岛素(1.6倍)或通过混合钒酸盐和H2O2产生的0.3 mM过氧钒酸盐(13倍)存在下孵育肝细胞，可增强氨氯地平敏感性Na+转运的速度。过氧钒酸盐使Na+/H+交换的表观Km(Na+)从5.9 mM增加到17.2 mM。在过氧钒酸盐存在下，分离膜的囊泡化和孵育对氨氯地平敏感性Na+转运的速度没有影响。结果表明，在肝质膜囊泡的分离过程中，激素受体介导的对(Na+,K+)ATP酶和Na+/H+交换的作用得以保留。似乎参与这种调节的是转运系统或其膜环境的稳定修饰，而不是需要细胞完整性的离子或代谢反应。
