Variable Effects of Nitrate on ATP-Dependent Proton Transport by Barley Root Membranes

The effects of NO(3) (-) and assay temperature on proton translocating ATPases in membranes of barley (Hordeum vulgare L. cv California Mariout 72) roots were examined. The membranes were fractionated on continuous and discontinuous sucrose gradients and proton transport was assayed by monitoring the fluorescence of acridine orange. A peak of H(+)-ATPase at 1.11 grams per cubic centimeter was inhibited by 50 millimolar KNO(3) when assayed at 24 degrees C or above and was tentatively identified as the tonoplast H(+)-ATPase. A smaller peak of H(+)-ATPase at 1.16 grams per cubic centimeter, which was not inhibited by KNO(3) and was partially inhibited by vanadate, was tentatively identified as the plasma membrane H(+)-ATPase. A step gradient gave three fractions enriched, respectively, in endoplasmic reticulum, tonoplast ATPase, and plasma membrane ATPase. There was a delay before 50 millimolar KNO(3) inhibited ATP hydrolysis by the tonoplast ATPase at 12 degrees C and the initial rate of proton transport was stimulated by 50 millimolar KNO(3). The time course for fluorescence quench indicated that addition of ATP in the presence of KNO(3) caused a pH gradient to form that subsequently collapsed. This biphasic time course for proton transport in the presence of KNO(3) was explained by the temperature-dependent delay of the inhibition by KNO(3). The plasma membrane H(+)-ATPase maintained a pH gradient in the presence of KNO(3) for up to 30 minutes at 24 degrees C.