The effect of glyceollin on soybean (Glycine max L.) tonoplast and plasma membrane vesicles

An improved method for the isolation of transport-competent plasma membrane and tonoplast vesicles from soybean (Glycine max L., Co. Harsoy) is presented. When resolved on linear sucrose gradients, the soybean membrane vesicles displayed two peaks of ATP-dependent H⁺-transport activity that were inhibited by either vanadate (plasma membrane) or nitrate (tonoplast). That the vanadate and nitrate sensitive H⁺-transport activities were representative of plasma membrane and tonoplast vesicles was further supported by their differing pH optima for ATP-dependent H⁺-transport. Membrane vesicles displaying vanadate sensitive H⁺ transport showed optimal activity at pH 6.5 while membrane vesicles displaying nitrate sensitive H⁺-transport showed optimal activity at pH 7.5. When ATP-dependent H⁺-transport was examined as a function of Mg:ATP concentration, both vesicle types displayed simple Michaelis-Menten type kinetics and K_m-values of 0.56 and 0.97 mM were observed for plasma membrane and tonoplast vesicles, respectively. Incubation of plasma membrane or tonoplast vesicles with glyceollin resulted in an inhibition of ATP-dependent H⁺-transport. While ninety percent inhibition of ATP-dependent H⁺-transport was observed with tonoplast vesicles when incubated with 25 μM glyceollin, a 4-fold higher concentration of the phytoalexin was required to produce a similar inhibition with plasma membrane vesicles. Although glyceollin had only small effects upon ATP hydrolytic activity, this compound increased the H⁺-conductance of the vesicles in a manner consistent with the relative inhibition of ATP-dependent H⁺-transport.