Body plasma membrane vesicles from Paramecium contain a vanadate-sensitive Ca(2+)-ATPase.
Paramecia are an excellent model system for studying the mechanisms involved in sensory transductions and intracellular Ca2+ regulation. These cells have two functionally distinct plasma membrane domains, body and cilia. The body plasma membrane is responsible for transduction of sensory stimuli into receptor potentials and the ciliary membrane is required for Ca2+ action potentials. Although ciliary membrane vesicles (cmv) have been purified and well characterized, body plasma membranes have not. We have generated body plasma membrane vesicles (bmv) by homogenization of deciliated cells and purified them from the microsome fraction by a two-phase aqueous polymer separation. The major criteria for purity of the bmv fraction are: (i) It is enriched 15-fold for a known plasma membrane marker (immobilization antigen) while the marker activities for other membranes were all decreased. The protein banding pattern of bmv is generally similar to cmv on SDS-PAGE. (ii) It contains a vanadate-sensitive Ca(2+)-ATPase activity that has been suggested to be a plasma membrane Ca2+ pump. The specific activity of this bmv Ca(2+)-ATPase is increased 4-fold over that of the homogenate. (iii) The phospholipid, fatty acid, and sterol composition of the bmv fraction are indicative of plasma membranes because they are qualitatively similar to cmv. The bmv also contains a membrane-bound NADPH-dependent cytochrome c reductase activity, suggesting that it may play a role in body plasma membrane function. This purified bmv preparation is useful for studying the role of the body plasma membrane in Ca2+ regulation, sensory transduction, protein and lipid trafficking, and plasma membrane fusion events.
Department of Biological Sciences, State University of New York, Buffalo 14260-1300.
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Smith
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Hennessey
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000032:analysis*; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology; D005227:Fatty Acids / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis; D016805:Paramecium tetraurelia / Q000201:enzymology* / Q000648:ultrastructure*; D010743:Phospholipids / Q000032:analysis; D012680:Sensitivity and Specificity; D013261:Sterols / Q000032:analysis; D014638:Vanadates / Q000494:pharmacology*
D005227:Fatty Acids; D008565:Membrane Proteins; D010743:Phospholipids; D013261:Sterols; D014638:Vanadates; D000252:Calcium-Transporting ATPases