The Drosophila acetylcholine receptor subunit D alpha5 is part of an alpha-bungarotoxin binding acetylcholine receptor.
The central nervous system of Drosophila melanogaster contains an alpha-bungarotoxin-binding protein with the properties expected of a nicotinic acetylcholine receptor. This protein was purified 5800-fold from membranes prepared from Drosophila heads. The protein was solubilized with 1% Triton X-100 and 0.5 M sodium chloride and then purified using an alpha-cobratoxin column followed by a lentil lectin affinity column. The purified protein had a specific activity of 3.9 micromol of 125I-alpha-bungarotoxin binding sites/g of protein. The subunit composition of the purified receptor was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This subunit profile was identical with that revealed by in situ labeling of the membrane-bound protein using the photolyzable methyl-4-azidobenzoimidate derivative of 125I-alpha-bungarotoxin. The purified receptor reveals two different protein bands with molecular masses of 42 and 57 kDa. From sedimentation analysis of the purified protein complex in H2O and D2O and gel filtration, a mass of 270 kDa was calculated. The receptor has a s(20,w) of 9.4 and a Stoke's radius of 7.4 nm. The frictional coefficient was calculated to be 1.7 indicating a highly asymmetric protein complex compatible with a transmembrane protein forming an ion channel. The sequence of a peptide obtained after tryptic digestion of the 42-kDa protein allowed the specific identification of the Drosophila D alpha5 subunit by sequence comparison. A peptide-specific antibody raised against the D alpha5 subunit provides further evidence that this subunit is a component of an alpha-bungarotoxin binding nicotinic acetylcholine receptor from the central nervous system of Drosophila.
Wu
Peipei
P
Department of Biological Sciences, Center for the Study of Gene Structure and Function, Hunter College and Graduate Center of the City University of New York, New York, New York 10021, USA.
Ma
Dongdong
D
Pierzchala
Marek
M
Wu
Jun
J
Yang
Lee-Chuan
LC
Mai
Xiaoping
X
Chang
Xiaoying
X
Schmidt-Glenewinkel
Thomas
T
D000109:Acetylcholine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000737:chemistry; D001665:Binding Sites; D015153:Blotting, Western; D002038:Bungarotoxins / Q000737:chemistry*; D002490:Central Nervous System / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003039:Cobra Neurotoxin Proteins / Q000737:chemistry; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D004591:Electrophoresis, Polyacrylamide Gel; D007096:Imidoesters / Q000737:chemistry; D020128:Inhibitory Concentration 50; D007477:Ions; D037102:Lectins; D027806:Lens Plant / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D017830:Octoxynol / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D010802:Phylogeny; D011485:Protein Binding; D011950:Receptors, Cholinergic / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology
D001386:Azides; D002038:Bungarotoxins; D003039:Cobra Neurotoxin Proteins; D029721:Drosophila Proteins; D007096:Imidoesters; D007477:Ions; D037102:Lectins; D008024:Ligands; D010455:Peptides; D011950:Receptors, Cholinergic; D011978:Receptors, Nicotinic; C113681:nicotinic acetylcholine receptor alpha-subunits, Drosophila; D012965:Sodium Chloride; C025154:alpha-cobratoxin; C026810:methyl 4-azidobenzimidate; D017830:Octoxynol; D014357:Trypsin; D000109:Acetylcholine