Plasma membranes from adrenal cells: Purification and properties

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Abstract
A method is reported for preparing surface (plasma) membranes from adrenal tumour (Y-l) cells. The procedure is based upon homogenization in hypotonic ZnCl2 followed by sedimentation through two sucrose density gradients. The purified membranes consist of large sheets of membrane. The identity and purity of the membranes was demonstrated by: (1) microscopy (phase contrast and electron); (2) enzyme markers; and (3) functional activities associated with plasma membranes (binding of ACTH and LDL and adenylate cyclase). Phase-contrast microscopy revealed the release of membrane ghosts free from cytoplasm and nuclei. Electron microscopy showed membranes with small fragments of cytoplasm attached to the inside. Binding of ACTH was found to be specific with KD 0·12nM and the equivalent of 2500 sites per cell. Binding of LDL was also specific with KD 0·5 nM and the equivalent of 4800 sites per cell. Specific activities of binding for ACTH and LDL were increased by 21-fold and 15-fold, respectively, relative to whole homogenate. Membranes were also prepared from beef fasciculata cells by the same method.