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Isolation of plasma membrane, Golgi apparatus, and endoplasmic reticulum fractions from single homogenates of mouse liver
Author(s) -
Croze Edward M.,
Morré D. James
Publication year - 1984
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041190109
Subject(s) - endoplasmic reticulum , golgi apparatus , vesicle , biochemistry , microsome , membrane , golgi membrane , cell fractionation , hepatocyte , biology , secretory pathway , chemistry , microbiology and biotechnology , enzyme , in vitro
Procedures to isolate plasma membrane, Golgi apparatus, and endoplasmic reticulum from a single homogenate of mouse liver are described. Fractions contain low levels of contaminating membranes as determined from morphometry and analyses of marker enzymes. The method requires only 2–3 gm of liver as starting material and yields approximately 0.7, 0.7, and 0.5 mg protein/gm liver, respectively, for endoplasmic reticulum, Golgi apparatus, and plasma membrane. Golgi apparatus fractions show high levels of galactosyltransferase activity and consist of cisternal stacks and associated secretory vesicles and tubules. Endoplasmic reticulum fractions are enriched in both glucose‐6‐phosphatase and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)‐cytochrome c reductase and contain membrane vesicles with attached ribosomes. K + ‐stimulated p‐nitrophenyl phosphatase and (Na + K + ) adenosine triphosphatase activity are enriched in the plasma membrane fraction. This fraction consists of membrane sheets, many with junctional complexes, and bile canaliculi that are representative of the total hepatocyte plasma membrane. The fractionation procedure is designed to utilize small amounts of tissue (e.g., with liver slices), to reduce the total time required for fractionation, and to permit comparisons of constituents of plasma membrane, Golgi apparatus, and endoplasmic reticulum prepared from the same starting homogenates.