The use of reconstitution and inhibitor/ion interaction assays to distinguish between Ca2+/H+and Cd2+/H+ antiporter activities of oat and tobacco tonoplast vesicles |
| |
Authors: | Victor D. Koren'kov Ryan W. Shepherd Georg J. Wagner |
| |
Affiliation: | Plant Physiology/Biochemistry/Molecular Biology Program, Department of Agronomy, University of Kentucky, Lexington, KY 40546-0091, USA |
| |
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 Ca2+/H+ and Cd2+/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 Cd2+/H+ antiport specific activity (at 10 µM Cd2+), and almost complete loss of Ca2+/H+ antiport activity. Similar results were found for vesicles from mature tobacco (Nicotiana tabacum) roots. Cd2+ concentration‐dependent proton efflux was similar and linear with both oat vesicles and proteoliposomes. In contrast, Ca2+ concentration‐dependent proton efflux of oat vesicles was easily observed while that with proteoliposomes was minimal and non‐linear. Cd2+ pre‐treatment of oat vesicles reduced verapamil inhibition of Cd2+/H+ activity and verapamil binding to vesicles, while Ca2+ pre‐treatment was much less protective of Ca2+/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 Cd2+/H+ and Ca2+/H+. Our observation that solubilization and reconstitution have similar effects on both Cd2+/H+ and Ca2+/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. |
| |
Keywords: | |
|
|