Purification and immunological characterization of a calcium pump from bovine brain synaptosomal vesicles.
In previous work ( Papazian , D., H. Rahamimoff , and S. M. Goldin (1979) Proc. Natl. Acad. Sci. U. S. A. 76: 3708-3712), an ATP-dependent calcium transport activity derived from rat brain synaptosomes was reconstituted into artificial lipid vesicles and substantially purified by transport-specific fractionation. When this procedure was applied to bovine brain synaptosomes, the approximately 70-fold purified, reconstituted Ca2+ uptake system contained two major polypeptides of Mr = 230,000 (" C230 ") and 94,000 (" C94 ") as observed on sodium dodecyl sulfate (SDS) gels. Evidence is presented here that these polypeptides are immunologically related to one another and that the synaptosomal Ca2+ pump is immunologically distinct from Ca2+ pumps in non-neuronal cells. Antisera and monoclonal antibodies to the purified, reconstituted protein did not significantly cross-react with the Ca2+ pumps or any other components of bovine sarcoplasmic reticulum or erythrocytes. However, these antibodies did cross-react with a component of bovine brain axolemma-enriched membranes. A monoclonal antibody was produced that immunoprecipitated the Ca2+ transport activity, both in native, synaptosomal vesicles and in liposomes containing the reconstituted transport system. This antibody bound C230 more prominently than C94 on Western blots of SDS gels. An antiserum raised against C94 alone, obtained by elution from SDS gels, was also found to bind most prominently to C230 on Western blots. These results suggest that this synaptosomal Ca2+ pump is specific to nerve tissue and that C94 and C230 are structurally homologous components of this transport activity.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D001693:Biological Transport, Active; D002118:Calcium / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D002417:Cattle; D011232:Chemical Precipitation; D003429:Cross Reactions; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D013574:Synaptosomes / Q000032:analysis* / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; D000255:Adenosine Triphosphate; D002118:Calcium