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Topological dispositions of lysine alpha 380 and lysine gamma 486 in the acetylcholine receptor from Torpedo californica



Topological dispositions of lysine alpha 380 and lysine gamma 486 in the acetylcholine receptor from Torpedo californica



Biochemistry 30(16): 4105-4112



The locations have been determined, with respect to the plasma membrane, of lysine .alpha.380 and lysine .gamma.486 in the .alpha. subunit and the .gamma. subunit, respectively of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the .alpha. subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the .gamma. subunit. They were used to isolate these peptides from proteolytic digests of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium [3H]-borohydride. Saponin was added to a portion of the vesicles prior to labeling to render them permeable to pyridoxal phosphate. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine .alpha.380 and lysine .gamma.486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The peptides bound and released by the immunoadsorbents were positively identified and quantified by high-pressure liquid chromatography. Modification of lysine .alpha.380 in the native acetylcholone receptor in sealed vesicles increased 5-fold in the presence of saponin, while modification of lysine .gamma.486 was unaffected by the presence of saponin. The conclusions that follow from these results are that lysine .alpha.380 is on the inside surface of a vesicle and lysine .gamma.486 is on the outside surface. Because a majority (85%) of the total binding sites for .alpha.-bungarotoxin bind the toxin in the absence of saponin, the majority of the vesicles are right side out with the inside of the vesicle corresponding to the cytoplasmic surface and the outside of the vesicle corresponding to the extracytoplasmic, synaptic surface. Because lysine .alpha.380 and lysine .gamma.486 lie on opposite sides of the membrane, a membrane-spanning segment must be located between the two positions occupied by these two amino acids in the common sequence of a polypeptide of the acetylcholine receptor. Within the 64 amino acids in the common amino acid sequence that separates the positions homologous to .alpha.380 and .gamma.486 is a hydrophobic segment of sufficient length to span the membrane.

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Accession: 007956080

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PMID: 1902116


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Topological dispositions of lysine .alpha.380 and lysine .gamma.486 in the acetylcholine receptor from Torpedo californica. Biochemistry 30(16): 4105-4112, 1991

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