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Plasma membranes from adrenal cells: purification and properties
Journal of Cell Science
|August 1, 1985
Summary
This study details a new method for isolating plasma membranes from adrenal tumor cells using homogenization and density gradient sedimentation. The purified membranes show high purity and specific binding activity for ACTH and LDL.
Area of Science:
- Cell Biology
- Biochemistry
Background:
- Adrenal tumor (Y-1) cells are a valuable model for studying cellular processes.
- Isolation of pure plasma membranes is crucial for understanding cell surface receptor function.
Purpose of the Study:
- To develop and validate a method for preparing surface (plasma) membranes from Y-1 cells.
- To characterize the purity and functional integrity of the isolated membranes.
Main Methods:
- Homogenization of Y-1 cells in hypotonic ZnCl2.
- Sedimentation through two sucrose density gradients.
- Characterization using microscopy, enzyme markers, and functional assays (ACTH/LDL binding, adenylate cyclase).
Main Results:
- Purified membranes obtained as large sheets, free from cytoplasm and nuclei.
- Specific binding of ACTH (KD 0.12 nM, 2500 sites/cell) and LDL (KD 0.5 nM, 4800 sites/cell).
- Increased specific binding activity for ACTH (21-fold) and LDL (15-fold) compared to whole homogenate.
Conclusions:
- The reported method effectively isolates pure and functional plasma membranes from Y-1 cells.
- The isolated membranes retain specific binding sites for ACTH and LDL, suitable for further biochemical studies.
- The method is also applicable to preparing membranes from beef fasciculata cells.