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

✍ Scribed by Lieve Vleurick; Eduard R. Kühn; Eddy Decuypere; Paul P. Van Veldhoven

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 48 KB
• Volume: 72
• Category: Article
• ISSN: 0730-2312
• DOI: 10.1002/(sici)1097-4644(19990301)72:3<349::aid-jcb4>3.0.co;2-h

✦ Synopsis

Chicken liver plasma membranes, minimally contaminated with Golgi apparatus-derived vesicles, were prepared from a low-speed (400 g) 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.