A simple biochemical approach to quantitate rough endoplasmic reticulum.
In this report we demonstrate that the changes in size of the rough endoplasmic reticulum (RER) can be determined by quantifying the membrane-bound ribosomal population separated by cell fractionation and sucrose density gradient analysis. Total cell membranes, rather than microsomes, were used as the source of membrane-bound ribosomes to eliminate potential losses during the preparation of microsomes. Bound ribosomes were assayed after quantitative release and recovery from total cell membranes using puromycin in the presence of high-salt buffer. Using this analysis, we demonstrate a 4.2-fold increase in RER in estrogen-treated male Xenopus laevis liver. Furthermore, we show that the ratio of the distribution of free to membrane-bound ribosomes in a nonsecretory cell line (HeLa) was 3.3, while this ratio in a secretory cell line (AR42J) was 1.2, indicating that cells active in secretion contain more RER. We suggest that this biochemical technique provides a simpler assay to detect changes in the size of the RER.
Rajasekaran
A K
AK
Department of Cell Biology and Anatomy, Cornell University Medical College, New York 10021.
Langhans-Rajasekaran
S A
SA
Gould
R M
RM
Rodriguez-Boulan
E
E
Morimoto
T
T
D000818:Animals; D001671:Biochemistry / Q000379:methods*; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure*; D006367:HeLa Cells; D006801:Humans; D008099:Liver / Q000166:cytology / Q000648:ultrastructure; D051379:Mice; D008854:Microscopy, Electron; D051381:Rats; D017207:Rats, Sprague-Dawley; D012270:Ribosomes / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014982:Xenopus laevis