Molecular forms and hydrodynamic properties of acetylcholine receptor from electric tissue.
We have studied purified acetylcholine receptor proteins from Electrophorus electricus and Torpedo marmorata which function in both binding and reconstitution experiments. The molecular properties of these receptor-channel complexes were analyzed under non-denaturing conditions by polyacrylamide gradient gel electrophoresis, ultracentrifugation sedimentation studies and laser light scattering. The purified receptor proteins exist in different interconvertible forms depending on both the type and concentration of detergent present, and the presence or absence of an intersubunit disulfide bridge. Receptor purified in the absence of sulfhydryl-blocking agents exists in two monomeric and two dimeric forms at very low detergent concentrations (0.01-0.05% Tween 80). At intermediate detergent concentrations (0.4% Triton X-100) one monomeric and one dimeric form are present. Only the monomeric form remains at high levels of detergent (2% Triton X-100). This form has a sedimentation coefficient of 9.29 S, as measured by ultracentrifugation using Schlieren optics. If receptor is purified in the presence of sulfhydryl-blocking agents, conversion of dimers into monomers by high concentrations of detergent does not occur. Disulfide-reducing agents convert dimers into monomers independent of whether the receptor's free sulfhydryl groups are blocked or not. These findings imply that dimer formation is primarily due to hydrophobic interactions between monomers. When these interactions are reduced by high levels of detergent the intersubunit disulfide bridge is dissociated at the expense of newly formed interasubunit ones, as long as the sulfhydryl group(s) have remained unblocked. Monomers and dimers bind alpha-cobratoxin with the same affinity and kinetics. Dimers of disulfide-linked monomers are not required for the reconstitution of a functional ion-translocation system. The presence of EDTA during the purification of receptor proteins (as recommended by many groups to inhibit proteolysis) adversely influences the activity of the receptor in channel gating. These observations are discussed in terms of the requirements for a purification procedure yielding receptor preparations unaffected by proteolysis and functioning in both binding and ion translocation. The molecular weight of the receptor monomer was determined by several independent techniques and yielded values in the range of 250-300 x 10(3). With a translational diffusion constant D20, w = 2.95 x 10(-7) cm2 s-1 and a sedimentation coefficient s20, w = 9.29 S, the frictional coefficient ratio f/f0 = 1.51 was calculated for the receptor monomer from Torpedo marmorata. This indicates a considerable asymmetry in the shape of the detergent-solubilized receptor.