A SIMPLE BIOCHEMICAL APPROACH TO QUANTITATE ROUGH ENDOPLASMIC-RETICULUM

In this report we demonstrate that the changes in size of the rough en doplasmic reticulum (RER) can be determined by quantifying the membran e-bound ribosomal population separated by cell fractionation and sucro se density gradient analysis. Total cell membranes, rather than micros omes, were used as the source of membrane-bound ribosomes to eliminate potential losses during the preparation of microsomes. Bound ribosome s 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-t reated male Xenopus laevis liver. Furthermore, we show that the ratio of the distribution of free to membrane-bound ribosomes in a nonsecret ory cell line (HeLa) was 3.3, while this ratio in a secretory cell lin e (AR42J) was 1.2, indicating that cells active in secretion contain m ore RER. We suggest that this biochemical technique provides a simpler assay to detect changes in the size of the RER.