Photolabeling of Tonoplast from Sugar Beet Cell Suspensions by [⁳H]5-(N-Methyl-N-Isobutyl)-Amiloride, an Inhibitor of the Vacuolar Na⁺/H⁺ Antiport

• Published in: Plant physiology (Bethesda) 1990-07, Vol.93 (3), p.924-930
• Main Authors: Barkla, Bronwyn J., Jeffrey H. M. Charuk, Cragoe, Edward J., Blumwald, Eduardo
• Format: Article
• Language: English
• Subjects: Barley; Binding sites; Biological and medical sciences; Cell membranes; Cell physiology; Fluorescence; Fundamental and applied biological sciences. Psychology; Membranes and Bioenergetics; Plant physiology and development; Plants; Plasma membrane and permeation; Protons; Sodium; Sugar beets; Tonoplast; Vacuoles
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.93.3.924

The effects of 5-(N-methyl-N-isobutyl)-amiloride (MIA), an amiloride analog, was tested on the Na+/H+ antiport activity of intact vacuoles and tonoplast vesicles isolated from sugar beet (Beta vulgaris L.) cell suspension cultures. MIA inhibited Na+/H+ exchange in a competitive manner with a Ki of 2.5 and 5.9 micromolar for ΔpH-dependent 22Na+ influx in tonoplast vesicles and Na+-dependent H+ efflux in intact vacuoles, respectively. Scatchard analysis of the binding of [3H]MIA to tonoplast membranes revealed a high affinity binding component with a Kd of 1.3 micromolar. The close relationship between the dissociation constant value obtained and the constants of inhibition for MIA obtained by fluorescence quenching and isotope exchange suggests that the high affinity component represents a class of sites associated with the tonoplast Na+/H+ antiport. Photolabeling of the tonoplast with [3H]MIA revealed two sets of polypeptides with a different affinity to amiloride and its analog.