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ATP‐driven Ca 2+ transport in sealed plasma membrane vesicles prepared by aqueous two‐phase partitioning from leaves of Commelina communis
Author(s) -
Gräf Peter,
Weiler Elmar W.
Publication year - 1989
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1989.tb05611.x
Subject(s) - oligomycin , protonophore , vesicle , trifluoperazine , chemistry , vanadate , proton transport , atpase , calmodulin , electrochemical gradient , ruthenium red , biophysics , nigericin , chromatography , membrane , biochemistry , calcium , biology , organic chemistry , enzyme
Sealed plasma membrane vesicles were obtained in high purity from leaves of Commelina communis L. by aqueous two‐phase partitioning. Based on the analysis of a range of markers, the preparations (U 3 +U 3 ′ phases) were shown to be devoid of tonoplast, Golgi and thylakoid membranes, and showed only trace mitochondrial contamination. One‐third of the vesicles were oriented inside out and exhibited ATP‐driven 45 Ca 2+ transport [? 15 pkat (mg protein) −1 ]. Ca 2+ uptake into the vesicles had a pH optimum of 7.2 and apparent K m values for Ca 2+ of 4.4 μ M and for Mg‐ATP of 300 μ M . Ca 2+ uptake, K + , Mg 2+ ‐ATPase (EC 3.6.1.3) activity as well as glucan synthase II (EC 2.4.1.34) activity were all maximal at the same equilibrium density (1.17 g cm −3 ) on continuous sucrose density gradients. The protonophore carbonylcyanide m ‐chlorophenylhydrazone (CCCP) did not inhibit the ATP‐dependent Ca 2+ transport into the vesicles, excluding a Ca 2+ /H + exchange driven by a proton gradient. ATP‐dependent Ca 2+ uptake was inhibited by erythrosin B (I 50 = 0.1 μ M ), ruthenium red (I 50 = 30 μ M ), La 3+ (I 50 = 10 μ M ) and vanadate (I 50 = 500 μ M ), but not by azide, cyanide and oligomycin. The calmodulin antagonists, trifluoperazine (I 50 = 70 μ M ) and W‐7 (I 50 = 100 μ M ) were also inhibitory, However, this inhibition was not overcome by calmodulin. Trifluoperazine and W‐7, on the other hand, stimulated Ca 2+ efflux from the vesicles rather than inhibit Ca 2+ uptake. Our results demonstrate the presence of a Ca 2+ ‐ATPase in the plasma membrane of C. communis . In the intact cell, the enzyme would pump Ca 2+ out of the cell. Its high affinity for Ca 2+ makes it a likely component involved in adjusting low cytoplasmic Ca 2+ levels. No indications for a secondary active Ca 2+ /H + transport mechanism in the plasma membrane of C. communis were obtained. Both, the nucleotide specificity and the sensitivity towards vanadate. distinguish the Ca 2+ ‐ATPase from the H + ‐translocating K + . Mg 2+ ‐ATPase in C. communis plasma membranes.