Kang M S, Au-Young J, Cabib E
J Biol Chem. 1985 Oct 15;260(23):12680-4.
Yeast protoplasts were coated with different amounts of concanavalin A. Upon subsequent lysis and centrifugation in isopycnic density gradients, it was found that the buoyant density of plasma membranes was progressively increased from 1.125 to 1.21, according to the amount of bound concanavalin A. Enzymes that are attached to the plasma membrane showed the same density modifications and could thus be distinguished from constituents of intracellular membranes and organelles. With this methodology, it was confirmed that about two-thirds of yeast chitin synthetase is associated with the plasma membrane. The remainder of the enzyme was found in a peak at a lower density. Vanadate-sensitive ATPase showed a similar pattern, whereas GDP-mannose dolichyl-phosphate mannosyltransferase, an enzyme attached to the endoplasmic reticulum, remained in the same position in the gradients, irrespective of the amount of concanavalin A associated with the plasma membrane. Potential applications of this technique to the determination of plasma membrane markers and to the separation of subcellular organelles are discussed.
酵母原生质体用不同量的伴刀豆球蛋白A包被。随后在等密度梯度中进行裂解和离心，结果发现，根据结合的伴刀豆球蛋白A的量，质膜的浮力密度从1.125逐渐增加到1.21。附着在质膜上的酶显示出相同的密度变化，因此可以与细胞内膜和细胞器的成分区分开来。通过这种方法，证实了约三分之二的酵母几丁质合成酶与质膜相关。其余的酶在较低密度的峰中被发现。钒酸盐敏感的ATP酶显示出类似的模式，而内质网附着的酶GDP-甘露糖二磷酸多萜醇磷酸甘露糖基转移酶，在梯度中保持在相同位置，与质膜结合的伴刀豆球蛋白A的量无关。讨论了该技术在质膜标记物测定和亚细胞器分离中的潜在应用。
