PURIFICATION AND RECONSTITUTION OF THE SODIUM-COUPLED AND POTASSIUM-COUPLED GLUTAMATE TRANSPORT GLYCOPROTEIN FROM RAT-BRAIN
PURIFICATION AND RECONSTITUTION OF THE SODIUM-COUPLED AND POTASSIUM-COUPLED GLUTAMATE TRANSPORT GLYCOPROTEIN FROM RAT-BRAIN
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DOI：
10.1021/bi00480a025
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发表时间：
1990-07-17
期刊：
影响因子：
2.9
通讯作者：
KANNER, BI
中科院分区：
文献类型：
--
作者：
DANBOLT, NC;PINES, G;KANNER, BI
假设失活和活性转运体共色谱，通过转运重构将大鼠脑中钠钾偶联的l -谷氨酸转运体纯化为接近均匀的转运体。纯化步骤包括用3-[（3-氯胺丙基）二甲酰胺]-1-丙磺酸（CHAPS）溶解膜蛋白的凝集素层析、羟基磷灰石分馏和离子交换层析。比活性提高了30倍。由于纯化过程中发生了3-5倍失活，因此实际纯化率更高。重构效率约为20%。纯转运体的性质被完全保留。它们包括离子依赖性、电原性、亲和性、底物特异性和立体特异性。十二烷基硫酸钠-聚丙烯酰胺电泳显示一个主带，表观分子质量约为80 kDa，还有几个小带。在整个分离过程中，多肽组成与l -谷氨酸转运活性的比较表明，只有80 kda波段与活性相关。GABA转运体具有相同的表观分子质量（Radian et al., 1986），在最后两个纯化步骤中与之分离。免疫印迹实验显示，抗GABA转运体的抗体仅与具有GABA转运活性的组分反应，而不与含有谷氨酸转运体的组分反应。我们得出结论，80 kda波段代表功能性钠和钾偶联的l -谷氨酸转运蛋白。
The sodium- and potassium-coupled L-glutamate transporter from rat brain has been purified to near homogeneity by reconstitution of transport as an assay, assuming that inactivated and active transporters cochromatograph. The purification steps involve lectin chromatography of the membrane proteins solubilized with 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), fractionation on hydroxylapatite, and ion-exchange chromatography. The specific activity is increased 30-fold. The actual purification is higher since 3-5-fold inactivation occurs during the purification. The efficiency of reconstitution was about 20%. The properties of the pure transporter are fully preserved. They including ion dependence, electrogenicity, affinity, substrate specificity, and stereospecificity. Sodium dodecyl sulfate-polyacrylamide electrophoresis revealed one main band with an apparent molecular mass of around 80 kDa and a few minor bands. Comparison of polypeptide composition with L-glutamate transport activity throughout the fractionation procedure reveals that only the 80-kDa band can be correlated with activity. The GABA transporter, which has the same apparent molecular mass (Radian et al., 1986), is separated from it during the last two purification steps. Immunoblot experiments reveal that the antibodies against the GABA transporter only reacted with fractions exhibiting GABA transport activity and not with those containing the glutamate transporter. We conclude that the 80-kDa band represents the functional sodium- and potassium-coupled L-glutamate transporter.