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Isolation of plasma membranes and Golgi apparatus from a single chicken liver homogenate

Isolation of plasma membranes and Golgi apparatus from a single chicken liver homogenate Chicken liver plasma membranes, minimally contaminated with Golgi apparatus‐derived vesicles, were prepared from a low‐speed (400g) pellet by means of flotation in isotonic Percoll solution, followed by a hypotonic wash and flotation in a discontinuous sucrose gradient. Based on the analysis of suitable marker enzymes, alkaline phosphatase and alkaline phosphodiesterase, two plasma membrane fractions were isolated with enrichments, depending on the equilibrium density and marker of 28–97 and with a total yield of 4–5%. Golgi apparatus fractions were prepared by flotation of microsomes, obtained from the same homogenate as the low‐speed pellet, in a discontinuous sucrose gradient. The trans‐Golgi marker galactosyltransferase was 27‐fold enriched in a fraction of intermediate density (d=1.077–1.116 g/ml). Approximately 12% of galactosyltransferase was recovered in the membranes equilibrating d=1.031–1.148 g/ml. Contamination with plasma membrane fragments was low in the light (d=1.031–1.077 g/ml) and intermediate density Golgi vesicles. The isolation of purified plasma membranes and Golgi vesicles from one liver homogenate will enable future studies on receptor cycling between these cell organelles. J. Cell. Biochem. 72:349–355, 1999. © 1999 Wiley‐Liss, Inc. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Cellular Biochemistry Wiley

Isolation of plasma membranes and Golgi apparatus from a single chicken liver homogenate

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Publisher
Wiley
Copyright
Copyright © 1999 Wiley‐Liss, Inc.
ISSN
0730-2312
eISSN
1097-4644
DOI
10.1002/(SICI)1097-4644(19990301)72:3<349::AID-JCB4>3.3.CO;2-8
Publisher site
See Article on Publisher Site

Abstract

Chicken liver plasma membranes, minimally contaminated with Golgi apparatus‐derived vesicles, were prepared from a low‐speed (400g) pellet by means of flotation in isotonic Percoll solution, followed by a hypotonic wash and flotation in a discontinuous sucrose gradient. Based on the analysis of suitable marker enzymes, alkaline phosphatase and alkaline phosphodiesterase, two plasma membrane fractions were isolated with enrichments, depending on the equilibrium density and marker of 28–97 and with a total yield of 4–5%. Golgi apparatus fractions were prepared by flotation of microsomes, obtained from the same homogenate as the low‐speed pellet, in a discontinuous sucrose gradient. The trans‐Golgi marker galactosyltransferase was 27‐fold enriched in a fraction of intermediate density (d=1.077–1.116 g/ml). Approximately 12% of galactosyltransferase was recovered in the membranes equilibrating d=1.031–1.148 g/ml. Contamination with plasma membrane fragments was low in the light (d=1.031–1.077 g/ml) and intermediate density Golgi vesicles. The isolation of purified plasma membranes and Golgi vesicles from one liver homogenate will enable future studies on receptor cycling between these cell organelles. J. Cell. Biochem. 72:349–355, 1999. © 1999 Wiley‐Liss, Inc.

Journal

Journal of Cellular BiochemistryWiley

Published: Mar 1, 1999

Keywords: subcellular fractionation; plasma membranes; Golgi apparatus; marker enzymes; chicken; liver

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