Solubilization and functional reconstitution of the human placental taurine transporter.
Solubilization and functional reconstitution of the human placental taurine transporter.
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人胎盘牛磺酸转运蛋白的溶解和功能重建。
DOI：
10.1016/0005-2736(93)90296-c
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Ganapathy,V
中科院分区：
文献类型：
--
作者：
Ramamoorthy,S;Kulanthaivel,P;Leibach,FH;Mahesh,VB;Ganapathy,V
从纯化的人胎盘刷状缘膜的牛磺酸转运蛋白溶解和重组成蛋白脂质体的功能形式。在4 M尿素存在下用2.5%胆酸盐进行增溶。用6%聚（乙二醇）沉淀增溶物中的蛋白质，并将沉淀的蛋白质以10：1的asolectin/蛋白质比例重构成脂蛋白体。在这些实验条件下，牛磺酸转运活动的蛋白脂质体是最大的。然而，蛋白质的SDS-PAGE分析显示，脂蛋白体仍然含有大部分最初存在于刷状缘膜中的蛋白质。重构蛋白脂质体中牛磺酸的摄取必然依赖于Na+和Cl−的存在。用其他一价阳离子如K+和Li+取代Na+会显著降低牛磺酸转运活性。类似地，用其他一价阴离子如SCN-、F-、I和NO3-取代Cl，只能支持最多为对照活性的30%的转运活性。在Cl−存在下，摄取速率与Na+浓度呈S形相关，导致Na+/牛磺酸偶联比为2：1。Na+的表观解离常数约为195 mM。在Na+存在的情况下，摄取速率与Cl−浓度呈双曲线关系，表明Cl−/牛磺酸偶联比为1：1。Cl−的表观解离常数约为205 mM。NaCl依赖的牛磺酸摄取受到内负膜电位的刺激，表明摄取过程是产电的。摄取系统对β-氨基酸具有特异性。该体系对牛磺酸有较高的亲和力，表观解离常数为2.7 ± 0.1μM。可以得出结论，牛磺酸转运蛋白可以从胎盘刷状缘膜和重组的催化活性从蛋白脂质体，其特性没有显着变化。
The taurine transporter from purified human placental brush-border membranes was solubilized and reconstituted into proteoliposomes in a functional form. Solubilization was done with 2.5% cholate in the presence of 4 M urea. The proteins in the solubilizate were precipitated with 6% poly(ethylene glycol) and the precipitated proteins were reconstituted into proteoliposomes with an asolectin/protein ratio of 10:1. Under these experimental conditions, the taurine transport activity in the proteoliposomes was maximal. SDS-PAGE analysis of proteins, however, revealed that the proteoliposomes still contained a majority of the proteins originally present in the brush-border membranes. Uptake of taurine in the reconstituted proteoliposomes was obligatorily dependent on the presence of Na+as well as Cl−. Substitution of Na+with other monovalent cations such as K+and Li+reduced the taurine transport activity drastically. Similarly, substitution of Cl with other monovalent anions such as SCN−, F−, I and NO3−could support the transport activity only to a maximum of 30% of the control activity. In the presence of Cl−, the uptake rate was sigmoidally related to Na+concentration, resulting in a Na+/taurine coupling ratio of 2:1. The apparent dissociation constant for Na+was about 195 mM. In the presence of Na+, the uptake rate was hyperbolically related to Cl−concentration, indicating a Cl−/taurine coupling ratio of 1:1. The apparent dissociation constant for Cl−was about 205 mM. The NaCl-dependent taurine uptake was stimulated by an inside-negative membrane potential, showing that the uptake process was electrogenic. The uptake system was specific for β-amino acids. The affinity of the system for taurine was high with an apparent dissociation constant of 2.7 ± 0.1μM. It is concluded that the taurine transporter can be dislodged from the placental brush-border membranes and reconstituted in a catalytically active from in proteoliposomes with no significant change in its characteristics.