Purification of a Presynaptic Membrane Protein that Mediates a Calcium-Dependent Translocation of Acetylcholine

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 83; no. 23; pp. 9226 - 9230
• Main Authors: Israël, M., Morel, N., Lesbats, B., Birman, S., Manaranche, R.
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.12.1986; National Acad Sciences
• Subjects: acetylcholine; Acetylcholine - metabolism; Animals; biochemical analysis; Biological and medical sciences; Calcium; Calcium - physiology; Carrier Proteins - isolation & purification; Cell membranes; Cell physiology; Centrifugation; Chemical precipitation; electric organs; Ethers; Fundamental and applied biological sciences. Psychology; Gels; Hydrogen-Ion Concentration; Lipids; Membrane proteins; Membrane Proteins - isolation & purification; Microscopy, Electron; Molecular and cellular biology; Molecular Weight; Nerve Tissue Proteins - isolation & purification; Neuromuscular Junction - analysis; neurosecretion; Neurotransmission; P branes; proteins; Solvents; Torpedo; Torpedo marmorata; Membrane protein; Purification; Torpedo marmorata; Electric organ; Mechanism; Characterization; Vertebrata; Proteoliposome; Pisces; Animal; Plasma membrane; Acetylcholine; Release; Presynaptic
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.83.23.9226

A protein, which we call ``mediatophore,'' that mediates calcium-dependent release of acetylcholine from proteoliposomes has been purified from the presynaptic plasma membrane. About 250 μ g of this material was obtained from 500 g of Torpedo marmorata electric organ. Precipitation of the protein and subsequent removal of associated lipids inactivated the protein, which then became water soluble; this permitted evaluation of its Stokes radius (52 angstrom) and its sedimentation coefficient (9.8 ± 0.75 S) and, hence, an approximate molecular mass of 210 ± 16 kDa could be determined. PAGE analysis showed that the protein is made of 17-kDa subunits, not linked by disulfide bonds. When this material was observed by electron microscopy after negative staining, the apparently pentameric structures had an average diameter of about 7 nm.