Preparation of liver microsomes with high recovery of endoplasmic reticulum and a low grade of contamination.
A modified procedure for preparing the microsomal fraction from rat liver was developed with the aim of increasing the recovery without increasing the degree of contamination. 87% of the membranes of the microsomal fraction isolated from the first mitochondrial (10 000 X g) supernatant originates from the endoplasmic reticulum, representing a 35% yield. By gentle resuspension of the 10 000 X g pellet followed by differential centrifugation a second crop of microsomes can be prepared which, together with the first crop, gives a 55% total recovery of microsomal markers. 87% of the protein in this second crop also originates from the endoplasmic reticulum and this fraction has properties similar to those of the first crop. Contaminating membranes include Golgi membranes (0.6% of the total protein), mitochondria (2.5%), lysosomes (5%) and plasma membranes (5%). Collecting further crops increases the contamination. Subfractionation studies revealed almost identical distributions of ribosome-rich, ribosome-poor and smooth membranes in the two crops of microsomal fractions. The results obtained after treatment of the animals with phenobarbital or methylcholantrene were similar to those obtained with control animals; but in the case of methylcholantrene treatment the second crop represents a larger portion of the total membranes of the endoplasmic reticulum.
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D004721:Endoplasmic Reticulum / Q000648:ultrastructure*; D008099:Liver / Q000648:ultrastructure*; D008297:Male; D008563:Membrane Lipids / Q000032:analysis; D008862:Microsomes, Liver / Q000648:ultrastructure*; D010743:Phospholipids / Q000032:analysis; D011506:Proteins / Q000032:analysis; D012313:RNA / Q000032:analysis; D051381:Rats
D008563:Membrane Lipids; D010743:Phospholipids; D011506:Proteins; D012313:RNA