Density gradient localization of plasma membrane and tonoplast from storage tissue of growing and dormant red beet : characterization of proton-transport and ATPase in tonoplast vesicles

Abstract

Membranes from homogenates of growing and of dormant storage roots of red beet (Beta vulgaris L.) were centrifuged on linear sucrose gradients. Vanadate-sensitive ATPase activity, a marker for plasma membrane, peaked at 38% to 40% sucrose (1.165-1.175 grams per cubic centimeter) in the case of growing material but moved to as low as 30% sucrose (1.127 grams per cubic centimeter) during dormancy.A band of nitrate-sensitive ATPase was found at sucrose concentrations of 25% to 28% or less (around 1.10 grams per cubic centimeter) for both growing and dormant material. This band showed proton transport into membrane vesicles, as measured by the quenching of fluorescence of acridine orange in the presence of ATP and Mg(2+). The vesicles were collected on a 10/23% sucrose step gradient. The phosphate hydrolyzing activity was Mg dependent, relatively substrate specific for ATP (ATP > GTP > UTP > CTP = 0) and increased up to 4-fold by ionophores. The ATPase activity showed a high but variable pH optimum, was stimulated by Cl(-), but was unaffected by monovalent cations. It was inhibited about 50% by 10 nanomolar mersalyl, 20 micromolar N,N'-dicyclohexylcarbodiimide, 80 micromolar diethylstilbestrol, or 20 millimolar NO(3) (-); but was insensitive to molybdate, vanadate, oligomycin, and azide. Proton transport into vesicles from the 10/23% sucrose interface was stimulated by Cl(-), inhibited by NO(3) (-), and showed a high pH optimum and a substrate specificity similar to the ATPase, including some proton transport driven by GTP and UTP.The low density of the vesicles (1.10 grams per cubic centimeter) plus the properties of H(+) transport and ATPase activity are similar to the reported properties of intact vacuoles of red beet and other materials. We conclude that the low density, H(+)-pumping ATPase of red beets originated from the tonoplast. Tonoplast H(+)-ATPases with similar properties appear to be widely distributed in higher plants and fungi.