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Isolation of plasma membranes and Golgi apparatus from a single chicken liver homogenate
L Vleurick1, E R Kühn, E Decuypere
1Leuven Poultry Research Group, Zoological Institute and Faculty of Agronomy, Belgium. vleurick.l@fsagx.ac.be
Abstract:
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.
Insights
Researchers purified chicken liver plasma membranes and Golgi vesicles using density gradient centrifugation. This isolation method enables future studies on cellular receptor trafficking between these organelles.
Area of Science:
- Cell Biology
- Biochemistry
- Membrane Biology
Background:
- Plasma membranes and Golgi apparatus are crucial organelles involved in cellular transport and signaling.
- Efficient isolation of these organelles is essential for studying their functions and interactions.
- Previous methods for isolating these organelles often resulted in cross-contamination, limiting further research.
Purpose of the Study:
- To develop a method for isolating purified chicken liver plasma membranes and Golgi vesicles.
- To characterize the isolated fractions using enzyme markers.
- To enable future investigations into receptor cycling between plasma membranes and the Golgi apparatus.
Main Methods:
- Chicken liver homogenates were fractionated using differential centrifugation.
- Plasma membranes were isolated via Percoll density gradient flotation and hypotonic wash.
- Golgi apparatus fractions were obtained by sucrose density gradient flotation of microsomes.
Main Results:
- Two plasma membrane fractions were isolated with 28-97 fold enrichment of alkaline phosphatase and alkaline phosphodiesterase.
- Golgi apparatus fractions showed a 27-fold enrichment of the trans-Golgi marker galactosyltransferase.
- Contamination of Golgi vesicles with plasma membrane fragments was minimal.
Conclusions:
- A robust method was established for the co-isolation of purified plasma membranes and Golgi vesicles from chicken liver.
- The isolated fractions are suitable for biochemical analysis and future studies on organelle interactions.
- This purification technique facilitates research on receptor trafficking pathways within the cell.