Calmodulin-stimulated Ca(2+)-ATPases in the vacuolar and plasma membranes in cauliflower.
The subcellular locations of Ca(2+)-ATPases in the membranes of cauliflower (Brassica oleracea L.) inflorescences were investigated. After continuous sucrose gradient centrifugation a 111-kD calmodulin (CaM)-stimulated and caM-binding Ca(2+)-ATPase (BCA1; P. Askerlund [1996] Plant Physiol 110: 913-922; S. Malmström, P. Askerlund, M.G. Plamgren [1997] FEBS Lett 400: 324-328) comigrated with vacuolar membrane markers, whereas a 116-kD caM-binding Ca(2+)-ATPase co-migrated with a marker for the plasma membrane. The 116 kD Ca(2+)-ATPase was enriched in plasma membranes obtained by aqueous two-phase partitioning, which is in agreement with a plasma membrane location of this Ca(2+)-ATPase. Countercurrent distribution of a low-density intracellular membrane fraction in an aqueous two-phase system resulted in the separation of the endoplasmic reticulum and vacuolar membranes. The 111-kD Ca(2+)-ATPase co-migrated with a vacuolar membrane marker after countercurrent distribution but not with markers for the endoplasmic reticulum. A vacuolar membrane location of the 111-kD Ca(2+)-AtPase was further supported by experiments with isolated vacuoles from cauliflower: (a) Immunoblotting with an antibody against the 111-kD Ca(2+)-ATPase showed that it was associated with the vacuoles, and (b) ATP-dependent Ca2+ uptake by the intact vacuoles was found to be CaM stimulated and partly protonophore insensitive.
Askerlund
P
P
Department of Plant Biochemistry, Lund University, Sweden. per.askerlund@plantbio.lu.se
D000252:Calcium-Transporting ATPases / Q000302:isolation & purification / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D004533:Egtazic Acid / Q000494:pharmacology; D014617:Vacuoles / Q000201:enzymology* / Q000648:ultrastructure; D014675:Vegetables / Q000201:enzymology*
D002147:Calmodulin; D004533:Egtazic Acid; D000252:Calcium-Transporting ATPases