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ATP‐dependent Ca 2+ transport in wheat root plasma membrane vesicles
Author(s) -
Olbe Malin,
Sommarin Marianne
Publication year - 1991
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1991.tb02465.x
Subject(s) - vesicle , membrane , chemistry , atpase , egta , biophysics , chromatography , substrate (aquarium) , biochemistry , cytoplasm , gtp' , calcium , enzyme , biology , organic chemistry , ecology
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 Ca 2+ transport system in the wheat plasma membrane. The capacity for 45 Ca 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 45 Ca 2+ transport system, similar to the H + ‐ATPase, is located on the inner cytoplasmic surface of the plant plasma membrane. The 45 Ca 2+ uptake was MgATP‐dependent with an apparent K m for ATP of 0.1 m M and a high affinity for Ca 2+ [K m (Ca 2+ /EGTA) = 3 μ 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 45 Ca 2+ uptake seen with ATP. The 45 Ca 2+ uptake was stimulated by monovalent cations; K − and Na + being equally efficient. Vanadate inhibited the 45 Ca 2+ accumulation with half‐maximal inhibitions at 72, 57 and 2 μ M 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 n M and total inhibition at 1μ M . Our results demonstrate the presence of a primary Ca 2+ 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 Ca 2+ within the cell.