Functional and Regulatory Properties of H+ Pumps at the Tonoplast and Plasma Membranes of Zea mays Coleoptiles

Abstract

Abstract A microsomal membrane fraction (6000 x g supernatant of a cell homogenate), isolated from coleoptiles of Zea mays, was separated by isopycnic sucrose density gradient centrifugation in the presence of EDTA and without a prior pelleting step to avoid irreversible sticking of different membrane species. The membrane fractions were characterized by assaying commonly used marker enzymes, and the levels of activity investigated of ATP hydrolysis, ATP-dependent H+ transport, and co- and countertransport of ions, such as Cl- , fumarate2-, K+ and Li+. The following results were obtained: (1) ATP hydrolysis is performed by different enzymes associated with different membranes: - vacuolar acid phosphatase (AP; inhibited by molybdate); - Golgi phosphatase, revealing IDPase, pNPase and ATPase activity (not inhibited by molybdate); - ATPase activity of residual submitochondrial particles (sensitive to azide); - a H+-translocating ATPase at tonoplast membranes (Km(ATP) = 0.29 mᴍ; pH 7.5; stimulated by uncouplers and completely inhibited by NO- 3); - the H+-translocating ATPase of the plasmalemma (Km(ATP) = 0.39 mᴍ at pH 6.5, inhibited by vanadate, but not by NO- 3). The latter activity is evident only after an osmotic shock, indicating that PL vesicles primarily exist as inside-in-vesicles. (2) ATP-fueled H+ pumps are localized at tonoplast (TO) and plasmalemma (PL) vesicles, they differ to some extent in their properties: (a) The PLH+ pump has a very narrow pH optimum and exhibits highest levels of activity at pH 6.5 with a pronounced increase of activity between pH 7.5 and 6.5 (properties, obviously important in vivo for the regulation of active H+-extrusion by certain growth substances, which affect the cytoplasmic pH (Hager and Moser, Planta 1984, in press); in contrast, TO H+ pumps show a considerably wider pH optimum with highest levels of activity around pH 7.5. (b) In variance to the PL H+ pump the activity of TO pumps (Km(ATP) = 0.24 mᴍ) is regulated via the oxidation state of essential thiol groups. Their oxidation to the S -S - form (e.g. by blue light in the presence of a flavin) causes an inactivation, whereas a re-reduction by GSH or cystein restores the activity [51]. (c) The ATP-fueled H+ transport into TO vesicles depends on an anion co-transport; most effective is Cl- , but there is also a stimulation by organic ions, C4 and C5 dicarboxylates, such as malate, succinate, fumarate, 2-oxoglutarate and aspartate; NO- 3 is inhibitory. (d) H+-transport into sealed PL vesicles is also anion dependent. In this case, however, NO- 3 is as effective as Cl-. (3) The TO membranes contain a H+/K+ exchange mechanism responsible for a secondary active K+ uptake into the vacuole. This mechanism could be the reason for a lower (ATP dependent) acidification of TO vesicles in the presence of K+ compared with Li+. - Similar effects are observed with plasmamembrane vesicles, but in this case there is still the question whether a H+/K+-exchange, a K+ channel, or both are acting.