Characterization of proton pumping and ATPase activities in microsomal fractions from barley roots

Abstract

Abstract The properties of proton pumping and ATPase activities of barley (Hordeum vulgare L. cv. Daisen-gold) roots were investigated using microsomal preparations and partially purified ATPase. Quenching of acridine orange fluorescence was used to measure the proton pumping activity of microsomal vesicles collected on Dextran T-70. Addition of ATP·Mg3+ caused the quenching of the fluorescence. The quenching returned to the original level by addition of protonophores ((NH4)2SO4, gramicidin). The proton pumping activity was dependent on KCl, and was inhibited by KNO3. The ATPase enzyme was solubilized from microsomal preparations with 0.2% Zwittergent 3–14, and partially purified by DEAE-cellulose column chromatography. The ATPase activity was dependent on Mg2+, and inhibited by vanadate. The activity of the purified ATPase which was derived from the plasma membranes in the microsomal preparations, was insensitive to K+, and stimulated by Ca2+ in the absence of Mg2+. These results suggest that the proton pumping activity measured was primarily derived from tonoplast vesicles, and the purified ATPase was solubilized from the plasma membrane.