Abstract
Subsynaptic membrane domains from Torpedo californica electroplax contain nicotinic cholinergic receptor molecules at densities as high as 20,000 micrometers-2. Intense homogenization of the electroplax releases membrane fragments enriched in nicotinic receptor from basal lamina and other synaptic cleft and presynaptic elements. Ideally, preparations of membrane fragments, highly enriched in nicotinic receptor, should approach 125I-alpha-bungarotoxin-specific binding activities near the levels observed after receptor dispersal in detergents and subsequent affinity chromatography. We report the application of affinity partitioning, combined with multiple extraction techniques, to yield preparations of virtually homogeneous membranes enriched in nicotinic receptor alpha, beta, gamma, and delta subunits as well as the 43,000-dalton peripheral protein subunit. The countercurrent distribution technique serves to resolve three populations of receptor-containing membranes. One fraction is refractory to affinity partitioning and may represent aggregates of receptor-rich membranes with fragments derived from nonsynaptic membranes. The second and third fractions contain membrane fragments derived from the subsynaptic membrane and are highly enriched in nicotinic receptor (5.1 to 7.8 nmol of alpha-bungarotoxin binding sites/mg of protein). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of all three fractions indicates that alpha, beta, and gamma subunits are present in stable stoichiometric ratios, while the 43,000-dalton peripheral protein content varies by 33% between the fractions. However, removal of 90% of the 43,000-dalton component by mild alkali treatment does not result in conversion of one fraction into the other. The combination of affinity partitioning and counter-current distribution techniques utilized in this study should prove useful in the resolution of a variety of subcellular particles that contain specific binding sites.
Highlights
Subsynaptic membrane domains from Torpedo cali- The precise functional and modulatory features of integral fornica electroplax contain nicotinic cholinergic recep- membrane proteins such as neurotransmitter receptors are tor molecules at densities as high as 20,000 pm-’
In- best studied in their native membrane environments
In the tense homogenization of the electroplax releases mem- case of the nicotinic cholinergic receptor, ion translocation brane fragments enriched in nicotinic receptor from phenomena and the influence of local anestheticsonthe basal lamina and other synaptic cleft and presynaptic elements
Summary
From the Division of Neurosciences, City of Hope Research Institute, Duarte, California91010. The countercurrent distribution technique serves to tors may be partially purified by affinity partitioning, a resolve three populations of receptor-containing mem- method for separation of membrane fragments that relies on branes. In order to introduce specificity into the separation procedure, ligands known to associate with discrete sites on ratios, while the 43,000-dalton peripheral protein con- the membrane are covalently attached toone of the polymers, tent varies by 33% between the fractions. In order to test these concepts and to secure highly specific affinity-partitioning separations, we have synthesized new bisquaternary ammonium ligandasttached to poly(Et0).Bisquaternary ammonium ligands bindto the nicotinic cholinergic receptor-binding sites with enhanced affinity over monoparallel asxayz were perfom & usinggelfiltrationto separatetoxin-receptorcmplcx fm f r e et o x i n ( I ).
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