Danbolt N C, Pines G, Kanner B I
Department of Biochemistry, Hadassah Medical School, Hebrew University, Jerusalem, Israel.
Biochemistry. 1990 Jul 17;29(28):6734-40. doi: 10.1021/bi00480a025.
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 include 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.
通过将转运功能重建作为一种检测方法，假定失活的和有活性的转运体共层析，大鼠脑内的钠钾偶联L-谷氨酸转运体已被纯化至接近均一。纯化步骤包括用3-[(3-氯氨丙基)二甲基铵]-1-丙烷磺酸盐（CHAPS）溶解膜蛋白后的凝集素层析、羟磷灰石分级分离以及离子交换层析。比活性提高了30倍。由于在纯化过程中发生了3至5倍的失活，实际纯化倍数更高。重建效率约为20%。纯转运体的特性得到了充分保留。这些特性包括离子依赖性、电生性、亲和力、底物特异性和立体特异性。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示一条主要条带，表观分子量约为80 kDa，还有几条次要条带。在整个分级分离过程中，将多肽组成与L-谷氨酸转运活性进行比较，结果表明只有80 kDa的条带与活性相关。具有相同表观分子量的GABA转运体（Radian等人，1986年）在最后两步纯化过程中与其分离。免疫印迹实验表明，抗GABA转运体的抗体仅与表现出GABA转运活性的组分发生反应，而不与含有谷氨酸转运体的组分发生反应。我们得出结论，80 kDa的条带代表功能性的钠钾偶联L-谷氨酸转运体。
