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A membrane preparation that contains proteins characteristic of the rough endoplasmic reticulum
A Amar-Costesec1, M Hortsch, C Turu
1International Institute of Cellular and Molecular Pathology, Université Catholique de Louvain, Brussels, Belgium.
Abstract:
We describe a procedure for disassembling rat liver rough microsomes, which allows the purification of the rough endoplasmic reticulum (ER) membrane. Membrane-bound ribosomes and adsorbed proteins are first detached by washing rough microsomes with 5 mM Na-pyrophosphate. In a second step, the vesicle membrane is opened by digitonin, with concomitant release of the luminal content. The purification is monitored at each step by electron microscopy, and by assaying chemical constituents (protein, phospholipid, RNA) and marker enzymes for the main subcellular organelles. The final membrane preparation is representative of the ER, since it contains 24.1% of the liver glucose 6-phosphatase with a relative specific activity of 14.2. Contaminants represent less than 5% of its protein content. SDS-polyacrylamide gel electrophoresis, followed by immunoblot analysis, reveals that the ribophorins I and II, two established markers of the rough (d) domain are still present in the final membrane preparation. It also contains the docking protein (or signal recognition particle receptor) and protein disulfide isomerase, and has conserved the functional capacity to remove co- and post-translationally the signal peptide of pre-secretory proteins. The membrane preparation is suitable for studies on the polypeptide composition of the d domain.
Insights
Researchers developed a new method to purify rat liver rough endoplasmic reticulum (ER) membranes. This technique isolates ER membranes for detailed studies on their protein composition and function.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Rough endoplasmic reticulum (ER) is crucial for protein synthesis and modification.
- Isolation of pure rough ER membranes is challenging but essential for studying its specific functions.
Purpose of the Study:
- To establish a reliable procedure for purifying rat liver rough ER membranes.
- To obtain a highly pure rough ER membrane preparation suitable for biochemical and molecular analyses.
Main Methods:
- Washing rough microsomes with sodium pyrophosphate to detach ribosomes and proteins.
- Using digitonin to open vesicle membranes and release luminal content.
- Monitoring purification via electron microscopy, chemical assays, and enzyme activity measurements.
Main Results:
- A highly purified rough ER membrane fraction was obtained, with minimal contamination (<5%).
- The preparation retained key rough ER markers like ribophorins I and II, docking protein, and protein disulfide isomerase.
- The purified membranes demonstrated conserved functional capacity for signal peptide processing.
Conclusions:
- The described procedure effectively isolates pure rough ER membranes from rat liver.
- This preparation is suitable for investigating the polypeptide composition and functional properties of the rough ER membrane.