ATP‐dependent Ca²⁺ transport in wheat root plasma membrane vesicles

Abstract

Plasma membrane preparations of high purity were obtained from roots of dark‐grown wheat (Triticum aestivum L. cv. Drabant) by aqueous polymer two‐phase partitioning. These preparations mainly contained sealed, right‐side‐out vesicles (ca 90% exposing the original outside out). By subjecting the preparations to 4 freeze/thaw cycles the proportion of sealed, inside‐out (cytoplasmic side out) vesicles increased to ca 30%. Inside‐out and right‐side‐out plasma membrane vesicles were then separated by partitioning the freeze/thawed plasma membranes in another aqueous polymer two‐phase system. In this way, highly purified, sealed, inside‐out (>60% inside‐out) vesicles were isolated and subsequently used for characterization of the Ca2+ transport system in the wheat plasma membrane. The capacity for 45Ca2+ accumulation, nonlatent ATPase activity and proton pumping (the latter two markers for inside‐out plasma membrane vesicles) were all enriched in the inside‐out vesicle fraction as compared to the right‐side‐out fraction. This confirms that the ATP‐binding site of the 45Ca2+ transport system, similar to the H+‐ATPase, is located on the inner cytoplasmic surface of the plant plasma membrane. The 45Ca2+ uptake was MgATP‐dependent with an apparent Km for ATP of 0.1 mM and a high affinity for Ca2+ [Km(Ca2+/EGTA) = 3 μM]. The pH optimum was at 7.4–7.8. ATP was the preferred nucleotide substrate with ITP and GTP giving activities of 30–40% of the 45Ca2+ uptake seen with ATP. The 45Ca2+ uptake was stimulated by monovalent cations; K− and Na+ being equally efficient. Vanadate inhibited the 45Ca2+ accumulation with half‐maximal inhibitions at 72, 57 and 2 μM for basal, total (with KCI) and net K+‐stimulated uptake, respectively. The system was also highly sensitive to erythrosin B with half‐maximal inhibition at 25 nM and total inhibition at 1μM. Our results demonstrate the presence of a primary Ca2+ transport ATPase in the plasma membrane of wheat roots. The enzyme is likely to be involved in mediating active efflux (ATP‐binding sites on the cytoplasmic side) to the plant cell exterior to maintain resting levels of cytoplasmic free Ca2+ within the cell.

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).…”

Polyphosphoinositide Phospholipase C in Plasma Membranes of Wheat (Triticum aestivum L.)

“…To examine further whether polyphosphoinositide phospholipase C is localized in the cytoplasmic leaflet of the plasma membrane, we used the recently developed method to produce and subsequently separate inside-out and rightside-out plasma membrane vesicles (11,19,21). The two vesicle populations were assayed for proton pumping and nonlatent ATPase activities, which are both considered to be markers for the cytoplasmic surface of the plasma membrane (27,34) and for polyphosphoinositide phospholipase C activity.…”

“…Highly purified plasma membranes were isolated from the microsomal fraction (10,000-30,000g pellet) of wheat shoots and roots by partitioning in aqueous polymer two-phase systems as described earlier (28) with minor modifications (22 (11,19,21). As when isolating the original plasma membrane fraction, the rightside-out vesicles partitioned to the upper phase.…”

Cadmium sorption to plasma membrane isolated from barley roots is impeded by copper association onto membranes

“…Several types of mineral sorption experiments employing PM preparations of 62 plant tissue have been conducted [1][2][3][4]. Isolated PM preparations consist of the lipid 63 bilayer with PM localized proteins, such as ATPase or transporters, and form into 64 vesicles [1,5].…”