Sagné C, Isambert M F, Henry J P, Gasnier B
CNRS URA 1112, Institut de Biologie Physico-Chimique, Paris, France.
Biochem J. 1996 Jun 15;316 ( Pt 3)(Pt 3):825-31. doi: 10.1042/bj3160825.
The vesicular monoamine transporter, which catalyses a H+/ monoamine antiport in monoaminergic vesicle membrane, is a very hydrophobic intrinsic membrane protein. After solubilization, this protein was found to have a high tendency to aggregate, as shown by SDS/PAGE, especially when samples were boiled in the classical Laemmli buffer before electrophoresis. This behavior was analysed in some detail. The aggregation was promoted by high temperatures, organic solvents and acidic pH, suggesting that it resulted from the unfolding of structure remaining in SDS. The aggregates were very stable and could be dissociated only by suspension in anhydrous trifluoroacetic acid. This SDS-resistant aggregation behaviour was shared by very few intrinsic proteins of the chromaffin granule membrane. Consequently, a purification procedure was based on this property. A detergent extract of chromaffin granule membranes enriched in monoamine transporter was heated and the aggregates were isolated by size-exclusion HPLC in SDS. The aggregates, containing the transporter, were dissociated in the presence of trifluoroacetic acid and analysed on the same HPLC column. This strategy might be of general interest for the purification of membrane proteins that exhibit SDS-resistant aggregation.
囊泡单胺转运体催化单胺能囊泡膜中的H⁺/单胺反向转运，是一种高度疏水的内在膜蛋白。经增溶后，如SDS/PAGE所示，该蛋白具有很高的聚集倾向，尤其是在电泳前将样品在经典的Laemmli缓冲液中煮沸时。对这种行为进行了较为详细的分析。高温、有机溶剂和酸性pH会促进聚集，这表明聚集是由SDS中残留的结构展开导致的。聚集体非常稳定，只有悬浮在无水三氟乙酸中才能解离。嗜铬粒细胞膜的极少数内在蛋白也具有这种抗SDS聚集行为。因此，基于这一特性建立了一种纯化方法。将富含单胺转运体的嗜铬粒细胞膜去污剂提取物加热，通过SDS中的尺寸排阻HPLC分离聚集体。含有转运体的聚集体在三氟乙酸存在下解离，并在同一HPLC柱上进行分析。这种策略对于纯化表现出抗SDS聚集的膜蛋白可能具有普遍意义。
