Leikin A, Shinitzky M
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Biochim Biophys Acta. 1994 Mar 3;1211(2):150-5. doi: 10.1016/0005-2760(94)90263-1.
A procedure using high hydrostatic pressure without detergent has been applied in this study to subfractionate the components of the delta 6-desaturase system from the rat liver endoplasmic reticulum. Microsomes were suspended in a buffer containing liposomes made of phosphatidylcholine. The mixture was placed in a sealed pressure bomb and subjected to hydrostatic pressure of up to 1500 bars. Under these conditions the total desaturase activity was found in the liposomal fraction thus indicating that the three components of the desaturase system, namely NADH-cytochrome b5 reductase, cytochrome b5 and the delta 6-desaturase co-extracted. Size chromatography and FPLC of the released proteins followed by SDS-PAGE confirmed the independent release of the three components corresponding to the delta 6-desaturase, system. delta 6-Desaturase activity could be fully regenerated by mixing the aqueous dispersions of the three components without further purification. Our results indicate that these components are physically facing a similar lipid environment in the microsomal membrane.
本研究采用了一种不使用去污剂的高静水压程序，对大鼠肝脏内质网中Δ6-去饱和酶系统的组分进行亚分级分离。微粒体悬浮于含有由磷脂酰胆碱制成的脂质体的缓冲液中。将混合物置于密封的压力弹中，并施加高达1500巴的静水压。在这些条件下，发现总去饱和酶活性存在于脂质体部分，这表明去饱和酶系统的三个组分，即NADH-细胞色素b5还原酶、细胞色素b5和Δ6-去饱和酶共同被提取出来。对释放的蛋白质进行尺寸排阻色谱和快速蛋白质液相色谱，随后进行SDS-PAGE，证实了与Δ6-去饱和酶系统相对应的三个组分的独立释放。通过混合这三个组分的水分散体，无需进一步纯化，Δ6-去饱和酶活性即可完全恢复。我们的结果表明，这些组分在微粒体膜中实际上面临相似的脂质环境。
