ISSN:
1399-3054
Source:
Blackwell Publishing Journal Backfiles 1879-2005
Topics:
Biology
Notes:
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 (U3+U3′ 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 45Ca2+ transport [? 15 pkat (mg protein)−1]. Ca2+ uptake into the vesicles had a pH optimum of 7.2 and apparent Km values for Ca2+ of 4.4 μM and for Mg-ATP of 300 μM. Ca2+ uptake, K+, Mg2+-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 Ca2+ transport into the vesicles, excluding a Ca2+/H+ exchange driven by a proton gradient. ATP-dependent Ca2+ uptake was inhibited by erythrosin B (I50= 0.1 μM), ruthenium red (I50= 30 μM), La3+ (I50= 10 μM) and vanadate (I50= 500 μM), but not by azide, cyanide and oligomycin. The calmodulin antagonists, trifluoperazine (I50= 70 μM) and W-7 (I50= 100 μM) were also inhibitory, However, this inhibition was not overcome by calmodulin. Trifluoperazine and W-7, on the other hand, stimulated Ca2+ efflux from the vesicles rather than inhibit Ca2+ uptake. Our results demonstrate the presence of a Ca2+-ATPase in the plasma membrane of C. communis. In the intact cell, the enzyme would pump Ca2+ out of the cell. Its high affinity for Ca2+ makes it a likely component involved in adjusting low cytoplasmic Ca2+ levels. No indications for a secondary active Ca2+/H+ transport mechanism in the plasma membrane of C. communis were obtained. Both, the nucleotide specificity and the sensitivity towards vanadate. distinguish the Ca2+-ATPase from the H+-translocating K+. Mg2+-ATPase in C. communis plasma membranes.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1111/j.1399-3054.1989.tb05611.x
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