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

Abstract

ABSTRACITThe effects of NO3-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 KN03 when assayed at 24°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 KN03 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 KNO3 inhibited ATP hydrolysis by the tonoplast ATPase at 12°C and the initial rate of proton transport was stimulated by 50 millimolar KNO3. The time course for fluorescence quench indicated that addition of ATP in the presence of KN03 caused a pH gradient to form that subsequently collapsed. This biphasic time course for proton transport in the presence of KN03 was explained by the temperature-dependent delay of the inhibition by KNO3. The plasma membrane H'-ATPase maintained a pH gradient in the presence of KN03 for up to 30 minutes at 24°C.The characteristic that is used most frequently to identify the tonoplast ATPase is that the enzyme is inhibited by NO3-but not by vanadate or azide (3, 5, 9, 19-21, 24, 26, 31). Other inhibitors ofthe tonoplast ATPase, such as DCCD, are less useful, because they affect all of the proton-translocating ATPases (31).However, the effects of NO3-on the tonoplast H+-ATPase are complex (4, 18) and NO3-inhibits other ATPases as well. For example, a putative Golgi membrane H+-ATPase was partially inhibited by N03 (7) and the mitochondrial F,F0-ATPase was inhibited by NO3- (27,31). It is important to clarify the effects of nitrate on the tonoplast ATPase for several reasons. The criteria for using NO3-to identify the tonoplast ATPase need to be defined, particularly since there is some confusion in the literature between the effects of NO3-as an inhibitor of the ATPase and as a substrate for a NO3-/H' symport (4). The effect of NO3 on the tonoplast ATPase in vivo also needs to be elucidated. The physiological significance of the N03 -inhibition is unclear and puzzling, because the vacuole stores nitrate (23,29). Nitrate inhibits the mitochondrial F,F0-ATPase and the tonoplast H+-ATPase with a Ki of only 5 to 10 mm (27,31) although it was estimated that the cytoplasmic concentration of nitrate was approximately 4 mM and the vacuolar concentration was greater than 40 mm for barley leaf protoplasts (23).Barley roots have been used extensively for studies of ion transport and nitrogen metabolism, but until recently it has been difficult to obtain membrane preparations from barley that are suitable for studies of ion...

Rapid Induction of Na+/H+ Exchange Activity in Barley Root Tonoplast

“…A tonoplast-enriched membrane fraction was isolated as described previously (6,9). Roots (10 gm) were excised into ice water, rinsed, and then ground in homogenization buffer (67 mL) containing 250 mm sucrose, 50 mm Tris, 8 mM EDTA, and 4 mm DTT (pH 8.0).…”

“…The generation and dissipation of pH gradients across the membrane vesicles were followed by measuring quenching and recovery of acridine orange fluorescence as described previously (6,9). The assay buffer contained 250 mM sorbitol, 50 mm choline-Cl, 1 mM MgCl2, 1 mm EGTA, 2.5 1M acridine orange, and 5 mM Mes adjusted to pH 7.8 with Tris.…”

“…The generation and dissipation of pH gradients across the membrane vesicles were followed by measuring quenching and recovery of acridine orange fluorescence as described previously (6,9 …”

Carbohydrate transport in discs of storage parenchyma of celery petioles

“…The fact that the permeant NEM (and CCCP) displayed a greater inhibition than the relatively non-permeant PCMBS allows the speculation that two different mannitol carriers might be operative in celery petiole parenchyma, one at the plasmalemma and one at the tonoplast. The observed inhibition by KNO3 (50 mM) on mannitol uptake (31 %) could be indirect support of such an additional (active) tonoplastlocated carrier as KNO3 is known to inhibit mainly tonoplast H+-ATPase (Sze, 1985;DuPont, 1987). Alternatively, the strong inhibition of NEM (and CCCP) as compared to PCMBS could also be interpreted as indicates the presence of SH-groups of a plasmalemma mannitol-carrier on both sides of the membrane.…”

Evidence for a Highly Specific K+/H+ Antiporter in Membrane Vesicles from Oil-Seed Rape Hypocotyls

“…No sensitivity to vanadate was observed. The antiporter activity observed in these fractions was thus likely to be associated with the tonoplast (9,17).…”