Determination of H+/ATP Stoichiometry for thePlasma Membrane … · the measurement of ApH. At this point, ATP-driven H+ transport wasstoppedbychelating Mg2` with 10 mmEDTA (BTPsalt,

Plant Physiol. (1991) 95, 242-2500032-0889/91/95/0242/09/$01 .00/0

Received for publication June 21, 1990Accepted September 29, 1990

Determination of H+/ATP Stoichiometry for the PlasmaMembrane H -ATPase from Red Beet (Beta vulgaris L.)

Storage Tissue1

Donald P. Briskin* and Ingrid Reynolds-Niesman2Department of Agronomy, University of Illinois, Urbana, Illinois 61801

ABSTRACT

The H+/ATP stoichiometry was determined for the plasmamembrane H+-ATPase from red beet (Beta vulgaris L., var DetroitDark Red) storage tissue associated with native vesicles. Thedetermination of H+/ATP stoichiometry utilized a kinetic approachwhere rates of H+ influx, estimated by three different methods,were compared to rates of ATP hydrolysis measured by thecoupled enzyme assay under identical conditions. These meth-ods for estimating H+ influx were based upon either determiningthe initial rate of alkalinization of the extemal medium from pH6.13, measuring the rate of vesicle H+ leakage from a steady-state pH gradient after stopping the H+-ATPase or utilizing amathematical model which describes the net transport of H+ atany given point in time. When the rate of H+ influx estimated byeach of these methods was compared to the rate of ATP hydrol-ysis, a H+/ATP stoichiometry of about 1 was observed. In consid-eration of the maximum free energy available from ATP hydrolysis(AGATP), this value for H+/ATP stoichiometry is sufficient to ac-count for the magnitude of the proton electrochemical gradientobserved across the plasma membrane in vivo.

Through in vitro studies with isolated plasma membranevesicles (1 1, 13) and reconstituted enzyme systems (7, 21, 30,32), it has been demonstrated that the plasma membrane H+-ATPase has the capability of coupling ATP hydrolysis tovectorial H+-translocation. In vivo, it is proposed that thisactivity results in the production of a proton electrochemicalgradient across the plasma membrane consisting of both an

interior-negative membrane potential and acid-exterior pHdifference (16, 18, 31 and references therein). This inwardlydirected proton electrochemical gradient can be utilized forthe transport of solutes across the plasma membrane bycarriers acting as either H+/solute symports, H+/solute anti-ports, or membrane potential-driven uniports (16 and refer-ences therein).

Studies on the biochemical characteristics of the plantplasma membrane H+-ATPase have indicated that this en-

zyme is representative of the E1E2 class of transport ATPase(23). As with other enzymes of this type, the plant plasma

' Supported by U. S. Department ofAgriculture Competitive GrantAG88-37261-3555 awarded to D. P. B.

2 Present address: Department of Physiology, Louisiana State Uni-versity School of Medicine, New Orleans, LA 70065.

membrane H+-ATPase forms a covalent phosphorylated in-termediate during the course of ATP hydrolysis (8, 9) on anessential aspartyl residue in the active site of the enzyme (10,34). A further similarity to other transport ATPases of theEIE2 type regards the structure ofthe plant plasma membraneH+-ATPase where the functional unit of ATPase activity isassociated with a 100 kD catalytic subunit (8, 9). Recentefforts by several research groups (5, 15, 22) have resulted inthe successful cloning of the plasma membrane H+-ATPasegene and the deduction of the primary amino acid sequencefor this protein. Of prime importance will be developing anunderstanding of structure/function relationships for themechanism of energy coupling to H+-transport as mediatedby this protein.An important parameter associated with the H+-transport

function of the plasma membrane H+-ATPase is the stoichi-ometric relationship between the number of H+ transportedand ATP molecules hydrolyzed. This H+/ATP value sets anupper limit to the magnitude of the proton electrochemicalgradient that can be established across the plasma membrane(25, 28) and would be an important consideration in thedevelopment of mechanistic models to explain the catalytic/transport function of this enzyme.

In the determination of H+/ATP stoichiometry for an H+-ATPase, either a thermodynamic or kinetic approach couldbe utilized (2 and references therein). The thermodynamicapproach relies upon a comparison of the free energy ofATPhydrolysis (AGATP) with the magnitude of the maximumproton electrochemical gradient (AuH+) established accordingto: AGATP 2 n (AAsH+) where n equals the H+/ATP stoichi-ometry (4, 20, 24). As this method requires an accurateestimate of AGATP and is strongly influenced by the passiveH+ conductance ofthe membrane (24 and references therein),kinetic approaches have frequently been used to determineH+/ATP stoichiometry for H+-ATPases associated with iso-lated membrane vesicle fractions where the passive H+ con-ductance can be substantial (4, 7, 24). In the kinetic approach,H+/ATP stoichiometry is estimated by a comparison of therate of H+-translocation to the rate of ATP hydrolysis. Esti-mates of H+-translocation rate have been based upon eithermeasuring external pH change in a weakly buffered mediumusing a pH electrode (4, 29) or pH sensing dye (27), measuringthe rate ofH+ leakage from vesicles after the establishment ofa steady-state pH gradient using a ApH sensing fluorescentprobe (24) or utilizing a mathematical model describing thetimecourse ofH+ transport from initiation (by ATP addition)

242

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H+/ATP STOICHIOMETRY OF PLASMA MEMBRANE ATPase

to establishment of a steady-state pH gradient (7 and refer-ences therein).

In this communication, H+/ATP stoichiometry was deter-mined for the plasma membrane H+-ATPase associated withred beet plasma membrane vesicles where the H+-transportrate was estimated by all three methods described above. Witheach method utilized, a stoichiometry of about 1 H+ trans-ported per ATP hydrolyzed was observed for this enzymepreparation.

MATERIALS AND METHODS

Plant Material

Red beets (Beta vulgaris L., cv Detroit Dark Red) werepurchased commercially. The tops ofthe plants were removedand the storage tissue was stored at 2°C for at least 10 d beforeuse to ensure uniformity in membrane isolation (26).

Isolation of Plasma Membrane Vesicles

Plasma membrane vesicles were isolated from red beetstorage tissue as previously described (13). Briefly, tissuesections were peeled, cut into cubes and then vacuum infil-trated for 5 min in ice cold homogenization medium (1.5:1,medium:tissue) containing 250 mm sucrose, 2 mM EDTA,2 mm Na2ATP, 1% (w/v) BSA (fraction V powder), 0.5%(w/v) polyvinylpyrolidone (40,000 mol wt), 0.2 mM PMSF,15 mm f3-mercaptoethanol, 4 mm DTE, 10% (v/v) glycerol,70 mM Tris-HCl (pH 8.0), and 250 mM KI. Dithioerythreitol,f,-mercaptoethanol, ATP, and PMSF were added to the me-dium just before use. Following vacuum infiltration, the tissuewas homogenized in the medium using a vegetable juiceextractor and the homogenate was filtered through four layersof cheesecloth prior to centrifugation at 13,000g (8,500 rpm)for 15 min in a Sorvall GSA rotor. The 13,000g pellet wasdiscarded and the supernatant was centrifuged at 80,000g(32,000 rpm) in a Beckman type 35 rotor for 30 min. Theresulting microsomal pellet was suspended in a suspensionbuffer consisting of 250 mm sucrose, 10% (v/v) glycerol, 2mM BTP/Mes (pH 7.2), and 1 mM DTE. The suspendedmembranes were then layered onto a discontinuous sucrosedensity gradient consisting of26% (w/w) sucrose layered over38% (w/w) sucrose. The gradient solutions were buffered with1 mm Tris/Mes (pH 7.2) and contained 1 mM DTE. Thegradients were centrifuged at 100,000g (25,000 rpm) for 2 hin a Beckman SW 28 rotor. Membranes recovered at the 26/38% interface were removed using a Pasteur pipet, dilutedwith suspension buffer to 70 mL, and then centrifuged at80,000g (32,000 rpm) for 30 min in a Beckman type 35 rotor.The pellet was suspended at a protein concentration of ap-proximately 4 mg/mL, frozen under liquid nitrogen, and thenstored at -80°C until use.

Measurement of the Initial Rate of H+ Transport

The initial rate of proton transport was determined in a 1mL reaction volume containing 250 mm sorbitol, 3.0 mMMgSO4, 3.0 mm ATP, 0.5 mM BTP3/Mes (pH 6.13), 50 mM

'Abbreviations: BTP, bistris propane; EPR, electron paramagneticresonance.

KNO3, 15 yM pyranine, and 220 jAg of membrane protein.For the routine measurement of H+ transport, 950 ,uL of anassay solution containing all components except ATP wascarefully adjusted to pH 6.13 by the addition of either 1 mMHCI or NaOH. To initiate H+ transport, 50,L of60 mM ATP(Na+ salt, pH to 6.13 with HCl) was added to the reactionmixture and the increase in optical density was determined at450 nm. Calibration ofH+ transport was carried out by adding10,uL aliquots of 2 mm HCI and observing the correspondingdecrease in optical density.

Measurement of ApH

The development ofa proton gradient (ApH) was measuredat 22°C by the quenching of acridine orange fluorescence.The standard assay contained 250 mM sorbitol, 25 mM BTP/Mes (pH 6.5), 50 mm KCI, 5 uM acridine orange, 3.0 mMMgSO4, 3.0 mm ATP (BTP salt, pH 6.5), and 70 ,ug ofmembrane protein in a 1 mL reaction volume. The reactionwas initiated by the addition ofATP and the fluorescence wasmonitored using a Perkin-Elmer model 203 spectrofluorom-eter with the excitation monochronometer set at 472 nm andthe emission monochronometer set at 525 nm. To determinethe magnitude of the steady-state pH gradient, 10 Mm grami-cidin D was added when fluorescence quenching reached thefinal steady-state value and the increase in fluorescence wasnoted. To calibrate fluorescence quenching with the magni-tude of ApH, plasma membrane vesicles in 250 mM sorbitol,1 mm BTP/Mes/formate, 1 mM DTE were equilibrated atdifferent pH values by using a slow freeze/thaw method (14).The vesicles were then added to the standard fluorescenceassay medium (-ATP) the magnitude of the fluorescencequench was noted following the addition of 20 MuL of 1 MNH4Cl.

Estimation of pH Gradient Relaxation Rate

To estimate the rate of pH gradient decay, plasma mem-brane vesicles were allowed to develop a steady-state pHgradient under the standard assay conditions described forthe measurement of ApH. At this point, ATP-driven H+transport was stopped by chelating Mg2` with 10 mm EDTA(BTP salt, pH 6.5) and the decay rate of the pH gradient wasmeasured as the increase in acridine orange fluorescence overtime. The calibration curve described above was then used toconvert the fluorescence decay rate into units of pH units/min assuming that the external pH remains constant at 6.5(see ref. 24).

Measurement of Vesicle Internal Buffering Capacity

Internal buffering capacity was determined by the methodof Maloney (17) as modified for the red beet plasma mem-brane vesicles. Plasma membrane vesicles were diluted 40-fold in unbuffered 100 mM KCI, and then centrifuged for 1 hat 100,000g (41,000 rpm) in a Beckman type 50 rotor. Thiswashing procedure was repeated two times and the vesicleswere suspended in 150 mm KC1 at a protein concentration of1.0 mg/mL. The final vesicle suspension was purged with N2gas and the pH adjusted to a range of values from 4.5 to 6.5.

243



BRISKIN AND REYNOLDS-NIESMAN

In a vessel with C02-free air blown over the surface, knownquantities of HCI (25-100 nmol) were added and the changein external pH was measured with a pH electrode. Followinganalysis of the data according to Maloney (17) the bufferingcapacity was calculated to be 0.237 ,umol/pH unit. mg proteinover this pH range (average of five separate determinationsover the pH range).

Measurement of Vesicle Internal Volume

The internal volume of red beet plasma membrane vesicleswas measured by EPR as previously described (19) usingperdeuterated tempone. Plasma membrane vesicles in suspen-sion buffer (1.3 mg/mL protein) were diluted with an equalvolume of 140 mm NaCl, 2 mM KCI, 10 mM Hepes (pH 7.4),1 mm deuterated tempone (300 osmolar) and EPR spectrawere obtained using a Varian 109 9 GHz spectrometer. Spec-tra were then measured following the addition of an equalvolume of paramagnetic broadening agent (110 mm potas-sium chromium oxalate, 290 osmolar) and internal volumeas a percent of the external solution volume was calculatedaccording to:

% volume=peak height after broadening

peak height before broadening x gain factor

x dilution factor x 100.

With this approach it was found that the internal volume ofthe red beet plasma membrane vesicles accounted for about0.43% of the solution volume. As this would correspond to4.3 ,sL/mL and the membrane vesicles were present at a

protein concentration of 0.65 mg/mL, a vesicle volume of6.6 ,tL/mg protein was estimated.

ATP Hydrolysis Assay

ATP hydrolytic activity was measured at 22°C using thecoupled assay (4). When used in the calculation of stoichi-ometry relative to pyranine optical density change, the assaywas conducted in a 1 mL reaction volume containing 250mM sorbitol, 3.0 mm MgSO4, 3.0 mM ATP (Na+ salt adjustedto pH 6.13), 0.5 mm BTP/Mes (pH 6.13), 50 mM KNO3, 5units pyruvate kinase, 12 units lactate dehydrogenase, 0.42mM phosphoenolpyruvate, and 0.15 mM NADH. When usedto calculate stoichiometry with estimates of H+ backflux or

with the kinetic model for the H+-transport timecourse, mod-ifications include buffering with 25 mM BTP/Mes (pH 6.5)and use of the BTP salt ofATP (pH 6.5). After establishmentof a baseline value for ATP-independent NADH oxidation,the reaction was initiated by the addition of ATP and thedecrease in optical density at 340 nm was monitored. Therate of NADH oxidation was determined and related to therate of ATP hydrolysis assuming that 1 NADH is oxidizedper ATP hydrolyzed in the assay.

Protein Assay

Protein was determined by the method of Bradford (6)using BSA as a standard. The Bradford assay reagent was

filtered just prior to use.

RESULTS

In the determination of H+/ATP stoichiometry for a H+-transporting ATPase associated with isolated membrane ves-icles using kinetic methods, the rate of H+-translocation intothe vesicles is compared to the rate of ATP hydrolysis whereboth assays are conducted under similar reaction conditions.While the measurement ofATP hydrolysis generally involvesroutine assay techniques, the measurement of the H+ trans-location rate can prove more difficult and can involve meth-ods requiring assumptions concerning passive H+ conduct-ance. For determination ofH+/ATP stoichiometry for the redbeet plasma membrane H+-ATPase, three methods for esti-mating H+-translocation were used and the calculated ratesof H+ translocation were then compared to rates of ATPhydrolysis determined by the coupled ATPase assay.

Method 1: Determination of H+/ATP Stoichiometry Usingan Initial Rate Approach

In the initial rate approach to measuring H+/ATP stoichi-ometry, both H+-transport and ATPase assays are conductedunder identical initial rate conditions where H+ gradientsacross the vesicle membrane are small. Under these condi-tions, it is assumed that passive H+ flux out of the vesicleswill be negligible compared to the measured rate ofH+ influxand thermodynamic limitations on the H+-ATPase will beminimal. Thus, H+/ATP stoichiometry can be calculated asthe quotient of the initial rates of H+ influx and ATP hydrol-ysis. In practice, this method has involved measuring alkalin-ization ofthe medium external to the vesicles, weakly bufferedat pH 6.13. At this pH, the ATP hydrolytic reaction isisoprotic (1, 4, 27, 29) so that any initial alkalinization of themedium should result from H+ transport into the vesicles.Measurement of this alkalinization has been conducted atrelatively high levels of membrane protein using either a pHelectrode (e.g. 4, 29) or a pH-sensing dye (e.g. 27).

In the use of an optical method for the measurement ofexternal alkalinization, a suitable dye probe should have highsensitivity, fast response time, and low membrane permeabil-ity. As pointed out by Dencher et al. (12), pyranine (8-hydroxy- 1 ,3,6-pyrenetrisulfonic acid) has proven quite usefulfor measuring small scalar pH changes in vesicular or lipo-some systems. This dye shows a prominent pH-dependentchange in optical density at 450 nm with greatest sensitivitynear its pKa of about 7.0 (12). Although external alkaliniza-tion relevant to determinations of H+/ATP stoichiometrymust be measured at pH 6.13, the addition of either 10 nmolof OH- (as KOH) or H+ (as HCI) produced a measurablechange in pyranine absorbance at 450 nm (AOD _ 0.01)under our assay conditions. This absorbance change waslinear since stepwise additions of either 10 nmol OH- or H+resulted in equal changes in absorbance. From these results,it would appear that changes in pyranine absorbance can beused to estimate the small amount of H+ transported intoplasma membrane vesicles for the calculation of the initialrate of H+ transport.When ATP was added to a vesicle suspension (220 ,ug

protein) weakly buffered at pH 6.13, alkalinization of theexternal medium occurred as indicated by an increase in

244 Plant Physiol. Vol. 95, 1991




1-4

H _

ATFase Coupled Assa A0D340

0~~~ ~ ~ ~~~~~3

TI \nmol

ATP

1.6 \la.

TIME

Figure 1. Determination of ATP-dependent H+ transport and ATPaseactivity for red beet plasma membrane vesicles under initial rateconditions. Both H+ transport and ATPase activity were measured at220C in the presence of 250 mm sorbitol, 3 mm MgSO4, 3 mmNa2ATP, 50 mm KNO3, and 0.5 mm BTP/Mes (pH 6.13). The loss ofH+ from the external medium was determined by pyranine absorbancechange and ATPase activity was determined by the coupled enzymeassay.

pyranine absorbance (Fig. 1). The increase in pyranine ab-sorbance was linear for about the first 1.5 min of reaction andbegan to level off with increasing time. This decrease in therate of pyranine absorbance change in the latter portion ofthe time course most likely reflects both the establishment ofa steady-state pH gradient across the vesicle membrane andthe external scalar production ofH+ by ATP hydrolysis as thepH of the medium increased from 6.13 (1). As the additionof a H+-conducting ionophore such as gramicidin D at 1.5min fully collapsed this increase in pyranine absorbance (datanot shown), it was concluded that this initial optical changeof the dye reflects ATP-driven H+ transport into the vesicles.In contrast to the trace for pyranine absorbance change, ATPhydrolysis as measured by coupled NADH oxidation re-

mained linear for the duration of the assay. As the rate ofATP hydrolysis remained linear over this time range, it can

be assumed that thermodynamic limitations upon the ATPase(backpressure) were minimal.When calibrated by the addition of aliquots of H+, pyranine

absorbance change over the first 1.5 min of reaction was usedto estimate the initial rate of H+ transport into the vesicles.

When compared to the initial rate of ATP hydrolysis esti-mated over the same time interval using the coupled ATPaseassay, a H+/ATP stoichiometry of about 1.0 was observed forthe red beet plasma membrane H+-ATPase (Table I).

Method II: Determination of H+/ATP Stoichiometry UsingH+ Leakage to Estimate the Rate of H+-Transport

As an alternative to measuring the initial rate of ATP-driven H+ uptake by vesicles in H+/ATP stoichiometry cal-culations, Perlin et al. (24) developed a method to estimateH+ transport based upon measurements of H+ leakage fromvesicles when the H+-ATPase was inhibited at steady-state. Incontrast to the initial rate approach, this method can be usedat any pH where the H+ pump is active and does not requirethe assumption of minimal passive H+ leakage. The methodassumes that the steady-state pH gradient in vesicles estab-lished by a H+-ATPase results from a balance of ATP-drivenH+ influx and passive H+ efflux (pump/leak steady-state).Therefore, if the H+-ATPase is allowed to produce a steady-state pH gradient and is then rapidly shut off by an inhibitor,the H+ leak rate should estimate the steady-state rate of H+uptake. These rates of H+ leakage from a steady-state pHgradient established in vesicles can be measured by changesin the fluorescence quenching of the ApH sensing dye, acri-dine orange. This method was utilized by Perlin et al. (24) toestimate H+/ATP stoichiometry for Neurospora plasma mem-brane and E. coli H+-ATPases associated with vesicles.When the timecourse of ATP-driven ApH formation was

measured using acridine orange fluorescence quenching andcompared with the time course ofATP hydrolysis, it appearedthat the steady-state pH gradient present in red beet vesiclesalso represented a balance of ATP-driven H+ influx andpassive H+ efflux (Fig. 2). When determined at pH 6.5, thetime course of ATP hydrolysis remained linear even whenthe ApH reached its steady-state value. This would indicatethat the magnitude of the pH gradient was not limited by therate of ATP hydrolysis but, instead, represented a balance ofpump and passive H+ fluxes (24). The addition of gramicidinD resulted in a rapid increase in fluorescence to the initialbaseline value, consistent with this dye response reporting the

Table I. H+/ATP Stoichiometry for the Red Beet Plasma MembraneVesicles using Pyranine Absorbance Change and the CoupledATPase Assay

Experiments 1 to 5 and 6 to 10 were conducted with differentmembrane preparations. The mean H+/ATP stoichiometry was 1.037.

Experiment H+-Transport ATPase Activity H+/ATP

,umol H+/min * mg protein .nol Pi/min * mg protein

1 0.237 0.216 1.0972 0.220 0.285 0.7723 0.208 0.156 1.3334 0.238 0.260 0.9155 0.258 0.229 1.1276 0.154 0.126 1.2227 0.146 0.141 1.0358 0.123 0.156 0.7889 0.162 0.139 1.165

10 0.138 0.151 0.914

245




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0

60 4e;

50 H

40 X

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Figure 2. Timecourse of ATP hydrolysis and pH gradient formationby red beet plasma membrane vesicles. The production of a pHgradient was determined by the quenching of acridine orange fluo-rescence while ATPase activity was determined using the coupledenzyme assay. Both assays were conducted at 220C in the presenceof 250 mm sorbitol, 3 mm MgSO4, 3 mm ATP (BTP salt, pH 6.5), 50mM KCI, and 25 mm BTP/Mes (pH 6.5). As indicated, gramicidin wasadded to a final concentration of 10 MM.

establishment of a ApH. When the degree of acridine orangefluorescence quenching was determined for vesicles whereknown acid-interior pH gradients were imposed, a linearrelationship was observed when the data were plotted asLog (%Quench/100 - %Quench) versus ApH (Fig. 3). Fromthis calibration, it was determined that the steady-state ApHestablished by the H+-ATPase in red beet plasma membranevesicles was about two units under typical reaction conditions.

It should be noted that such a linear relationship betweenamine dye fluorescence quenching and ApH would be pre-dicted from the relationship:

Log(%Q/l00 - %Q) = ApH + log(V,/V0) (1)derived previously (2, 3, and references therein) where %Q isthe percent of fluorescence quenching, Vi is the interior vol-ume of the vesicle, and V0 is the volume exterior to the vesicle(Fig. 3) (2, 3 and references therein). This relationship assumesthat the unprotonated form of the amine dye is membranepermeable and that the distribution of dye is based upon theApH present across the vesicle membrane (3 and referencestherein). According to this relationship, the Y-intercept of aplot of log(%Q/l00 - %Q) versus ApH should give an esti-mate of the vesicle internal volume (i.e. from log Vi/V0). ForFigure 3, the extrapolated Y-intercept would be about -1.7which would mean that the Vil/V( would be 0.02. This wouldcorrespond to 20 AL of vesicle internal volume per 1 mLassay containing about 70 Mg protein or 285 ML/mg protein.However, when the internal volume for the red beet plasmamembrane vesicles was determined using electron paramag-netic resonance with a nitroxide spin probe, a value of about6.6 ,L/mg protein was found. As this value is much smaller

than the internal volume predicted from Equation 1, thiswould imply that the dye is taken up by the vesicles to agreater extent than expected for a free amine in solution.Although this indicates that the dye may be displaying non-ideal behavior with respect to Equation 1, this does not disputethe observation that the optical change (i.e. Log[%Q/100 -%Q]) occurs in proportion to ApH.A key requirement for the estimation of H+ uptake rate by

the method of Perlin et al. (24) is a means to rapidly (andreproducibly) stop the H+-ATPase at steady-state. As in theirstudies, it was found that Mg2> chelation by the addition ofEDTA provided the most satisfactory means to rapidly stopthe red beet plasma membrane H+-ATPase (Fig. 4). Whenthis was conducted with a range of EDTA concentrations, 10mM EDTA or greater provided the most immediate stoppingof the transport pump after the establishment of a steady-state pH gradient. This method was found to provide a morerapid means of stopping the H+-ATPase than either addingorthovanadate or removing the ATP substrate by addition ofglucose + hexokinase (data not shown). An additional require-ment for this method of estimating H+/ATP stoichiometry ismeasurement of the vesicle internal buffering capacity. Whenthis was determined using the method ofMaloney (17) a valueof 0.237 umol H+/pH unit. mg protein was found for a pHrange of 4.5 to 6.5. This was similar to the internal bufferingcapacity observed for Neurospora plasma membrane vesicles(0.260 ,umol H+/pH unit-mg protein, ref. 24) but greater thanthe internal buffering capacity observed for a reconstitutedvesicle preparation from maize roots (0.100 ,mol H+/pHunit mg protein, ref. 7).When red beet plasma membrane vesicle preparations were

allowed to establish a steady-state pH gradient in the presenceof ATP and the H+-ATPase was stopped by the addition of10 mm EDTA, the average (n = 8 determinations) initial rateof H+-leakage was found to be about 0.815 pH units/min(Table II). This value was calculated by noting the rate of

0.80-%

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of

0

0.4 -

-O.0 -

-0 .4 -

-0 8 -

-1 2 -

-1 6

-2.00 2 3

INPOSED ApH

Figure 3. Calibration curve for the fluorescence quenching of acridineorange in response to an imposed acid-interior ApH. An acid-interiorApH was produced across the vesicle membrane as described in"Materials and Methods" and the corresponding magnitude of fluo-rescence quenching was determined at three protein amounts in thefluorescence assay.

* 74 1L9* 70 ugo 65 A9

I


-r1




L)

u

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TIME

Figure 4. Effect of EDTA on the steady-state pH gradient producedby the red beet plasma membrane ATPase associated with vesicles.The production of a pH gradient was initiated by the addition ofMg:ATP and quantitated by acridine orange fluorescence quenchingas indicated in "Materials and Methods." As indicated, various con-centrations of EDTA were added to the vesicles once a steady statewas established and the decline in the pH gradient was determined.

fluorescence increase from the steady-state value of fluores-cence quenching following EDTA addition and converting topH units/min according to the calibration curve shown inFigure 3. By the method of Perlin et al. (24), this value, whenmultiplied by the vesicle internal buffering capacity, yields anestimate ofthe rate ofATP-driven H+ influx. When comparedto the average rate (n = 8 determinations) ofATP hydrolyticactivity as determined by the coupled assay under theseconditions, a H+/ATP stoichiometry of about 0.814 wasobserved with this method (Table II).

Method III: Determination of H+/ATP Stoichiometry Usinga Kinetic Analysis of the H+ Transport Timecourse

As discussed by Brauer et al. (7 and references therein) a

kinetic model can be used to describe the timecourse of ApHproduction in vesicles driven by a H+-translocating ATPaseand to estimate the stoichiometry of the transport pump. Intheir model, it is assumed that the magnitude of the pHgradient at any point in time represents the balance of ATP-driven H+-influx and both passive H+ leakage from the vesi-cles and any thermodynamic limitation upon the transportpump. Thus, proton transport (di/dt) can be described by:

d6/dt = m.R - (kL kbp).6 (2)

where 6 is the net amount of H+ transported, m is the H+/ATP stoichiometry, R is the rate of ATP hydrolysis, kL is therate constant for H+ leakage, and khp is the rate constant

associated with any thermodynamic backpressure upon thetransport pump. Since at steady-state the net H+-transportrate approaches zero, Equation 2 can reduce to:

m.R, =ki-6 (3)

where R, is the steady-state rate ofATP hydrolysis, k1 is a rateconstant representing a sum of the two rate constants (kL andkhp) above, and 6, is the net amount of H+ transported atsteady-state. Since the ATP hydrolysis rate for the red beetvesicles remains constant through the establishment of asteady-state pH gradient (Fig. 2) and the magnitude of thisgradient (in pH units) can be estimated by acridine orangefluorescence quenching (Fig. 3), Equation 3 provides a meansto calculate the H+/ATP stoichiometry provided that k, canbe estimated.As shown by Brauer et al. (7 and references therein), the

use of Equation 3 and the assumption that the rate of ATPhydrolysis remains constant through the establishment of a

steady-state pH gradient allows Equation 2 to be reduced to:

d3/dt = k, (6 - 6,)which upon integration yields:

ln(l - 6/6,) = -k, * t

(4)

(5)

From this relationship, k, can be estimated by a plot ofln(1 - 6/6,) as a function of time (t). Since the optical change(fluorescence or absorbance) of ApH sensing dyes such as

acridine orange can be used to estimate k, by this functionwhere 3/6, represents the ratio of dye optical change at time(t) to the final dye optical change at steady-state (7), ananalysis of fluorescence quenching data for the red beet vesi-cles could yield an estimate for this value. When a plot of thistype was constructed for a timecourse of ATP-driven ApHproduction by red beet plasma membrane vesicles, a linearrelationship was observed where the value of k, could bedetermined from the slope of this plot (Fig. 5). When thisanalysis was conducted for eight separate fluorescence quenchcurves of H+ transport, an average k, of 0.353 min-' was

observed.With this value of k, and Equation 3, the H+/ATP stoichi-

ometry was estimated for the red beet plasma membraneH+-ATPase according to the method of Brauer et al. (7)(Table III). To estimate 6,, the average steady-state pH gra-

dient in pH units was converted into units of,mol H+/mg

Table II. Estimation of H+-Transport/ATP Hydrolysis StoichiometryBased upon Measurements of H+-Leakage and ATP HydrolyticActivity

Parameter Value

H+-leakage rate (pH units/min) 0.815(n = 8)

Internal buffering capacity (Mmol H+/pH unit- 0.237mg protein)

Estimated H+ transport (Mmol H+/min * mg pro- 0.193tein)

ATP hydrolytic activity (,umol/min - mg protein) 0.237(n = 8)

H+/ATP stoichiometry 0.814

2i

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10.15

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TIME (min.)Figure 5. Determination of ki during the production of a pH gradientin red beet plasma membrane vesicles. The production of a pHgradient was determined according to "Materials and Methods"in the presence of 250 mm sorbitol, 3 mm MgSO4, 3 mm ATP,50 mM KCI, and 25 mm BTP/Mes (pH 6.5). The data for acridineorange fluorescence quenching was transformed as according toBrauer et al. (7).

protein by multiplying this value by the average internalbuffering capacity of the vesicles as estimated by the methodof Maloney (17). When the product k1 * 6, was calculated andthen compared with the rate of ATP hydrolysis according toEquation 3, a H+/ATP stoichiometry (m) of about 0.743 wasfound for the red beet plasma membrane H+-ATPase.

DISCUSSION

Using three different methods to estimate the rate of ATP-driven H+-influx and the coupled enzyme assay to estimateATP hydrolysis, an H+/ATP stoichiometry approaching 1

was observed for the plasma membrane H+-ATPase associ-ated with native membrane vesicles. The methods used toestimate the H+ transport rate differ with respect to initialassumptions, sources of possible error (see below), and entaildifferent types of measurements for H+-transport parameters.In the initial rate method (method I), H+-efflux was assumedto be minimal and H+-influx was estimated over the first 1.5min of reaction. In contrast, the method involving H+-efflux(method II) assumed that the steady-state ApH was basedupon a balance ofH+ pumping and passive efflux (pump-leaksteady-state) so that measurement ofH+ efflux (from a steady-state ApH) should estimate H+-influx. Finally, the methodutilizing a kinetic model to describe net H+ transport (methodIII) considered the relationship of H+ influx and efflux at anygiven point in time and H+ influx was estimated consideringthe entire timecourse of ApH production. A value of about 1

for the H+/ATP stoichiometry of the red beet plasma mem-brane H+-ATPase would be similar to that calculated for theNeurospora plasma membrane H+-ATPase (24) and close tothe value of 0.8 determined by Brauer et al. (7) for a recon-stituted plasma membrane H+-ATPase preparation from

maize roots. In contrast, the tonoplast H+-ATPase from redbeet storage tissue has been reported to have a H+/ATPstoichiometry of about 2 when measured by an initial ratemethod using a pH electrode (4).There are several possible sources of error which need to

be considered in the determination ofH+/ATP stoichiometryfor the plasma membrane H+-ATPase using these kineticmethods. If a substantial number of the plasma membranevesicles are leaky to H+, this would tend to underestimate theH+/ATP stoichiometry for methods I and III. These vesicleswould have lower rates ofH+ influx relative to ATP hydrolysisand would achieve a smaller steady-state pH gradient. Tosome extent, H+ influx estimation by method III would becompensated for H+ leakage since the k, value used in thecalculation of H+ influx would take into consideration H+leakage. In contrast, method II would be least affected bypassive H+ leakage (if a ApH can be measured) since the H+leak rate is actually used to estimate H+ influx under condi-tions ofa pump-leak steady-state. These considerations wouldapply to the situation where H+ leakage is due to a finite H+conductance of the vesicle membrane as opposed to a totallack of vesicle sealing. For this latter situation, all methodswould underestimate H+/ATP stoichiometry since ATP hy-drolysis would occur without measurable H+ transport.As methods II and III require use of the internal buffering

capacity in the estimation ofH+ influx, error in measurementof this parameter would affect calculation of the H+/ATPstoichiometry by these two approaches. While the methodgenerally used in estimation of internal buffering capacity (seerefs. 7, 17, 24) only considers buffer activity ofthe membranesalone (i.e. the vesicles are salt washed several times) internal-ized buffer normally present in transport assays could alsocontribute to the total buffering capacity of the vesicles.Therefore, it is possible that the effective buffering capacitypresent under conditions of transport assays might be under-estimated. This would, in turn, lead to an underestimation ofH+ influx and H+/ATP stoichiometry.The H+/ATP stoichiometry would also be underestimated

for methods II and III if there were a substantial number ofsealed vesicles that were present in the rightside-out orienta-tion. Both methods require conversion between fluorescenceand pH units based upon the use of a standard curve con-

Table l1l. Estimation of H+ TransportIATP Hydrolysis StoichiometryBased upon Analysis of the Fluorescence Profile and ATP HydrolyticActivity

Parameter Value

Steady-state ApH (pH units) 2.1(n = 8)

Internal buffering capacity (gmol H+/pH unit- 0.237mg protein)

3, (,mol H+/mg protein) 0.498K1 (min-1) 0.353

(n = 8)Estimated H+ transport (Amol/min -mg protein) 0.176ATP hydrolytic activity (limol/min - mg protein) 0.237

(n = 8)H+/ATP stoichiometry 0.743





structed by imposing pH gradients and noting the correspond-ing changes in fluorescence. Since imposition of artificial pHgradients would occur for all sealed vesicles and only inside-out vesicles are active in transport assays, the pH gradient willbe underestimated for the active vesicles when significantamounts of sealed, rightside-out vesicles are present. However,method I would not be subjected to this error since standard-ization is based upon addition of H+ to an external dye notsensing ApH. Finally, an overestimate of the H+/ATP stoi-chiometry could result by method II if the reagent used torapidly stop the H+ pump enhanced subsequent H+ effluxrelative to the level associated with the maintenance of thesteady-state ApH. Although these methods for estimating H+influx clearly differ in possible sources of error, a generalcorrespondence in the estimated H+/ATP stoichiometry wasobserved when utilized with the red beet plasma membraneH+-ATPase present in native vesicles.A major consideration in the estimation of H+/ATP stoi-

chiometry for the plasma membrane H+-ATPase is whetherthis value would be sufficient to account for the magnitudeof the proton electrochemical gradient established across theplasma membrane in vivo. From the thermodynamic relation-ship between the free energy available from ATP hydrolysis(AGATP) and the proton electrochemical gradient:

1/n (AGATP) 2 FAJ - 2.303 RT ApH (6)

it is possible to estimate the maximum pH gradient (ApH)which can be produced by a H+-ATPase of n stoichiometryat a given membrane potential (A'I) assuming that the ATP-ase is operating near thermodynamic equilibrium (see ref. 4for discussion). However, in order to conduct this calculation,a value for AGATP under cytoplasmic conditions will need tobe estimated according to:

AGATP = AGATP + RT ln[ADP] [Pi] (7)[ATP]

where AG>TP is the standard free energy of ATP hydrolysis(about -8.8 kcal mol-' at pH 7.0, 1 mM Mg2", and 298°Kfrom ref. 1). It should be noted that for Equations 6 and 7, Ris the gas constant (1.987 cal mol-' K-'), T is the absolutetemperature (298°K), and F is the Faraday constant (23.06kcal mol-' eV-'). For plant cells, ADP/ATP ratios are rela-tively constant at 0.3 and cytoplasmic ATP and Pi concentra-tions have been estimated to be about 1 mm and 1 to 5 mM,respectively (4 and references therein). Using this informationwith Equation 7, the free energy available from ATP hydrol-ysis would be about -13.2 kcal mol-' at 25°C.From this value for AGATP and the range of membrane

potentials observed for higher plant cells (-110 to -197 mVfrom ref. 33), it can be calculated from Equation 6 that a H+-ATPase with H+/ATP stoichiometry of 1 could produce amaximum ApH of 6.4 to 7.8 pH units. While this thermo-dynamic argument demonstrates that a H+/ATP stoichiom-etry of 1 can easily account for the magnitude of the protonelectrochemical gradient established across the plasma mem-brane (see ref. 33), it cannot be used to exclude a H+/ATPstoichiometry of 2 for the plasma membrane H+-ATPase. Ifthe H+/ATP stoichiometry is assumed to be 2 under theseconditions, a maximum ApH of 1.5 to 3.0 pH units could

still be produced by the H+-ATPase and this predicted rangeof ApH values can also account for estimates of ApH acrossthe plasma membrane (33 and references therein). On theother hand, a H'/ATP stoichoimetry of 1 would be moreconsistent with the very steep proton electrochemical gra-dients observed across the plasma membrane of plant cellswhen H+ efflux is stimulated by the addition of fusicoccin (18and references therein). If the plasma membrane H+-ATPaseoperates with a H+/ATP stoichoimetry of 1 in vivo as indi-cated from kinetic analysis, this would suggest that the H+-pump is functioning far from thermodynamic equilibriumand under kinetic control by cellular factors other than energysupply.

ACKNOWLEDGMENTS

The authors wish to acknowledge Dr. Mike Niesman for technicalassistance in the measurement of vesicle internal volumes by electronparamagnetic resonance and Dr. Jose Ruiz-Cristin for valuablediscussion.

LITERATURE CITED

1. Alberty RA (1969) Effect of pH and metal ion concentration onthe equilibrium hydrolysis of adenosine triphosphate to aden-osine diphosphate. J Biol Chem 243: 1337-1343

2. Azzone GF, Pietrobon D, Zoratti M (1984) Determination of theproton electrochemical gradient across biological membranes.Curr Topics Bioenerg 13: 1-77

3. Bennett AB, Spanswick RM (1982) Optical measurements ofApH and z' in corn root membrane vesicles. Kinetic analysisofC I - effects on a proton translocating ATPase. J Membr Biol71: 95-107

4. Bennett AB, Spanswick RM (1984) H+-ATPase activity fromstorage tissue of Beta vulgaris. II. H+/ATP stoichiometry of ananion-sensitive H+-ATPase. Plant Physiol 74: 545-548

5. Boutry M, Michelet B, Goffeau A (1989) Molecular cloning of afamily ofplant genes encoding a protein homologous to plasmamembrane H+-translocating ATPases. Biochem Biophys ResCommun 162: 567-574

6. Bradford MM (1977) A rapid and sensitive method for thequantification of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal Biochem 72: 243-254

7. Brauer D, Tu S, Hsu AF, Thomas CE (1989) Kinetic analysis ofproton transport by the vanadate-sensitive ATPase from maizeroot microsomes. Plant Physiol 89: 464-471

8. Briskin DP, Leonard RT (1982) Partial characterization of aphosphorylated intermediate associated with the plasma mem-brane ATPase of corn roots. Proc NatI Acad Sci USA 79:6922-6926

9. Briskin DP, Poole RJ (1983) Plasma membrane ATPase of redbeet forms a phosphorylated intermediate. Plant Physiol 71:507-512

10. Briskin DP, Poole RJ (1983) Evidence for a 3-aspartyl phosphateresidue in the phosphorylated intermediate of the red beetplasma membrane ATPase. Plant Physiol 72: 1133-1135

11. DeMichaelis MI, Spanswick RM (1986) H+-pumping driven bythe vanadate-sensitive ATPase in membrane vesicles from cornroots. Plant Physiol 81: 542-547

12. Dencher NA, Burghaus PA, Grzesiek S (1986) Determination ofnet proton-hydroxide ion permeability across vesicular lipidbilayers and membrane proteins by optical probes. MethodsEnzymol 127: 746-760

13. Giannini JL, Gildensoph LH, Briskin DP (1987) Selective pro-duction of sealed plasma membrane vesicles from red beet(Beta vulgaris L.) storage tissue. Arch Biochem Biophys 254:621-630

14. Giannini JL, Briskin DP (1988) Pyridine nucleotide oxidationby a plasma membrane fraction from red beet (Beta vulgarisL.) storage tissue. Arch Biochem Biophys 260: 653-660

249




15. Harper JF, Surowy TK, Sussman MR (1989) Molecular cloningand sequence ofcDNA encoding the plasma membrane protonpump (H+-ATPase) of Arabidopsis thaliana. Proc Nati AcadSci USA 86: 1234-1238

16. Leonard RT (1984) Membrane-associated ATPase and nutrientabsorption by roots. In PB Tinker, A Lauchli, eds, Advancesin Plant Nutrition, Vol I. Praeger Scientific, New York,pp 209-240

17. Maloney PC (1979) Membrane H+ conductance of Streptococcuslactis. J Bacteriol 140: 197-205

18. Marre E, Ballarin-Denti A (1985) The proton pumps of theplasmalemma and the tonoplast of higher plants. J BioenergBiomembr 17: 1-21

19. Mehlhorn RJ, Candau P, Packer L (1982) Measurement ofvolumes and electrochemical gradients with spin probes inmembrane vesicles. Methods Enzymol 88: 752-761

20. Nicholls DG (1982) Bioenergetics. An Introduction to the Chem-iosmotic Theory. Academic Press, London

21. O'Neill SD, Spanswick RM (1984) Solubilization and reconsti-tution of a vanadate-sensitive H+-ATPase from the plasmamembrane of Beta vulgaris. J Membr Biol 79: 231-243

22. Pardo JM, Serrano R (1989) Structure of a plasma membraneH+-ATPase gene from the plant Arabidopsis thaliana. J BiolChem 264: 8557-8563

23. Pederson PL, Carifoli E (1987) Ion motive ATPases. I. Ubiquity,properties and significance to cell function. Trends BiochemSci 12: 146-150

24. Perlin DS, San Francisco MJD, Slayman CW, Rosen BP (1986)H+/ATP stoichiometry of proton pumps from Neurospora

crassa and Escherichia coli. Arch Biochem Biophys 248:53-61

25. Poole RJ (1978) Energy coupling for membrane transport. AnnuRev Plant Physiol 29: 437-460

26. Poole RJ, Briskin DP, Kratky Z, Johnstone RM (1984) Densitygradient localization of plasma membrane and tonoplast fromstorage tissue of growing and dormant red beet. Characteriza-tion of proton transport and ATPase activity in tonoplastvesicles. Plant Physiol 74: 549-556

27. Rabon E, Chang H, Sachs G (1978) Quantitation of hydrogenion and potential gradients in gastric plasma membrane vesi-cles. Biochemistry 17: 3345-3353

28. Raven JA, Smith FA (1979) Intracellular pH and its regulation.Annu Rev Plant Physiol 30: 289-311

29. Reenstra WW, Forte JG (1981) H+/ATP stoichiometry for thegastric (K+ + H+)-ATPase. J Membr Biol 61: 55-60

30. Singh SP, Kesav BVS, Briskin DP (1987) Reconstitution andrapid partial purification of the red beet plasma membraneATPase. Physiol Plant 69: 617-626

31. Sze H (1985) H+-translocating ATPases: advances using mem-brane vesicles. Annu Rev Plant Physiol 36: 175-208

32. Vara F, Serrano R (1982) Partial purification and properties ofthe proton translocating ATPase of plant plasma membranes.J Biol Chem 257: 12826-12830

33. Villalobo A (1988) Energetic efficiency of different mechanisticmodels for potassium ion uptake in lower eucaryotic cells.Folia Microbiol 33: 407-424

34. Walderhaug MO, Post RL, Saccomani G, Leonard RT, BriskinDP (1985) Structural relatedness of three ion-transport aden-osine triphosphatases around their active sites of phosphoryl-ation. J Biol Chem 260: 3852-3859




Documents

Determination of H+/ATP Stoichiometry for thePlasma Membrane … · the measurement of ApH. At this point, ATP-driven H+ transport wasstoppedbychelating Mg2` with 10 mmEDTA (BTPsalt,