ATP‐driven Ca²⁺ transport in sealed plasma membrane vesicles prepared by aqueous two‐phase partitioning from leaves of Commelina communis

Abstract

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.

The fusicoccin receptor of plants is a member of the 14‐3‐3 superfamily of eukaryotic regulatory proteins

“…by partitioning between aqueous dextran and polyethylene glycol phases [13]. FC-binding assays were performed using [3H]9'-nor-8'-hydroxyfusicoccin as radioligand [14], plasma membrane vesicles were photoaffinity-labeled with a biologically active azido-derivative of FC, 9'-nor-8'-[(3,5-[3H]4-azidobenzoyl)diaminoethy I]-fusicoccin [6,8].…”

Identification of the Plasma Membrane Ca2+-ATPase and of Its Autoinhibitory Domain

“…So, we decided to exploit the characteristic of the PM Ca2'-ATPase as being specifically inhibited by low concentrations of fluorescein derivatives (Rasi-Caldogno et al, 1987Graf and Weiler, 1989;Williams et al, 1990;Olbe and Sommarin, 1991;Carnelli et al, 1992;De Michelis et al, 1993) and to use FITC to specifically label the PM Ca2+-ATPase. Because this modified dye binds to Lys residues of the protein in an alkali-stable covalent manner, it could serve to identify the PM Ca2+-ATPase also on SDS gels run under standard (Laemmli, 1970) conditions.…”

“…In this work, to study the effect of controlled proteolysis on the PM Ca2+-ATPase at the molecular le\rel, besides following the migration of its phosphorylated intermediate in acidic gels, we have developed methods suitable also for SDS-PAGE according to Laemmli (1970), exploiting some characteristics of the enzyme. First, based on its high sensitivity to inhibition by fluorescein tlerivatives (Rasi-Caldogno et al, 1987Graf and Weiler, 1989;Williams et al, 1990;Olbe and Sommarin, 1991;Carnelli et al, 1992;De Michelis et al, 1993), we have selectively labeled the PM Ca2+-ATPase with FITC (Dux et al, 1986). Second, based on the high affinity of the PM Ca2+-ATPase for CaM, we have labeled it with lz5I-CaM (James et al, 1989).…”

Membrane potential‐dependent calcium transport in right‐side‐out plasma membrane vesicles from Zea mays L. roots

“…Thus far, however, the majority of studies with plasma membrane vesicles have been concerned with the processes of active Ca2+ extrusion, rather than the pathways of Ca2+ influx (e.9. Evans eta/., 1991;Giannini etal., 1987;Graf and Weiler, 1989).…”