Smith G D, Peters T J
Eur J Biochem. 1980 Feb;104(1):305-11. doi: 10.1111/j.1432-1033.1980.tb04429.x.
A method for the subcellular fractionation of rat liver using whole homogenates of rat liver and analytical sucrose density gradient centrifugation is presented. The distributions in the sucrose gradients of marker enzymes for all organelles have been determined for control homogenates and for homogenates prepared in the presence of selective membrane perturbants. This technique is not subject to potential loss of information inherent in the use of postnuclear supernatants as starting material for fractionation experiments. Particular attention has been paid to the distributions of putative plasma membrane marker enzymes, up to 50% of which may be found in the nuclear pellet. Gamma-Glutamyltransferase has been found to be entirely plasma membrane in location but has a different distribution pattern when compared with other plasma membrane markers. Particulate alkaline phosphatase and alkaline phosphodiesterase are shown to have bimodal distribution, one peak of which is coincident with 5'-nucleotidase. The other peak is coincident with that of the golgi marker, galactosyltransferase, but the membrane structure containing these activities shows characteristics of plasma membrane rather than golgi apparatus.
本文介绍了一种利用大鼠肝脏全匀浆和分析性蔗糖密度梯度离心法对大鼠肝脏进行亚细胞分级分离的方法。已测定了对照匀浆以及在选择性膜扰动剂存在下制备的匀浆中所有细胞器标记酶在蔗糖梯度中的分布。该技术不存在将核后上清液用作分级分离实验起始材料时固有的潜在信息损失问题。特别关注了假定的质膜标记酶的分布，其中高达50%可能存在于核沉淀中。已发现γ-谷氨酰转移酶完全位于质膜，但与其他质膜标记物相比，其分布模式不同。颗粒性碱性磷酸酶和碱性磷酸二酯酶呈现双峰分布，其中一个峰与5'-核苷酸酶重合。另一个峰与高尔基体标记物半乳糖基转移酶的峰重合，但含有这些活性的膜结构显示出质膜而非高尔基体的特征。
