Mg ²⁺ /KCl-ATPase of plant plasma membranes is an electrogenic pump

Abstract

The function of the Mge+-requiring KCI-stimuIated ATPase (ATP phosphohydrolase, EC 3.6.1.3) ofhigher plants in active ion transport was investigated by using a purified microsomal fraction containing sealed plasma membrane vesicles. Proc. NatL AcaS Sci USA 77, [5904][5905][5906][5907][5908]. A transmembrane electrical potential (+30 to +44 mV), monitored by uptake of a permeant anion (3SCN-), was generated specifically by ATP in Purified microsomal vesicles of tobacco callus. ATPdependent JaSCN-uptake required Mg2+, was optimal at pH 6.75, and showed similar ATP concentration dependence as the Mg2+-requiring KCI-stimulated ATPase activity. Plasma membrane ATPase inhibitors (N,N'-dicyclohexylcarbodiimide and vanadate) inhibited generation of the ATP-dependent electrical potential. A proton conductor (carbonyl cyanide m-chlorophenyihydrazone), but not a K+ ionophore (valinomycin), completely collapsed the electrical potential. The results provide in vitro evidence that the Mg24/KCI-ATPase of higher plants is an electrogenic pump. These results are consistent with the hypothesis that an electrogenic H' pump is catalyzed by the plasma membrane ATPase of plants.In higher plants, the primary active transport process is believed to be an electrogenic H+ pump (1, 2). The proton motive force generated could provide the driving force for transport of cations, anions, amino acids, and sugars, according to Mitchell's chemiosmotic principle (3). This H+ pump may be important in auxin-enhanced cell elongation, as the initial response ofcells to auxins is a stimulation of H+ secretion (4, 5). Several workers have postulated that the H+ pump is dependent on the hydrolysis ofATP catalyzed by a plasma membrane ATPase (ATP phosphohydrolase, EC 3.6.1.3) (6, 7). This hypothesis is supported by correlations observed between ATPase activities in vitro and changes of H+ fluxes and membrane potentials in intact cells (8)(9)(10)(11)(12)(13) MATERIALS AND METHODSPreparation of Sealed Microsomal Vesicles. Sealed microsomal vesicles were prepared by flotation of a microsomal fraction over a dextran pad, as described, by using autonomous tobacco callus, Nicotiana tabacum Linnaeus cv. Wisconsin no. 38 (16). These vesicles were relatively free of mitochondria, as judged by the low activity of mitochondrial enzymes (cytochrome c oxidase and malate dehydrogenase). Therefore, this fraction was referred to as purified microsomal vesicles. Protein was measured by the Lowry method using bovine serum albumin as the standard (17).ATPase Assay. ATPase activity was measured as described (16, 18) KCl-stimulated ATPase activity (KCl-ATPase) was determined as the difference in activity between Mg2+/KCl-ATPase and Mg2+-ATPase. Neither Mg2+-ATPase nor KCl-ATPase activity was affected by the presence of 1 mM KSCN (data not shown).35SCN-Uptake. mSCN-uptake was measured by a Millipore filtration method (19). SCN-uptake was initiated by adding an aliquot ofpurified microsomal vesicles to a reaction mixture. The final reaction mixture contained 25 mM ...

Localization of the Proton Pump of Corn Coleoptile Microsomal Membranes by Density Gradient Centrifugation

“…On the basis of density, it seems that all of the electrogenic proton transport activities we have measured reside on the same light membrane fraction. DISCUSSION ATP-driven proton pumping or electrogenic activity has now been detected in the microsomal membranes of a number of different plant tissues (4,6,7,14,18,25,26,28), but the subcellular origin of this activity remains unresolved. Hager and Helmle (7) speculated that the microsomal vesicles involved may actually be native vesicles derived either from the golgi and destined for the plasma membrane or from the ER in the form of 'prevacuoles.'…”

“…Hager and Helmle (7) speculated that the microsomal vesicles involved may actually be native vesicles derived either from the golgi and destined for the plasma membrane or from the ER in the form of 'prevacuoles.' Sze and Churchill (28) and Rasi-Caldogno et al (18) proposed a plasma membrane origin for the electrogenic pump. In the preceding paper (15), we used the accumulation of ['4CJmethylamine in microsomal membranes from corn coleoptiles to show that proton accumulation was insensitive to vanadate and oligomycin, stimulated by chloride, and had a pH optimum of 7.5.…”

“…5). Inasmuch as there have been recent reports of microsomal preparations containing ATP-dependent electrogenic pumps (15,18,25,26,28), the uptake of thiocyanate ([14CJSCN-) was also monitored in fractions from the dextran gradient. ATP-driven SCN-uptake was detected on the dextran gradient, and the peak of activity coincided exactly with methylamine uptake (Fig.…”

“…tissues have recently been shown to possess ATP-dependent proton pumping or electrogenic activity (4,6,7,14,18,25,26,28). In two cases, the activity was attributed either to plasma membranes (18,28) or to plasma membrane precursors (6,7).…”

“…In two cases, the activity was attributed either to plasma membranes (18,28) or to plasma membrane precursors (6,7). However, microsomal membrane preparations are enriched in a great diversity of light membrane types, including endoplasmic reticulum, golgi, and tonoplasts.…”