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- Title
ATP-dependent Ca<sup>2+</sup> transport in wheat root plasma membrane vesicles.
- Authors
Olbe, Malin; Sominarin, Marianne
- 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 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 vCa2+ 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.
- Subjects
ADENOSINE triphosphatase; CELL membranes; WHEAT; POLYMERS; PLANT plasma membranes; PHOSPHATASES
- Publication
Physiologia Plantarum, 1991, Vol 83, Issue 4, p535
- ISSN
0031-9317
- Publication type
Article
- DOI
10.1111/j.1399-3054.1991.tb02465.x