Ewalt K L
Department of Chemistry, University of California, San Diego, La Jolla 92093-0506.
Biochemistry. 1994 May 3;33(17):5077-88. doi: 10.1021/bi00183a011.
The location, with respect to the membrane, of Lys 165 in the folded beta polypeptide of native nicotinic acetylcholine receptor has been determined by site-directed immunochemistry. Sealed, right-side-out vesicles rich in acetylcholine receptor were modified with pyridoxal phosphate and sodium [3H]-borohydride. Saponin was added to one portion of the vesicles to make them permeable to the pyridoxal phosphate and sodium borohydride; the other portion was modified in the absence of saponin. Both samples were then exhaustively succinylated and digested with trypsin and thermolysin to produce the peptide LDAKGER, which contains Lys beta 165. The digests were passed over an immunoadsorbent specific for peptides with the sequence LDAXGER, where X represents any modified or unmodified amino acid, and specifically bound peptides were eluted with 0.1 M sodium phosphate, pH 2.5. The eluates were submitted to high-pressure liquid chromatography, and two peptides, N epsilon-phospho[3H]pyridoxalLDAKGER and N epsilon-succinylLDAKGER, modified at the epsilon amino group of lysine with pyridoxal phosphate and sodium [3H]-borohydride or succinic anhydride, respectively, were identified by comparison to standards. The relative specific radioactivity of N epsilon-phospho[3H]pyridoxalLDAKGER modified in the presence or absence of saponin, respectively, was 0.9 +/- 0.4. The incorporation of phospho[3H]pyridoxyl groups into Lys alpha 380, a residue located on the cytoplasmic surface of acetylcholine receptor, was also monitored. The relative specific radioactivity of the peptide that contains the modified Lys alpha 380, N epsilon-phospho[3H]pyridoxalGVKYIAE, increased 3.6-fold when the modification was performed in the presence of saponin. This result verifies that the vesicles used in these experiments were sealed and right-side-out. Because the incorporation of [3H]pyridoxyl groups into Lys beta 165 is the same in the presence or absence of saponin, Lys beta 165 must have been located on the outside surface of the sealed, right-side-out vesicles, and therefore on the extracytoplasmic surface of native acetylcholine receptor.
