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A simple biochemical approach to quantitate rough endoplasmic reticulum
A K Rajasekaran1, S A Langhans-Rajasekaran, R M Gould
1Department of Cell Biology and Anatomy, Cornell University Medical College, New York 10021.
The American Journal of Physiology
|February 1, 1995
Summary
Quantifying membrane-bound ribosomes offers a new method to measure rough endoplasmic reticulum (RER) size. This technique revealed a significant RER increase in estrogen-treated frog liver cells.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The rough endoplasmic reticulum (RER) is crucial for protein synthesis and modification.
- Accurate measurement of RER size is important for understanding cellular function.
- Existing methods for RER assessment can be complex or lead to sample loss.
Purpose of the Study:
- To develop and validate a simpler biochemical assay for quantifying changes in RER size.
- To assess the RER size in response to estrogen treatment in Xenopus laevis liver.
- To compare RER abundance in secretory versus non-secretory cell lines.
Main Methods:
- Utilized cell fractionation and sucrose density gradient analysis to isolate membrane-bound ribosomes.
- Employed puromycin and high-salt buffer for quantitative release and recovery of bound ribosomes from total cell membranes.
- Quantified membrane-bound ribosomal populations as a proxy for RER size.
Main Results:
- Demonstrated a 4.2-fold increase in RER in estrogen-treated male Xenopus laevis liver.
- Showed a lower free-to-membrane-bound ribosome ratio (1.2) in secretory cells (AR42J) compared to non-secretory cells (HeLa, 3.3).
- Indicated a correlation between high secretory activity and increased RER abundance.
Conclusions:
- The developed method provides a simpler and more direct assay for measuring RER size changes.
- Estrogen significantly increases RER in Xenopus laevis liver.
- Cellular secretory activity correlates with the amount of RER present.