Calmodulin-stimulated Ca2+-ATPases in the vacuolar and plasma membranes in cauliflower

• The subcellular locations of Ca2+-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 Ca2+-ATPase (BCA1; P. Askerlund [1996] Plant Physiol 110: 913–922; S. Malmstrom, P. Askerlund, M.G. Palmgren [1997] FEBS Lett 400: 324–328) comigrated with vacuolar membrane markers, whereas a 116-kD CaM-binding Ca2+-ATPase co-migrated with a marker for the plasma membrane. The 116-kD Ca2+-ATPase was enriched in plasma membranes obtained by aqueous two-phase partitioning, which is in agreement with a plasma membrane location of this Ca2+-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 Ca2+-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 Ca2+-ATPase was further supported by experiments with isolated vacuoles from cauliflower: (a) Immunoblotting with an antibody against the 111-kD Ca2+-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.

Subject headings

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Keyword

Calcium-Transporting ATPases/isolation & purification/*metabolism

Calmodulin/*pharmacology

Cell Fractionation

Cell Membrane/enzymology/ultrastructure

Centrifugation; Density Gradient

Egtazic Acid/pharmacology

Vacuoles/*enzymology/ultrastructure

Vegetables/*enzymology

Publication and Content Type

ref (subject category)

art (subject category)