Characterization of acetylcholine receptor-rich and acetylcholinesterase-rich membrane particles from electroplax (original) (raw)
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Protease effects on the structure of acetylcholine receptor membranes from Torpedo californica
The Journal of Cell Biology, 1980
Protease digestion of acetylcholine receptor-rich membranes derived from Torpedo californica electroplaques by homogenization and isopycnic centrifugation results in degradation of all receptor subunits without any significant effect on the appearance in electron micrographs, the toxin binding ability, or the sedimentation value of the receptor molecule. Such treatment does produce dramatic changes in the morphology of the normally 0.5-to 2-lm-diameter spherical vesicles when observed by either negative-stain or freeze-fracture electron microscopy . Removal of peripheral, apparently nonreceptor polypeptides by alkali stripping (Neubig et al., 1979, Proc. Natl. Acad. Sci. U. S. A. 76:690-694) results in increased sensitivity of the acetylcholine receptor membranes to the protease trypsin as indicated by SDS gel electrophoretic patterns and by the extent of morphologic change observed in vesicle structure. Trypsin digestion of alkali-stripped receptor membranes results in a limit degradation pattern of all four receptor subunits, whereupon all the vesicles undergo the morphological transformation to minivesicles . The protein-induced morphological transformation and the limit digestion pattern of receptor membranes are unaffected by whether the membranes are prepared so as to preserve the receptor as a disulfide bridged dimer, or prepared so as to generate monomeric receptor.
European Journal of Biochemistry, 1978
The acetylcholine receptor from Torpedo californicu electric tissue consisting of polypeptide chains of molecular weight 42 000 (k 2000) is part of a protein complex. Cross-linking experiments with bifunctional reagents have shown that this complex has possibly a pentameric structure with a molecular weight of 270 000 (2 30 000). Besides the receptor subunit (a-chain), at least three further classes of polypeptide chains are part of the complex : (M , 48 000), y (M , 62 000) and 6 (M , 68 000). This can be shown by cross-linking the proteins extracted from receptor-enriched membrane fractions with a cleavable reagent : From the 270 000 molecular weight particle the four predominant polypeptide chains of the membrane, a, p, y, and 6, can be obtained. The &polypeptide chains appear to form a dimer connected by an inter-chain disulphide bridge.
Neurochemistry International, 1985
A rapid method for the isolation of acetylcholine receptor-rich membranes from Torpedo marmorata electric organ, using a Percoll density gradient, is presented. The preparation of purified membranes appeared on electron microscope examination as a homogeneous population of sealed vesicles, covered with the characteristic rosettes identified as acetylcholine receptor clusters. Biochemical characterization revealed an ~t-bungarotoxin specific binding activity of 1.6-2.1 nmol/mg of protein, low acetylcholinesterase specific activity, a protein:lipid ratio (w/w) of 2.1 with high cholesterol content. Polyacrylamide gel electrophoresis under denaturing conditions showed the polypeptide bands characteristic of the receptor (~t, 8, ~ and 6), together with 43 kDa and ~ 100 kDa proteins (already described as v and e).
1981
The rates of inactivations of the acetylcholine receptor-controlled ion flux were measured with membrane vesicles prepared from the electric organ of Torpedo californica. A flow quench technique with a time resolution of 2 msec was used. The vesicles were preincubated with carbamyleholine for various periods of time and then the rate of 86Rb+ influx was measured. The influx rate decreased progressively with the length of preincubation time. This decrease (inactivation) in influx rate occurred in two different time zones. A fast inactivation process with a half-time of ~300 msec resulted in at least a 60-fold decrease of ion flux rate. The remaining ion flux activity decreased to an undetectable level in a slow inactivation process with a halftime of 6-7 seconds. Previously, only one inactivation process has been observed by ion flux measurements with receptor-rich vesicles from Electrophorus electrieus or T__orpedo species.
Biochemical Journal, 1980
A rapid method for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-iodosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures. Electric organs of Torpedo californica have been used as an excellent source of postsynaptic membranes containing nicotinic acetylcholine receptor (Duguid & Raftery, 1973; Reed et al., 1975). ACh receptor isolated from T. californica and purified by affinity-chromatographic procedures (Schmidt & Raftery, 1972) consists of four types of polypeptide subunits having mol.wts. 40000, 50000, 60000 and 65000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (Raftery et al., 1974; Weill et al., 1974; Vandlen et al., 1976). The agonist-binding site has been shown by affinity labelling to be located on the mol.wt.-40000 subunit (Moore & Raftery, 1979). In addition to the four ACh-receptor subunits the other major polypeptides found in ACh-receptor-enriched membrane fragments have mol.wts. 43000 and 90000 on sodium dodecyl sulphate/polyacrylamide gels. Eldefrawi et al. (1977) reported that in 1% Triton X-100 extracts of T. ocellata membrane fragments the agonist-binding site could be separated from an H12-HTX-binding component by gel filtration. Sobel et al. (1978) suggested that a 43000-mol.wt. polypeptide contains the binding site for histrionicotoxin and local anaesthetics in membranes isolated from T.
The biochemistry of an acetylcholine receptor
Journal of supramolecular structure, 1974
The acetylcholine receptor from Torpedo californica electroplax has been studied at three levels of molecular organization: receptor-rich membrane fragments, solubilized and purified receptor, and reconstituted receptor in phospholipid vesicles. The binding of cholinergic ligands t o the membrane-bound and the solubilized material is not cooperative, and the number of ligand sites is less than the number of toxin sites. In addition, the Durified macromolecule contains the molecular features necessary for ion-translocation during postsynaptic depolarization, since a chemically excitable membrane can be formed from purified receptor and Torpedo phospholipids.