O'Toole K, Pollak J K
Biochem J. 1974 Mar;138(3):359-71. doi: 10.1042/bj1380359.
A major difficulty in studying quantitative changes in free and membrane-bound ribosomes in a tissue under different physiological conditions is that membrane-bound ribosomes are not usually recovered quantitatively in a conventional microsomal fraction. This problem was resolved for developing chick liver by determining the conditions for the isolation of a microsomal fraction containing the highest practicable yield of rough vesicles, and then separating it into free-ribosome- and rough-vesicle-containing fractions. With the aid of a marker enzyme for the microsomal membranes and the RNA content of the recovered membrane-bound ribosomes, it was possible to correct for the recovery of rough vesicles and hence to determine the concentration of membrane-bound ribosomes in the homogenate. Despite the fact that morphological studies have suggested that most of the cellular ribosomes are not bound to membrane in chick liver cells at the earliest developmental age studied (6 days of egg incubation), 49% of the total ribosomes were found to be membrane-bound by using the new fractionation technique. This fraction increased (to 66%) during development. The discrepancy between the cell-fractionation and morphological approaches could not be attributed to artifacts of the separation method but rather to difficulties inherent in the morphological approach.
在研究不同生理条件下组织中游离核糖体和膜结合核糖体的定量变化时，一个主要困难在于，在常规微粒体组分中，膜结合核糖体通常无法被定量回收。通过确定分离含有最高可行产量的粗面小泡的微粒体组分的条件，然后将其分离为含游离核糖体和含粗面小泡的组分，这个问题在发育中的鸡肝脏中得到了解决。借助微粒体膜的标记酶以及回收的膜结合核糖体的RNA含量，可以校正粗面小泡的回收率，从而确定匀浆中膜结合核糖体的浓度。尽管形态学研究表明，在所研究的最早发育阶段（孵化6天的鸡蛋），鸡肝细胞中的大多数细胞核糖体并未与膜结合，但使用新的分级分离技术发现，49%的总核糖体是膜结合的。在发育过程中，这一比例增加（至66%）。细胞分级分离法和形态学方法之间的差异不能归因于分离方法的假象，而应归因于形态学方法固有的困难。
