Solubilization and partial characterization of [3H]choline mustard-labeled high-affinity choline carrier from presynaptic plasma membrane of Torpedo electric organ.
The objective of the present study was to characterize the hydrodynamic properties of the detergent-solubilized high-affinity choline carrier from presynaptic plasma membranes of electric organ of Torpedo marmorata following radioaffinity labelling with [3H]choline mustard aziridinium ion [( 3H]ChM Az). Membrane proteins were solubilized with 0.8% CHAPS, then analyzed by gel permeation chromatography and equilibrium density sedimentation in sucrose gradients made in H2O and D2O. The radiolabeled protein eluted from the gel filtration column with a distribution coefficient of 0.36 allowing calculation of a Stokes radius of 4.4 nm. Distribution of the [3H]ChM Az-labeled proteins differed in the H2O and D2O sucrose gradients, with apparent sedimentation coefficients of 5.2 and 4.7, respectively, indicating that detergent may be bound to hydrophobic regions of the protein. The calculated partial specific volume for the protein complex was 0.76. The estimate of molecular mass for the complex was 115,000 Da, with the protein portion having a molecular mass of about 83,000 Da. Analysis of the peak fractions from the gel filtration column and sucrose gradients by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a 3H-labeled polypeptide at 0,000-42,000 Da. The choline carrier may exist as a dimer of this polypeptide.
Rylett
R J
RJ
Department of Physiology, University of Western Ontario, London, Canada.
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification*; D002462:Cell Membrane / Q000737:chemistry; D002499:Centrifugation, Density Gradient; D002793:Cholic Acids; D002794:Choline / Q000031:analogs & derivatives / Q000378:metabolism*; D002850:Chromatography, Gel; D004557:Electric Organ / Q000737:chemistry*; D008970:Molecular Weight; D012995:Solubility; D014101:Torpedo / Q000378:metabolism*
D002352:Carrier Proteins; D002793:Cholic Acids; C013457:cyclocholine; D002794:Choline; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate