Identification of the sodium-calcium exchanger as the major ricin-binding glycoprotein of bovine rod outer segments and its localization to the plasma membrane.
After neuraminidase treatment the Na+/Ca2+ exchanger of bovine rod outer segments was found to specifically bind Ricinus communis agglutinin. SDS gel electrophoresis and Western blotting of ricin-binding proteins purified from rod outer segment membranes by lectin affinity chromatography revealed the existence of two major polypeptides of Mr 215K and 103K, the former of which was found to specifically react with PMe 1B3, a monoclonal antibody specific for the 230-kDa non-neuraminidase-treated Na+/Ca2+ exchanger. Reconstitution of the ricin affinity-purified exchanger into calcium-containing liposomes revealed that neuraminidase treatment had no significant effect on the kinetics of Na+/Ca2+ exchange activation by sodium. We further investigated the density of the Na+/Ca2+ exchanger in disk and plasma membrane preparations using Western blotting, radioimmunoassays, immunoelectron microscopy, and reconstitution procedures. The results indicate that the Na+/Ca2+ exchanger is localized in the rod photoreceptor plasma membrane and is absent or present in extremely low concentrations in disk membranes, as we have previously shown to be the case for the cGMP-gated cation channel. Previous reports describing the existence of Na+/Ca2+ exchange activity in rod outer segment disk membrane preparations may be due to the fusion of plasma membrane components and/or the presence of contaminating plasma membrane vesicles.
Reid
D M
DM
Department of Biochemistry, University of British Columbia, Vancouver, Canada.
Friedel
U
U
Molday
R S
RS
Cook
N J
NJ
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000032:analysis / Q000648:ultrastructure; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D007474:Ion Exchange; D007700:Kinetics; D008854:Microscopy, Electron; D009439:Neuraminidase / Q000378:metabolism; D010786:Photoreceptor Cells / Q000378:metabolism*; D011233:Precipitin Tests; D011863:Radioimmunoassay; D012276:Ricin / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism* / Q000648:ultrastructure; D012964:Sodium / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger
D002352:Carrier Proteins; D019831:Sodium-Calcium Exchanger; D012276:Ricin; D012964:Sodium; D009439:Neuraminidase; D002118:Calcium