Abstract
Tonoplast, ion antiport activities are critical to ion homeostasis and sequestration in plants. The biochemical properties of these activities, and the enzymes that catalyse them, are little characterized. Here we applied biochemical approaches to study some characteristics and to distinguish between Ca 2+ /H + and Cd 2+ /H + antiporter activities of tonoplast vesicles from non-transformed, wild-type plants. Solubilization and reconstitution of oat-seedling (Avena sativa L.) root tonoplast vesicles resulted in about a 6-fold loss of protein, about a 6-fold enhancement of Cd 2+ /H + antiport specific activity (at 10 μM Cd 2+ ), and almost complete loss of Ca 2+ /H + antiport activity. Similar results were found for vesicles from mature tobacco (Nicotiana tabacum) roots. Cd 2+ concentration-dependent proton efflux was similar and linear with both oat vesicles and proteoliposomes. In contrast, Ca 2+ concentration-dependent proton efflux of oat vesicles was easily observed while that with proteoliposomes was minimal and nonlinear. Cd 2+ pre-treatment of oat vesicles reduced verapamil inhibition of Cd 2+ /H + activity and verapamil binding to vesicles, while Ca 2+ pre-treatment was much less protective of Ca 2+ /H + activity and verapamil binding. Results show the usefulness of reconstitution, and also inhibitor/ion interaction assays for distinguishing between transporter activities in vitro, but they do not resolve the question of whether there are separate enzymes for Cd 2+ /H + and Ca 2+ /H + . Our observation that solubilization and reconstitution have similar effects on both Cd 2+ /H + and Ca 2+ /H + activities of root tonoplast vesicles from immature oat and mature tobacco roots suggests that the transporters involved are similar in young and mature roots, and in roots of different species.
Published Version
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