Plant Physiol. (1990) 93, 1128-11330032-0889/90/93/11 28/06/$01 .00/0

Received for publication July 17, 1989Accepted January 31, 1990

An Essential Arginyl Residue in the TonoplastPyrophosphatase from Etiolated Mung Bean Seedlings'

Soong Yu Kuo and Rong Long Pan*Institute of Radiation Biology and of Nuclear Sciences, College of Nuclear Sciences, National Tsing Hua University,

Hsin Chu, 30043 Taiwan, Republic of China

ABSTRACT

Tonoplast membrane of etiolated mung bean (Vinga radiata.L.) seedlings contained HW-translocating pyrophosphatase (PPase).Modification of tonoplast vesicles and partially purified PPasefrom etiolated mung bean seedlings with arginine-specific re-agents, phenylglyoxal (PGO) and 2,3-butanedione (BD), resultedin a marked decline in HW-translocating PPase activity. The half-maximal inhibition was brought about by 20 millimolar PGO and50 millimolar BD for membrane bound and 1.5 millimolar PGO and5.0 millimolar BD for soluble PPase, respectively. The substrate,Mg2 -pyrophosphate, provided partial protection against inacti-vation by these reagents. Loss of activity of partially purifiedPPase followed pseudo-first order kinetics. The double logarithmplots of pseudo-first order rate constant versus reagent concen-trations gave slopes of 0.88 (PGO) and 0.90 (BD), respectively,suggesting that the inactivation may possibly result from reactionof at least one arginyl residue at the active site of H+-translocatingPPase.

The H+-translocating enzymes of the tonoplast play anessential role in the maintenance and regulation of cell turgorand in the storage and transport of ions and metabolites (26).A line of evidence has revealed the presence of H+-translocat-ing ATPase and PPase2 on the tonoplast membrane (12).Recent reports demonstrated that the tonoplast associatedPPase and ATPase activities are distinct on the membrane(12). The tonoplast ATPase from several sources has beenpurified and characterized by many workers (26, 29). How-ever, information on the structure and function of tonoplastPPase is still limited.

Group-specific chemical probes, which react covalently andcause changes in enzymatic activity, have been widely usedto provide more insight into structure and function at theactive site (27). Many essential amino acid residues involvedin the active site of yeast and Escherichia coli PPase weredemonstrated (3, 11, 15, 23). However, the amino acid resi-dues involved in the active oftonoplast PPase of higher plantsstill remains to be elucidated (8).

Several reports using guanidium-specific modifiers (eitherPGO or BD), have revealed the involvement of arginine

' This work was supported by the grant from National ScienceCouncil, Republic of China (NSC77-0201-B007-1 1) to R.L.P.

2Abbreviations: PPase, pyrophosphatase; BD, 2,3-butanedione;OG, octylglucopyranoside; PGO, phenylglyoxal.

residues in the binding of anionic substrate or cofactor atactive site (22). In this manner, an essential arginyl residuewas found for enzymes such as ATPases which hydrolyze theanionic substrate ATP (8, 14, 19). The physiological substrateof PPase, inorganic pyrophosphate, is also anionic in naturesimilar to ATP. In this paper we determine, using argininemodifier PGO and BD, whether the arginine residue(s) isinvolved in tonoplast PPase activity of higher plants. Theresults indicate that PPase in the vacuolar membraneof etiolated mung bean seedlings contains at least onearginyl residue essential for enzymatic activity and protontranslocation.

MATERIALS AND METHODS

Plant Material

Seeds of Vigna radiata L. (mung bean), purchased from alocal market, were soaked for 3 h in tapwater and thengerminated at room temperature in the dark using a com-mercial seedling incubator. The hypocotyls of 4-d-old etio-lated seedlings were excised, chilled on ice, and then used asstarting materials.

Membrane Preparation

Tonoplast vesicles were prepared from etiolated seedlingsas previously described (29). All procedures were carried outat 4°C. The tonoplast vesicles were isolated at the interface ofa 0 to 4% (w/v) dextran (79,000) gradient following centrifu-gation of the microsomal fraction at 70,000g for 2 h.

Partial Purification of PPase

Soluble tonoplast PPase was prepared from resealed tono-plast vesicles depleted of ATPase using detergent solubiliza-tion. The tonoplast vesicles were pretreated with low salt at4°C for 30 min to deplete peripheral ATPase (16) in a mediumcontaining 0.1 mM Mops-KOH (pH 7.9), 15% (w/v) glyceroland final membrane protein concentration 1 mg/mL. Afterincubation, the tonoplast vesicles were then centrifuged at120,000g for 1 h and the pellet was resuspended in the buffercontaining 0.1 mM Mops-KOH (pH 7.9), 30% (w/v) glycerol,and a protein concentration of 2 mg/mL. OG was addeddropwise from a 200 mm stock solution to a final concentra-tion of 20 mm. After 30 min incubation at 4°C, OG-treatedtonoplast vesicles were centrifuged at 120,000g for 1 h. Analiquot (0.8 mL) of supernatant containing PPase activity was

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ESSENTIAL ARGINYL RESIDUE IN TONOPLAST PYROPHOSPHATASE

then layered onto 4 mL 7 to 27% linear sucrose gradientcontaining 10 mm Tris-Cl (pH 7.3), 0.25 M sorbitol, 1 mMEGTA, 1 mm DTT, 0.2 mM PMSF, 15% (w/v) glycerol, and7 to 27% sucrose. The gradient was centrifuged at 200,000gfor 5 h. Aliquots (0.28 mL) were collected from the bottomof gradient and the fraction with highest PPase activity wasused for this study.

Treatment of PPase with BD and PGO

The tonoplast vesicles or partially purified PPase wereincubated with PGO in a medium containing 50 mm Mops-KOH (pH 7.9) at 37°C for the time periods indicated. Simi-larly, modification of tonoplast vesicles and soluble PPasewith BD was conducted in 50 mm borate buffer (pH 7.9).MgCl2 and protection agents were added (when present) asdescribed in figure legends. The final protein concentrationof tonoplast vesicles was 0.8 to 1.0 mg/mL while that ofpartially purified PPase, 0.4 to 0.6 mg/mL.

Enzyme Assay and Protein Determination

PPase activities of the modified and control membranepreparations were determined by measuring the released Piand PPi. Since 2 mol of Pi are released from hydrolysis of 1mol PPi, we have expressed the activity as moles PPi con-sumed. Aliquots of resealed vesicles or soluble PPase wereassayed in a 1.0 mL volume containing 25 mM Mops-KOH(pH 7.9), 3 mM MgSO4, 50 mM KCl, 3 mM K4 PPi, 16 to 20,ug/mL membrane protein or 8 to 12 ,ug/mL partially purifiedPPase, with 0.5 mm NaN3, 0.1 mm Na-orthovanadate, 50 mMKNO3, and 0.1 mM ammonium molybdate to inhibit theATPase of mitochondria, plasma membrane and tonoplast,and other possible acid phosphatase, respectively (26, 29).Assays were carried out at 37°C for 15 to 30 min and termi-nated by adding a solution containing 1.7% (w/v) ammoniummolybdate, 2% (w/v) SDS and 0.02% (w/v) l-amino-2-naph-thol-4 sulfonic acid. The released Pi was determined spectro-photometrically (SLM-AMINCO U2000) as described else-where (29).

Protein concentration was measured according to the mod-ified Lowry method (29) using BSA as the standard.

Measurement of Proton Translocation

Proton translocation was measured as fluorescence quench-ing of acridine orange (excitation wavelength 495 nm, emis-sion wavelength 530 nm) with a Hitachi F-4000 fluorescencespectrophotometer. The reaction mixture contained 5 mMMops-KOH (pH 7.9), 250 mM sorbitol, 3 mM MgSO4, 50 mMKCl, 3 mm PPi, 0.1 mm Na-orthovanadate, 50 mM KNO3,0.1 mM ammonium molybdate, 0.5 mM NaN3, 5 juM acridineorange, and 16 to 20 ,ug/mL membrane protein. The fluores-cence quenching was initiated by adding 3 mM MgSO4. Theionophore gramicidin (2 ,ug/mL) was added at the end of eachassay.

Kinetic Analysis

The t(/2 values, time required for 50% inhibition of activity,at various concentration of modifiers were measured accord-

-6L-c0

-A-0 5.0

PGO (mM) -°,-

1004

8076

40

2 0

0 25 50 75 100 125 150

BD (mM) -°090

Figure 1. Inactivation of H+-PPase activity as a function of concen-tration of PGO and BD. Membrane protein (1.0 mg/mL) and partiallypurified PPase (0.6 mg/mL) were incubated at 370C in Mops-KOH(pH 7.9) (PGO) or borate-KOH, pH 7.9 (BD) with various concentra-tions of modifiers as indicated. The incubation periods for tonoplastvesicles were 30 min (BD) and 15 min (PGO); for partially purifiedPPase, 15 min (BD) and 5 min (PGO), respectively. After treatments,aliquots (20 4L) were removed and assayed for pyrophosphataseactivities and H+-translocation, expression as a percentage of residualactivities with modifiers. The assay medium for pyrophosphatasehydrolysis contained: 25 mM Mops-KOH (pH 7.9), 3 mM MgSO4, 50mM KCI, 3 mm potassium pyrophosphate, 0.1 mm Na orthovanadate,50 mM KNO3, 0.1 mm ammonium molybdate, 0.5 mm NaN3 and 16to 20 ,Ag/mL membrane protein or 8 to 12 Ag/mL partially purifiedPPase. The control activities were 7.48 and 30.2 Amol PPi con-summed/mg protein/h for membrane bound and soluble PPase,respectively. The reaction conditions for H+-translocation were de-scribed as shown in Figure 2. (A) PGO; (B), BD; (O-O), H+-translocation; (-4*), pyrophosphate hydrolysis activity of mem-brane bound PPase; (A i), pyrophosphate hydrolysis activity ofpartially purified PPase.

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ing to semilogarithmic plots ofpercent residual activity versustime as described by Levy et al. (17). The reaction order (n)with respect to modifier was determined from double-log plotsof 1000/t1/2 versus concentration of modifiers (17). The K3,dissociation constant for the modifier and T1/2, t1I2 at excessconcentration of modifiers, were calculated from the plots oft112 as a function of the reciprocal of modifier concentrationaccording to Carlson (4).

Chemicals

PGO and BD were purchased from Sigma. ATP was ob-tained from Merck. All other chemicals were of analytic gradeand used without further purification.

lines were obtained with slopes equal to the number of mol-ecules of modifier labeled at each active site of PPase. Slopesof 0.90 and 0.88 were determined for BD and PGO asmodifier, respectively (insets of Fig. 3). It is likely that theinactivation of H+-translocating PPase was primarily due tothe modification of at least one arginyl residue at active siteof enzyme by PGO and BD. It might be possible that morethan one arginyl group exists at the active site. However, thispossibility seems unlikely since the semi-logarithm of percentresidual activity shows a simple exponential function of in-cubation time.

Furthermore, a plot of tTI12 versus reciprocal of modifierconcentration gives Ki and minimum t/2 (4, 6). The mini-mum t1/2 values (T) of 3.0 min and 4.0 min at excess concen-

RESULTS AND DISCUSSION

Inactivation of PPase by PGO and BD

The effects of PGO and BD on the enzymatic activity ofpartially purified and membrane bound tonoplast H+-PPasewere examined. Incubation of the vesicles and partially puri-fied protein with various concentrations of PGO and BDresulted in a loss of enzymatic activity of the H+-PPase (Fig.1 [ - ; A-i]). The degree of inactivation ofH+-PPaseactivity was at least twofold higher by PGO than by BD asjudged from their concentration dependence. For instance,20 mM PGO inhibited approximately 50% of membranebound H+-PPase activity. However, more than 50 mm ofBDwas required to cause similar loss of enzymatic activity. Thegreater hydrophobicity of PGO might offer the advantage forthe modifier to access the active site. Furthermore, the I50values are approximately 10-fold higher for the membranebound than partially purified tonoplast PPase. Recently, Bai-kov et al. (1) demonstrated the differential reactivities ofsulfhydryl reagents to free and membrane bound inorganicPPase from submitochondria particles of rat liver. They spec-ulated that the active site of PPase was deeply embedded inthe membrane. The same conclusion could be drawn fromthe sensitivities to BD and PGO that inorganic PPase of plantvacuoles might be also submerged in the membrane. Thus,PGO was used preferentially as the selective modifier ofarginyl residues in this report.

In parallel experiments, PGO and BD inhibited the PPase-supported H+-translocation across the tonoplast vesicle asdetermined by fluorescence quenching ofApH probe, acridineorange (Fig. 2). The concentration dependence of inactivationof H+-translocation of PGO and BD coincides with that ofenzymatic activity (Fig. 1 [O O]), implying the possibleinvolvement of arginyl residues in PPase activity as well as itsassociated H+-translocation.

Kinetic Analysis

Inactivation kinetic studies of partially purified PPase activ-ity as a function of time revealed to be pseudo-first orderaccording to semilogarithmic plots of percent residual activityversus time (Fig. 3). The reaction order with respect to PGOand BD was determined from double-log plots of 1000/tI/2 aSfunction of modifier concentrations. Accordingly, straight

Mg2 +

101.I

a) .

cEon

i:

2min

Mg2+

t oa)(OwU.

22min

A

(PGO) mM50

30

0

Gramicidin

B

(BD) mM10050

0

GramicidinFigure 2. Inactivation of PPase-mediated H+-translocation by PGOand BD. The treatment of tonoplast vesicles with PGO and BD wereas described in Figure 1. The reaction medium for fluorescencequenching contained: 5 mm Mops-KOH (pH 7.9), 250 mm sorbitol, 3mM MgSO4, 50 mm KCI, 3 mm potassium pyrophosphate, 0.1 mmNa-orthovanadate, 50 mm KNO3, 0.1 mm ammonium molybdate, 0.5mM NaN3, and 5 AM acridine orange. The reaction was initiated byadding 3 mm Mg2+. The initial rates of fluorescence quenching weredetermined from the changes in fluorescence observed in first 1 min.Excitation wavelength was 495 nm, while emission wavelength was530 nm. Two ug/ml of gramicidin were added to stop the fluorescencequenching. (A) PGO; (B) BD.

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10%

I

ESSENTIAL ARGINYL RESIDUE IN TONOPLAST PYROPHOSPHATASE

0

-

0

;-

0-

(I

Time (min)

_ 6001

4_0

-60, 2.1 1

u~~~Tm (mn

1

001.5

-1.3t

0 -.7 1.0 1.3 1.6

Iog(lOXEBDJmM)

4 1 2 1 6 2 0 24

Time (min)

Figure 3. Inactivation of enzymatic activity of partially purified PPaseby PGO and BD. The treatment and assay conditions were asdescribed in Figure 1. The pseudo-first order rate constant wasestimated from the slopes of the plots according to the equation, Inactivity = -kt + C. Insets show the estimated reaction order withrespect to modifier employed. Values of t112 = n = log [modifier] -log K2, where n is the reaction order, [modifier] is the concentrationof modifiers. (A) PGO; (B) BD.

tration of modifier PGO and BD respectively were obtainedfrom intercepts at coordinate. Ki values of 3.12 and 4.10 mMfor PGO and BD were also calculated, respectively, from theslopes (4). The lower T1/2 and Ki values for PGO than BDagain indicate the PGO is more effective to inhibit the enzy-matic activity of pyrophosphatase.

The effects of Vmax and Km by PGO were also investigated(Fig. 4) after inactivating PPase with or without 5.0 mM PGOfor 6 min at 37°C. In the control, the Km for PPase was 2.5mm and Vmax 57.1 umol PPi consummed/mg protein/h. Inthe presence ofPGO the Vmax was not changed, while the Kmincreased three-fold to 7.7 mm. The mode of inhibition ofpartially purified PPase by PGO is competitive as determinedby its effect on Km but not Vmax. Thus, it is confirmedkinetically that the modified arginine residue locates at activesite.

Protection against PGO Inactivation

Protection against modifier-induced inactivation was stud-ied. The PPase was preincubated 5 min with protectors in thepresence and absence of 5.0 mM MgCl2. After addition ofPGO to a final concentration of 5.0 mm for 12 min at 37°C,the enzymatic activity of PPase was determined as mentionedabove. Table I summarizes the protection effects of its sub-strate (pyrophosphate), inorganic phosphate, and substrateanalogs, such as p-nitrophenyl phosphate, phosphoserine,phosphothreonine, and imidodiphosphate. Substantial pro-tection was provided by the physiological substrate of theenzyme, pyrophosphate, with 5 mM MgCl2. The presence ofMg2' is absolutely crucial for the partial protection againstmodifiers. In its absence, pyrophosphate exerted almost noprotection effect. It is believed that native substrate is Mg2+-pyrophosphate. However, we can not exclude the possibilitythat Mg2' induces the conformational change of PPase re-sulting in the less accessibility of a-dicarbonyl reagents. Inaddition, inorganic phosphate has a fairly reasonable partialprotection (42.9%). Inorganic phosphate is the product ofPPase. It can also bind to the active site and exert partialprotection effect. Substrate analogs such as p-nitrophenylphosphate and imidodiphosphate provided 51.8% and 60.6%protection, respectively. However, phospho-derivatives ofamino acid residues like phosphoserine and phosphothreo-nine had negligible protection effect. The stereohindrance ofamino acid moieties might make them less accessible to theactive site of PPase.ATP, ADP, and AMP contain a moiety similar to pyro-

phosphate at one end of the molecule. It is interesting toinvestigate their possible protection against PGO at native siteof PPase. The hydrolytic activities of ATP, ADP, and AMPwere low compared with pyrophosphate. ATP offered theprotection against PGO as well as pyrophosphate. However,ADP and AMP had hardly any protection effects. SinceATPase and PPase use the anionic substrates, the possiblesimilarity at active site of ATPase and PPase deserves specialattention. Several studies revealed that at least one arginylresidue locates at the catalytic site of ATPase from varioussources (6, 19, 24). For ATPase, the degree of protection ofarginyl residue against a-dicarbonyl reagents highly dependedon the number of phosphate, as well as on the species ofnucleotide moieties (6). It is believed (8) the arginyl residuesin question bridged the a- and ,B-phosphate of ATP at theATP binding site of these ATPases. In our study, ATP, whichcontains three phosphate moieties, had a reasonable protec-

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51_'-5

I _6E

0

cE0

2.51

I1-0.5 - 0.25

PGO

control

0 0.25

U/S

0.5

[mm1]Figure 4. Double reciprocal plot of partially purified PPase in thepresence and absence of PGO. Partially purified PPase (0.4 mg/ml)was modified by 5.0 mm PGO in 240 AL of 50 mm Mops-KOH buffer(pH 7.9) for 6 min at 370C. The pyrophosphate hydrolysis activitiesof aliquots (20 ,L) of control (@) or modified (0) soluble PPase were

measured in the assay medium as described in Figure 1.

sources contain essential lysyl (15, 25), histyl (23), tyrosyl (2,10, 20, 23), sulfhydryl (1, 3, 21, 28), arginyl (5, 9), methionine(30), and carboxylates (9) residues. The lysine residue was

supposed to be involved in the protonation in the process ofenzymatic activity and in the stabilization of transition state(15). The histyl and tyrosyl residues probably resided in theactive site and participated in the mechanism of stabilityagainst heat inactivation (23). Circular dichroism studiesshowed yeast inorganic pyrophosphatase contained 5 to 10residues of tyrosine and 1.4 to 2.5 residues of tryptophanexposed to the solvent (20). The modification of severalresidues of tyrosine and tryptophan resulted in total loss ofactivity indicting their important roles on the enzyme (10,20). More than one carboxyl group was demonstrated to beessential in the mechanism of pyrophosphatase, probably bydirect interaction with the substrates (9). The involvement ofmethionine and cysteine in pyrophosphatase activity were

also studied and the roles of these residues in enzymaticprocess were also proposed (2, 21, 30).A conserved essential arginine residue was suggested in the

enzymes such as ATPase using anionic substrates (6, 7, 13).It is not surprising that PPase, as another example ofenzymesusing anionic substrates, contains an essential arginine residue(9). The arginine residue might participate in the binding ofpyrophosphate in the active cleft. The absolute requirementof Mg2' for protection suggests its role in the anchoring ofsubstrate in active site, probably through the carboxylates ofthe enzyme (9, 27).

This paper is first to report that H+-translocating pyrophos-phate oftonoplast vesicles from mung bean etiolated seedlings

tion effect for PPase. We speculate that active pocket ofPPasehas room enough only for the binding of A- and 'y-phosphateof ATP. The adenine and ribose moieties of ATP were leftout of the active cleft of enzyme. Thus, ATP still offeredprotection while nucleotides such as ADP and AMP, withrelatively higher stereohindrance at active cleft, had very littleprotection effect.From the protection studies above, it is confirmed that the

essential arginyl residues are in the active site of PPase.However, there might be other alternatives to explain theprotection effect against modifiers. For instance, protectorscould directly block the modification reaction with essentialarginine. No spectrophotometrical evidence indicates the pos-sibility of complex formation between protectors and modi-fiers (data not shown). On the other hand, we cannot excludethe possibility that the presence of protectors could cause a

conformational change which would make the arginine lesssusceptible to attack. The later possibility requires furtherelucidation.

PPases from various sources were purified and molecularmasses of their subunits were measured ranging from 20 to70 kD (3, 11, 18). However, little is known on amino acidcomposition, sequence, and possible homology in active do-main. Even under such conditions, many reports demon-strated by using chemical modifiers that PPases from these

Table I. Protection of PPase Activity against Inactivation by PGOThe partially purified tonoplast PPase (0.6 mg/mL) was preincu-

bated with 5.0 mm protectors for 5 min at 370C in the presence orabsence of 5.0 mm MgCl2. After addition of 5.0 mm PGO for 12 minat 370C, aliquots (20 ,L) were assayed for PPase activity as de-scribed in "Materials and Methods." The percentage protection wascalculated according to the equation:

Percentage protection = [SAproted- SA(unprotected)] x 100[SA(t- SAunpmtted)]SA, specific activity.

Protection against PGOProtectors Activity as (5 mM)

(5 mM) Substrate5 mM MgCI2 0 mM MgC12

,umol PPi consumedmg protein-h

K4PPi 32.12 (100.0) 113.0 <1.0p-Nitrophenyl phos- 2.55 (8.0) 51.8 <1.0

phateInorganic phos- 42.8 3.5

phateImidodiphosphate 4.19 (13-1) 60.6 <1.0Phosphothreonine 3.06 (9.5) <1.0 1.2Phosphoserne 3.02 (9.4) <1.0 6.7ATP 6.57 (20.5) 95.4 10.7ADP 4.74 (14.8) 25.0 <1.0AMP 0.0 (0.0) 28.6 <1.0

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ESSENTIAL ARGINYL RESIDUE IN TONOPLAST PYROPHOSPHATASE

was inhibited by incubation with two a-dicarbonyl com-

pounds, PGO and BD. The inactivation by PGO and BD ispossibly due to the specific modification of positively chargedguanidium group of arginine residue at active site of pyro-phosphatase. The kinetic analysis reveals that one essentialarginyl residue is involved at each site of enzyme. The partialprotection of enzymatic activity against PGO inhibition inthe presence of Mg2" indicates the binding of Mg2+-pyrophos-phate at an active site. We conclude from the evidence abovethat H+-PPase of mung bean tonoplast contains at least one

arginyl residue essential to its enzymatic activity.

ACKNOWLEDGMENTS

We thank Dr. G. G. Chang for the discussion on kinetic analysisand Ms. Y. M. Tsai for her expert secretarial assistance.

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