Isolation from Cholinergic Synapses of a Protein That Binds to Membranes in a Calcium‐Dependent Manner

Abstract

A protein that binds to membranes in a Ca-dependent manner between Ca concentrations of 10-5 and 10-6 M was isolated in large amounts (20 mg/kg tissue) from the entirely cholinergic electric organ of Torpedo marmorata. The protein bound reversibly to membrane fractions in a Ca-specific and saturable manner. The protein also bound to lipids isolated from Torpedo electric organ, to clathrin-coated vesicles prepared from pig brain and to a Triton X-100-sensitive site. It had an apparent subunit MW of 32,000 by polyacrylamide gel electrophoresis and of 35,900 by amino acid analysis; a broad isoelectric range of 4.8-5.5; and 27% of its amino acids after hydrolysis were observed to be aspartic and glutamic acids. Synaptosomes derived from electric organ were enriched in the protein which is probably localized within the nerve ending. It was localized in the synaptic region of the electric organ by immunofluorescence. In the electric lobe, discrete patches of fluorescence were seen within the cell bodies that innervate the electric organ. The protein may be involved in the recognition of membranes within the cholinergic neurons. Proteins with similar purification properties were found in all tissues investigated so far, and polypeptides of subunit MW 32,000 were identified in bovine adrenal medulla and guinea pig brain synaptosomes.