ATP-DEPENDENT CA2+ TRANSPORT IN WHEAT ROOT PLASMA-MEMBRANE VESICLES

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 Ca-45(2+) 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 Ca-45(2+) transport system, similar to the H+-ATPase, is located on the inner cytoplasmic surface of the plant plasma membrane. The Ca-45(2+) uptake was MgATP-dependent with an apparent K(m) for ATP of 0.1 mM and a high affinity for Ca2+ [K(m)(Ca2+/EGTA) = 3-mu-M]. 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 Ca-45(2+) uptake seen with ATP. The Ca-45(2+) uptake was stimulated by monovalent cations; K+ and Na+ being equally efficient. Vanadate inhibited the Ca-45(2+) accumulation with half-maximal inhibitions at 72, 57 and 2-mu-M for basal, total (with KCl) 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 1-mu-M. 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.