Gordon A M, Kanner B I
Department of Biochemistry, Hadassah Medical School, Hebrew University, Jerusalem, Israel.
Biochim Biophys Acta. 1988 Sep 15;944(1):90-6. doi: 10.1016/0005-2736(88)90320-3.
The sodium- and potassium-coupled L-glutamate transporter from rat brain has been solubilized with cholate and 10-20-fold purified using Wheat Germ Agglutinin-Sepharose 4B. Transport activity--as determined upon reconstitution of the fraction into liposomes--was retained on the column and eluted by N-acetylglucosamine. When the glycoprotein fraction was depleted of the N-acetylglucosamine and applied to a second round of lectin-chromatography, the L-glutamate transport activity was retained and again could be eluted by the sugar. The transporter activity reconstituted from the glycoprotein fraction exhibited the same features as that in synaptic plasma membranes, including electrogenicity, an absolute dependence on external sodium and internal potassium, affinity and stereospecificity. Furthermore, efflux and exchange properties of the reconstituted preparation were also unchanged by the solubilisation and lectin-chromatography. These observations indicate that the sodium- and potassium-coupled L-glutamate transporter is a glycoprotein and is predominantly reconstituted in the 'right-side-out' conformation.
大鼠脑内的钠钾偶联L-谷氨酸转运体已用胆酸盐溶解，并使用麦胚凝集素-琼脂糖4B进行了10至20倍的纯化。在将该组分重构成脂质体后测定的转运活性保留在柱上，并被N-乙酰葡糖胺洗脱。当糖蛋白组分去除N-乙酰葡糖胺并进行第二轮凝集素色谱分析时，L-谷氨酸转运活性得以保留，并且再次可以被该糖洗脱。从糖蛋白组分重构的转运体活性表现出与突触质膜中相同的特征，包括电生性、对外部钠和内部钾的绝对依赖性、亲和力和立体特异性。此外，重构制剂的流出和交换特性也不因溶解和凝集素色谱分析而改变。这些观察结果表明，钠钾偶联L-谷氨酸转运体是一种糖蛋白，并且主要以“外翻”构象重构。
