The Ca2+ Pump of the Plasma Membrane of Arabidopsis thaliana: Characteristics and Sensitivity to Fluorescein Derivatives

Abstract

AbstractThe transport and hydrolytic activities of the plasma membrane (PM) Ca2+ pump were characterized in a PM fraction purified from seedlings of Arabidopsis thaliana by the aqueous two‐phase partitioning technique.Ca2+ uptake could be energized by ATP and by ITP (at about 70% the rate sustained by ATP). This characteristic was used to measure the hydrolytic activity of the enzyme as Ca2+‐dependent ITPase activity. The PM Ca2+ pump displayed a broad pH optimum around pH 7.2, was drastically inhibited by erythrosin B (EB), and was half‐saturated by 60 μM ITP. It was stimulated by CaM, specially at low, non‐saturating Ca2+ concentrations. All of these characteristics closely resemble those of the PM Ca2+ pump in other plant materials.Analysis of the effects of EB and other fluorescein derivatives (eosin Y and rose bengal) showed that: i) EB behaved as a competitive inhibitor with respect to ITP; ii) the PM Ca2+ pump was drastically inhibited by concentrations of fluorescein derivatives (submicromolar), much lower than those required to inhibit the PM H+‐ATPase; iii) the different fluorescein derivatives were diversely efficient in inhibiting the activities of the Ca2+ pump and of the H+‐ATPase of the PM (eosin Y was about 10000‐fold, EB 1000‐fold and rose bengal only 50‐fold more active on the Ca2+ pump than on the H+‐ATPase); and iv) the effectiveness of EB in inhibiting the Ca2+ pump was strongly affected by the protein concentration in the assay medium.