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A membrane preparation that contains proteins characteristic of the rough endoplasmic reticulum
Author(s) -
AmarCostesec Alain,
Hortsch Michael,
Turu Christine
Publication year - 1988
Publication title -
biology of the cell
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.543
H-Index - 85
eISSN - 1768-322X
pISSN - 0248-4900
DOI - 10.1111/j.1768-322x.1988.tb00731.x
Subject(s) - endoplasmic reticulum , digitonin , biology , biochemistry , membrane protein , microsome , organelle , protein disulfide isomerase , signal peptide , secretory protein , membrane , signal recognition particle , pyrophosphate , ribosome , enzyme , rna , secretion , recombinant dna , gene
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‐phosphate 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 containes 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.