Affinity partitioning of membranes. Evidence for discrete membrane domains containing cholinergic receptor.

摘要: Subsynaptic membrane domains from Torpedo californica electroplax contain nicotinic cholinergic receptor molecules at densities as high 20,000 micrometers-2. Intense homogenization of the releases fragments enriched in basal lamina and other synaptic cleft presynaptic elements. Ideally, preparations fragments, highly receptor, should approach 125I-alpha-bungarotoxin-specific binding activities near levels observed after dispersal detergents subsequent affinity chromatography. We report application partitioning, combined with multiple extraction techniques, to yield virtually homogeneous membranes alpha, beta, gamma, delta subunits well 43,000-dalton peripheral protein subunit. The countercurrent distribution technique serves resolve three populations receptor-containing membranes. One fraction is refractory partitioning may represent aggregates receptor-rich derived nonsynaptic second third fractions subsynaptic are (5.1 7.8 nmol alpha-bungarotoxin sites/mg protein). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis all indicates that gamma present stable stoichiometric ratios, while content varies by 33% between fractions. However, removal 90% component mild alkali treatment does not result conversion one into other. combination counter-current techniques utilized this study prove useful resolution a variety subcellular particles specific sites.