CHARACTERIZATION OF PROTON PUMPING AND ATPase ACTIVITIES IN MICROSOMAL FRACTIONS FROM BARLEY ROOTS

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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・Mg^<2+> caused the quenching of the fluorescence. The quenching returned to the original level by addition of protonophores ((NH_4)_2SO_4, gramicidin). The proton pumping activity was dependent on KCl, and was inhibited by KNO_3. 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 Mg^<2+>, 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 Ca^<2+> in the absence of Mg^<2+>. 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.
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