Reductive methylation as a tool for the identification of the amino groups in alpha-bungarotoxin interacting with nicotinic acetylcholine receptor.
alpha-Bungarotoxin (alpha Bgt) is a postsynaptic neurotoxin which blocks cholinergic transmission at the neuromuscular junction by binding tightly to the acetylcholine receptor (AcChR). The number of methylation sites in alpha Bgt has been shown to decrease significantly upon binding of the toxin to the AcChR [Soler, G., Farach, M. C., Farach, H. A., Mattingly, J. R., & Martinez-Carrion, M. (1983) Arch. Biochem. Biophys. 225, 872-878]. We have compared the chemical reactivities of amino groups in free and AcChR-bound alpha Bgt in an attempt to identify the regions in the alpha Bgt molecule that become masked upon binding to the AcChR. Free alpha Bgt and AcChR-bound alpha Bgt were reductively methylated with formaldehyde and sodium cyanoborohydride, and the rate of modification of each one of the available amino groups was followed by cleaving the methylated toxin with V8 protease and resolving the resulting peptides by reversed-phase, high-performance liquid chromatography. Under conditions of limited reagent availability, five of seven amino groups in free alpha Bgt reacted readily, whereas two other amino groups, probably those corresponding to Lys-51 and Lys-70, displayed lower reactivity. Upon binding to the AcChR, the rates of reductive methylation of residues Ile-1, Lys-26, and Lys-38 were considerably reduced (although to differing extents). The degree of protection was most pronounced for Lys-26. The rates of methylation of the amino groups in all other positions remained unchanged. These results allow further definition of the minimal binding surface of a representative neurotoxin.
Garcia-Borron
J C
JC
Department of Biochemistry, Virginia Commonwealth University, Richmond 23298.
Bieber
A L
AL
Martinez-Carrion
M
M
D000588:Amines; D000595:Amino Acid Sequence; D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004557:Electric Organ / Q000378:metabolism; D007700:Kinetics; D008745:Methylation; D011487:Protein Conformation; D011950:Receptors, Cholinergic / Q000302:isolation & purification / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000302:isolation & purification / Q000378:metabolism*; D014101:Torpedo; D064569:alpha7 Nicotinic Acetylcholine Receptor
D000588:Amines; D002038:Bungarotoxins; D011950:Receptors, Cholinergic; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor