Modification of the Red Beet Plasma Membrane

Plant Physiol. (1990) 94, 696-7030032-0889/90/94/0696/08/$01 .00/0

Received for publication March 12, 1990Accepted June 11, 1990

Modification of the Red Beet Plasma MembraneH+-ATPase by Diethylpyrocarbonate192

Lynne H. Gildensoph3 and Donald P. Briskin*Department of Agronomy, University of Illinois, 1102 S. Goodwin Avenue, Urbana, Illinois 61801

ABSTRACT

Incubation of the red beet (Beta vulgaris L.) plasma membraneH+-ATPase with micromolar concentrations of diethylpyrocarbon-ate (DEPC) resulted in inhibition of both ATP hydrolytic and protonpumping activity. Enzyme activity was restored when DEPC-modified protein was incubated with hydroxylamine, suggestingspecific modification of histidine residues. Kinetic analyses ofDEPC inhibition performed on both membrane-bound and solu-bilized enzyme preparations suggested the presence of at leastone essential histidine moiety per active site. Inclusion of eitherATP (substrate) or ADP (product and competitive inhibitor) in themodification medium reduced the amount of inhibition observedin the presence of DEPC. However, protection was not entirelyeffective in returning activity to noninhibited control values. Theseresults suggest that the modified histidine does not reside directlyin the ATP binding region of the enzyme, but is more likelyinvolved in enzyme regulation through subtle conformationaleffects.

The H+-ATPase associated with the plant plasma mem-brane transports H+ from the cytoplasm to the cell exterior,producing an inwardly directed proton electrochemical gra-dient (AjH+). Secondary solute transport occurs in responseto this gradient which consists of a negative-interior electricalpotential (At) and an acid-exterior proton gradient (ApH).In this fashion, the H+-ATPase is proposed to have a vitalrole in providing the driving force for solute transport and tobe involved in cellular processes including regulation of cy-toplasmic pH; nutrient status and turgor driven responsessuch as stomatal movements and IAA-mediated growth re-sponses (2 1).The plant plasma membrane H+-ATPase is representative

of the EjE2 class of transport enzyme (also called P-type).During ATP hydrolysis this enzyme forms a phosphorylatedintermediate at an active site aspartyl residue and analysis ofthe phosphorylated protein by polyacrylamide gel electropho-resis indicates the presence of a 100 kilodalton catalyticsubunit (3). Recently, the amino acid sequence of this proteinhas been deduced from cDNA clones produced from Arabi-

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

2 This paper is dedicated to the memory of Dr. Robert F. Davisfor his help and encouragement to one of the authors (L.H.G.).

' Current address: Department of Biology, Hamline University,1536 Hewitt Ave., St. Paul, MN 55104.

dopsis thaliana (16) and Nicotiana plumbaginifolia (2). Hy-dropathy analysis of the deduced sequence (16) suggestedeight probable transmembrane segments separated by threehydrophilic domains that extend into the cytoplasm. Withina large cytoplasmic domain are the aspartyl residue, whichbecomes phosphorylated, and a lysine, which in other E1E2-type ATPases binds the fluorescent probe fluoroscein-5'-iso-thiocyanate. This latter residue is proposed to play an impor-tant role in binding the adenine portion of the ATP substrateto the enzyme (7). Chemical modification studies have indi-cated the presence of an essential arginine (13, 19) that maybe involved in stabilizing binding of the anionic phosphateportion of the ATP substrate (31).

Results from a previous kinetic study on the plasma mem-brane H+-ATPase from red beet suggested that histidine resi-dues might also play a role in the catalytic cycle ofthe enzyme(6). When the rate of phosphoenzyme formation was deter-mined as a function of assay pH, the generated curve resem-bled a pH titration curve for histidine, with an inflection pointat approximately pH 6.5. This could be explained by theimidazole side chain of a histidine moiety having some rolein the process of transferring the a-phosphate from ATP tothe aspartyl residue of the enzyme. Alternatively, this resultcould occur as the net effect of a number of residues actingin concert. In this study, the technique of specifically modi-fying histidine residues with the chemical reagent DEPC4 wasused in order to determine if histidine moieties have anessential role within the mechanism of the red beet plasmamembrane H+-ATPase. Both membrane-bound and solubi-lized ATPase fractions were used in order to determine ifthere was a difference in accessibility or sensitivity of histidineresidues to DEPC with these two enzyme preparations.

MATERIALS AND METHODSPlant Material

Fresh red beets (Beta vulgaris L., cv Detroit Dark Red)were purchased commercially, detopped, and then stored indark, moist conditions at 0 to 4°C for at least 10 d prior touse to ensure dormancy and tissue uniformity as previouslydescribed (1 1).

Isolation of Plasma Membrane Fractions and DetergentSolubilization of the H+-ATPasePlasma membrane fractions were obtained following the

procedure of Giannini et al. (1 1) except that the homogeni-4 Abbreviations: DEPC, diethylpyrocarbonate; BTP, bis-tris-pro-

pane; LDS, lithium dodecyl sulfate.696

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HISTIDINE RESIDUE IN RED BEET PLASMA MEMBRANE ATPase

zation buffer contained 0.2 mM PMSF. Plasma membraneH+-ATPase was solubilized using the procedure of Briskinand Poole (4). This procedure involves extraction of theplasma membrane fraction with 0.1% (w/v) sodium deoxy-cholate to remove proteins other than the plasma membraneH+-ATPase and then solubilization of the enzyme using 0.1%(w/v) Zwittergent 3-14. Both detergent treatments were car-

ried out at a detergent to protein ratio of 1:1 (mg/mg).

Modification of H+-ATPase with DEPC

Treatment of red beet plasma membrane H+-ATPase prep-

arations with DEPC was carried out for 20 min at 37°C in a

reaction solution containing 30 mM BTP/Mes (pH 6.5), 50mM KCI, 3 mM MgSO4, and various concentrations of DEPC(freshly prepared in absolute ethanol). Ethanol concentrationsnever exceeded 3% (v/v) in the incubation media and 0.3%(v/v) in the ATPase assay. At this level, no effect upon ATPaseactivity was observed. At specified times, aliquots were with-drawn and diluted into an ice-cold ATPase assay solutioncontaining 30 mm imidazole to block unreacted DEPC. Fortime course studies, 10 mM L-histidine was also included tofurther inactivate any DEPC carried over from the incubationmedium. Any deviations from this procedure are indicated in"Results and Discussion."

Measurement of ATPase Activity

ATPase activity was measured as described previously (1 1),in a reaction solution containing 3 mM ATP, 3 mM MgSO4,50 mM KCI, and 30 mm imidazole/HCl (pH 6.5).

Optical Measurement of Vesicle pH Gradient

Acid-interior pH gradients were measured by the decreaseof acridine orange absorbance at 490 nm as described byGiannini et al. (12). The standard assay (1 mL volume)contained various concentrations of freshly prepared DEPCin ethanol (ethanol concentration never exceeded 2%), 250mM sorbitol, 25 mM BTP/Mes (pH 6.5), 100 mm KNO3, 3.75mM MgSO4, 3.75 mm BTP/ATP, and 10 Mm acridine orange.The reaction was started by the addition of ATP to vesiclesequilibrated with dye and followed by absorbance decreaseover time (at room temperature) using a Beckman DU-40spectrophotometer fitted with a kinetics program.

Protein Measurement

Protein concentrations were determined as previously de-scribed (11) by the method of Bradford using BSA as a

standard.

RESULTS AND DISCUSSION

DEPC was used to determine whether histidine plays an

essential role in the mechanism of the plasma membrane H+-ATPase. This reagent generally modifies the imidazole sidechain of histidine at near neutral pH conditions (9, 24, 25).The reaction is covalent and irreversible under appropriateconditions. However, when subsequently treated with rela-

tively high concentrations of hydroxylamine, this reagent can

be discharged from the protein by deacylation of the DEPC-histidine moiety (24, 25). Determination ofessential histidineswith this reagent has been accomplished for a number ofproteins isolated from bacterial and animal tissues, includingactins (29), tyrosine phenyl lyase (20), lysyl oxidase (10), andan organic anion exchange protein isolated from renal mem-brane vesicles (32). Recently, DEPC was used to demonstratethe role of an essential histidine in the plasma membrane H+-ATPase of the fungus Neurospora crassa (28). Our initialeffort was directed towards optimizing the conditions forreaction of this reagent with the H+-ATPase associated withthe red beet plasma membrane.

Optimization of Conditions for Modification of the H+-ATPase by DEPC

Since DEPC has not been used previously in studies involv-ing the H+-ATPase associated with the higher plant plasmamembrane, the reaction between the enzyme and DEPC was

examined under various conditions. Miles (27) reported thatTris buffer accelerated the decomposition of DEPC and it isalso possible for Tris to react with this reagent as a nucleophile(1). In either case, Tris buffer could potentially reduce theeffective DEPC concentration during incubation with theenzyme preparation. Likewise, boric acid buffer is known tostabilize the reaction between butanedione and arginine resi-dues (13). However, when red beet plasma membrane H+-ATPase was treated with DEPC in any of the three buffersystems tested, very little difference was observed for enzymeinhibition (Table I). Although a slight increase in inhibitionwas observed in the presence of BTP/Mes buffer, this was theresult of increased activity of controls incubated withoutDEPC. BTP/Mes buffer was used for DEPC-enzyme incuba-tion in order to maximize relative activities and eliminate anypossibility of a Tris-DEPC reaction. Magnesium and potas-sium were included in the incubation medium since they are

necessary for optimal ATPase functioning and did not de-crease inhibition of the enzyme by DEPC.

Reported temperatures for DEPC modification of proteins

Table I. Effects of ATPase Reaction Ligands and Various Buffers atpH 6.5 on Inhibition of Plasma Membrane H+-ATPase by 5 mMDEPC

Incubation took place at 0°C for 20 min in the indicated buffersystems.

ATPaseAdditions

Tris/Mes BTP/Mes Boric acid/Mes

pumolPi*mg-'*h-'-DEPC, +Mg, +KCI8 24.4 27.8 25.7+DEPC, -Mg, -KCI 5.8 (24)b 4.7 (17) 6.8 (27)+DEPC, +Mg, -KCI 4.6 (19) 4.3 (16) 4.6 (18)+DEPC, -Mg, +KCI 5.0 (21) 3.6 (13) 5.2 (20)+DEPC, +Mg, +KCI 4.8 (20) - 4.9 (19)a When added, ligand concentrations were 3 mm MgSO4 and 50

mM KCI. b Numbers in parentheses represent percent controlincubated with 3 mm MgSO4 and 50 mm KCI in the absence of DEPC.

697



GILDENSOPH AND BRISKIN

have varied from 0°C (20, 29) to room temperature (22-25°C)( 10 32). Berger (1) reported that temperatures exceeding 60°Ccaused DEPC to decompose while low temperature greatlyenhanced the stability of this reagent. When both membrane-bound (Fig. 1A) and detergent-solubilized (Fig. 1B) ATPasepreparations were incubated with DEPC at temperatures rang-ing from 0°C to 37°C inhibition relative to a control sample(incubated in the absence of inhibitor) increased as the tem-perature was increased. As incubation with DEPC at 370Cresulted in a maximal degree of inhibition for both the plasmamembrane and solubilized preparations, this reaction tem-perature was utilized in further experiments (except wherenoted).The effect ofpH on DEPC reaction with plasma membrane

and solubilized ATPase preparations is shown in Figure 2.Inhibition by 2.0 mm DEPC for membrane-bound (Fig. 2A)and 1.0 mm for solubilized (Fig. 2B) ATPase was nearlycomplete at all pH values tested following a 20 min incubationperiod at 370C. Enzyme activity in the absence of DEPC wasinhibited when enzyme was preincubated at pH values below6.5. To ensure specificity of DEPC for histidine moieties andmaintain high activity in unmodified control samples, all

30

20

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10

0

50

100

50

0

0 5 1 0 1 5 20 5 30 37

Texperature (degrees Celsius)

Figure 1. Effect of temperature on inhibition of membrane-bound (A)and detergent solubilized (B) red beet plasma membrane ATPase byDEPC. Enzyme preparations were incubated with or without 2.5 mMDEPC for 20 min at various temperatures in a reaction solutioncontaining 30 mm BTP/Mes (pH 6.5), 50 mm KCI, and 3 mm MgSO4.Aliquots were withdrawn and then assayed for ATPase activity as

described in "Materials and Methods."

5.5 6.0 6.5 7.0 7.5 8.0

pH

Figure 2. Effect of incubation pH on inhibition of membrane-bound(A) and solubilized (B) red beet plasma membrane ATPase by DEPC.Treatment with DEPC was conducted for 20 min at 370C as describedin "Materials and Methods" in the presence of 30 mm BTP/Mes atthe indicated pH values. Aliquots were then withdrawn and assayedfor ATPase activity.

further incubations were performed at pH 6.5. This wouldalso serve to stabilize the AN-carbethoxy-histidine residuewhich is reported to be most stable at near neutral pH values(26, 27). The effect of pH on the rate of inhibition by DEPCwas also investigated using a solubilized enzyme preparationand a lower DEPC concentration. Under these conditions,measurement of the time course of inhibition could be madeand the rate of inhibition exhibited a pseudo-first order kineticrelationship. As observed in Figure 3. when the enzyme was

incubated with 800 Mm DEPC at pH values ranging from 5.5to 9.0, a series of straight lines were observed when log percentresidual activity was plotted as a function of time. Whenapparent first order rate constants were calculated and thenplotted as a function of pH, a tripartite pattern of rate changewas observed (Fig. 3B). Between pH 5.5 and 6.5 the rate ofinhibition was relatively unchanged while a linear increase inrate was observed between pH 7.0 and 8. At pH values above8.0 a dramatic increase in inhibition rate was observed withthe increase in pH. This is similar to what was observed byMorjana and Scarborough (28) in their investigation on therole of histidine in the N. cra.ssa plasma membrane H+-ATPase. This effect could be due to complex factors, reflecting

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Plant Physiol. Vol. 94, 1990698




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Figure 3. Effect of pH on the rate of ATPase inactivation by 800 /M

DEPC for the solubilized enzyme preparation. Treatment of thesolubilized plasma membrane H+-ATPase with 800 gM DEPC was

conducted over time at 370C and at the indicated pH. Values for ATPhydrolysis at zero time were based upon a -DEPC control sampleassayed immediately. Rates calculated from regression analyses ofinhibition data observed over 8 min (A) were then plotted as a functionof pH (B).

the pH dependence of the histidine group and the environ-ment surrounding this histidine, or effects upon DEPC itself,and/or conformational changes in the protein. At the more

alkaline pH values, it is also possible for DEPC to react withlysine moieties (ref. 24 and references therein), and this in-crease in inactivation above pH 8.0 may reflect binding ofDEPC to both an essential histidine and essential lysine.Preliminary studies conducted in this laboratory have sug-

gested the presence of an essential lysine associated with thered beet plasma membrane H+-ATPase (14).

Kinetics of DEPC Inhibition of Plasma Membrane ATPaseActivity

When membrane-bound red beet plasma membrane H+-ATPase preparations were incubated for 20 min with DEPC

at various concentrations in the range from 0 to 20 mM, itwas evident that this reagent was a potent inhibitor of phos-phohydrolase activity, even when incubation took place at0°C (Fig. 4). At reagent concentrations exceeding 2 mm, asharp decrease in the activity of DEPC-modified enzyme wasobserved, although inhibitor concentrations as low as 600 AMwere effective in inhibiting enzyme activity to less than halfof the control value. With higher concentrations of DEPC(i.e. 5, 10, and 20 mM), the enzyme was inhibited by 90 to98% and no significant increase in the degree of inhibitionwas shown as the DEPC concentration exceeded 5 mM. DEPCconcentrations in the low millimolar range have been reportedfor inactivation of enzymes such as the organic anion exchan-ger isolated from renal tissue (32) and lysyl oxidase catalasefrom calf aorta (10), but concentrations below 1 mm have

10 been effective when used with Escherichia coli phenol lyase(20) and pig dopa decarboxylase (8). The plant plasma mem-brane H+-ATPase appears to fall into this latter category sinceit is effectively inhibited by low DEPC concentrations. As wasobserved in Figure 1, the inhibition percentage was increasedwhen incubation with DEPC took place at 37°C, where activ-ity in the presence of 2.5 mm DEPC was inhibited 97 to 99%in both the membrane-bound and solubilized preparations.When 0 to 800 Mm DEPC was used to modify the red beet

plasma membrane H+-ATPase, a pseudo-first-order kineticrelationship for inhibition was observed for both the mem-brane-bound (Fig. 5A) and solubilized (Fig. SB) preparationsof enzyme. Incubation of the enzyme at 37°C for up to 8 minyielded a series of straight lines when data were plotted as logresidual activity (percent control) versus time. Use of irrevers-ible modifiers (such as DEPC under slightly acid conditions)and modifier concentrations in excess of the enzyme concen-tration allows for calculation of the reaction order number,n, according to the equation of Levy et al. (22):

Log kob, = log k + n log [inhibitor]

100

0

4-,

0

'4

80

60

40

20

0

0.0 5.0 10.0 15.0 20.0

DEPC Concentration (nfl)

Figure 4. Effect of DEPC concentration on inhibition of membrane-bound ATPase. Treatment with the indicated concentration of DEPCwas conducted for 20 min, at 00C in the presence of 30 mm BTP/Mes (pH 6.5), 50 mm KCI, and 3 mm MgSO4. Aliquots were withdrawnand then assayed for ATPase activity. The data are presented as a

percent of the control activity for samples treated identically exceptin the absence of DEPC.

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2.0

1 .8

2.2

2.0

1 .8

0 2 4 6 8

Incubation Tiae (ain)

Figure 5. Time course of DEPC inhibition for membrane-bound (A)and solubilized (B) red beet plasma membrane ATPase. Enzymepreparations were incubated with the indicated concentration ofDEPC as described in "Materials and Methods." At various times,aliquots were removed and then assayed for ATPase activity. Dataare presented as the percentage of control samples treated identicallybut without DEPC. The value for ATP hydrolytic activity at zero timewas based upon the immediate assay of a control sample withoutDEPC.

where k equals the true inhibition rate constant and k0b, equalsthe apparent first-order rate constant estimated from slopevalues (multiplied by 2.3) of inhibition time course plots suchas those shown in Figure 5. Plotting the log of these apparentfirst-order rate constants as a function of log inhibitor con-

centrations yields a straight line with a slope value equal tothe reaction order number, n. According to Levy et al. (22),the reaction order number calculated through use of thisequation estimates the number of molecules of inhibitorbound per molecule of enzyme. As shown in Figure 6, bothenzyme preparations had n values close to one when analyzedin this fashion. This suggests that at least one histidine residueactively participates in the ATPase reaction. However, care

must be taken with interpretation ofthese data, as it is possiblethat more than one essential histidine, with similar reactioncharacteristics, might be taking part in the enzyme catalyticcycle (33). Therefore, it is not possible to conclude that onlyone histidine is essential for the ATPase reaction, but rather

that one or more histidine residues might be essential forcatalytic activity.

Effect of DEPC on the Proton Pumping Activity of thePlasma Membrane ATPase

The effect of increasing concentrations of DEPC on theproton pumping activity associated with the red beet plasmamembrane H+-ATPase in sealed plasma membrane vesicleswas tested. For these initial assays, the enzyme was notpreincubated with DEPC and DEPC was simply included inthe transport reaction mixture. As shown in Figure 7, protonpumping activity was progressively inhibited in the presenceof 0 to 400 gM DEPC. At DEPC concentrations above 400Mm inhibition was relatively constant, generally around 60%.However, when plasma membrane vesicles were preincubated(10 min at 22°C with 400 or 800 gM DEPC), inhibition ofproton transport was increased to 89% and 99%, respectively.As preincubation with DEPC represented reaction conditionscloser to those used for examining effects upon ATP hydro-lytic activity (i.e. Fig. 4), this would suggest a greater sensitivityof proton pumping activity to DEPC.As the measured proton pumping activity associated with

the red beet plasma membrane vesicles can be a reflection ofboth the rate of H+ influx driven by the ATPase and the rateof passive H+ efflux from the vesicles ("leakage"), effects of

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2.2 2.4 2.6 2.8

Log DEPC Concentration

Figure 6. Transformation and regression analysis of apparent rateconstants (obtained from slope data from Figure 4 [x 2.303]) plottedaccording to the equation: log K., = log K + n log [inhibitor].

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700 Plant Physiol. Vol. 94, 1990

1 ,

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0

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110

90

70

50

300 200 400 600

DEPC Concentration (ipfl)

800

Figure 7. Effect of increasing concentrations of DEPC on protonpumping activity of plasma membrane vesicles. DEPC was includeddirectly in the assay which took place in a buffer containing 250 mMsorbitol, 25 mm BTP/Mes (pH 6.5), 100 mm KNO3, 3.75 mm MgSO4,10 /iM acridine orange, and 3.75 mm ATP (BTP salt, pH 6.5), and therate of proton movement was determined by observing the changein absorbance at 490 nm of acridine orange over a 4 min period atroom temperature.

DEPC on overall proton pumping activity could be due tothis reagent modifying either one or both of these parameters.To examine DEPC effects upon passive H+ efflux from thevesicles, proton pumping was allowed to proceed for 5 minto establish a pH gradient, and then either DEPC, gramicidinD, EDTA, or orthovanadate were added. As shown in Figure8, when gramicidin D (channel forming ionophore) wasadded, the pH gradient was collapsed as shown by the rapidreversal of acridine orange absorbance decrease. In contrast,treatments which stop the plasma membrane H+-ATPase suchas the addition of EDTA to chelate Mg2" or addition of theinhibitor orthovanadate result in a more gradual decrease inthe pH gradient over time. Clearly, the addition of 800 yMDEPC results in a gradual decrease in the pH gradient similarto these treatments which stop the H+-pump. Thus, it wouldappear that DEPC effects upon proton pumping activity arerelated to modification of the H+-ATPase rather than increas-ing passive H+ conductance. The greater sensitivity of protonpumping to DEPC as compared to ATP hydrolytic activitymay reflect some effect on the process of energy coupling toH+ transport or that histidine moieties may have an importantrole in the process of H+ translocation across the membrane.

Reversal of DEPC Modification by Hydroxylamine

Although DEPC has been shown to be a relatively specificmodifier of histidine residues, some investigations have indi-cated possible reaction with other residues under certainconditions (24, 27). Treatment ofthe DEPC-modified enzymewith hydroxylamine can result in deacylation of derivatizedhistidine moieties and provide a restoration of previouslyinhibited enzyme activity (24, 25, 27). This ability to restore

enzyme activity following hydroxylamine treatment is gener-ally considered to be specific for derivatization of histidineand is used to confirm histidine essentiality (24, 25).When red beet plasma membrane ATPase preparations

previously inhibited by DEPC were subsequently incubatedwith increasing amounts of hydroxylamine for 1 h at roomtemperature, a corresponding increase in the percentage ofenzyme activity relative to controls occurred (Table II). Whenenzyme modified with 800 ,M DEPC was treated with 0.9 Mhydroxylamine, inhibition was reduced from 72% to 20%,consistent with the involvement ofhistidyl moieties in enzymeactivity. Although hydroxylamine treatment did result in anelevation ofcontrol activities (no DEPC treatment), inhibitiondata are expressed on a percent inhibition basis relative tosuch controls. This effect may be due to stabilization of theenzyme during incubation since inclusion of 0 to 6 mMhydroxylamine (maximum possible after repeated washing)did not change absorbance readings of control tubes (notshown). While it is possible that complete restoration ofenzyme activity might be accomplished with a longer expo-sure to NH2OH, this could not be tested as the ATPaseloses activity over prolonged incubation periods at roomtemperature.

Protection against DEPC Inhibition by NucleosidePhosphates

If the modified histidine residue is present in the active siteof the plasma membrane H+-ATPase, incubation with DEPCin the presence of saturating amounts of substrate ATP, orthe initial reaction product (and competitive inhibitor) ADP,

0.00o

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-0.04

-0.06

-0.08

-0.1 0

-0.12 +-0.0 2.0 4.0 6.0 8.0 10.0

Time Elapsed (min)

Figure 8. Effects of various reagents upon the rate of proton pump-ing observed in red beet plasma membrane vesicles. The dye acridineorange was allowed to equilibrate across the vesicle membrane in abuffer containing 250 mm sorbitol, 25 mm BTP/Mes (pH 6.5), 100 mMKNO3, 3.75 mm MgSO4, and then 3.75 mm ATP was added to initiatethe proton-pumping reaction. After 5 min, either gramicidin D, EDTA,sodium vanadate, or DEPC was added and quickly mixed. Thereaction was then allowed to proceed for a subsequent 5 min. Allabsorbance changes were monitored by use of a Beckman DU-40spectrophotometer fitted with a kinetics program.

701




should greatly decrease the amount of inhibition of ATPaseactivity produced by DEPC treatment. When solubilizedATPase was incubated with increasing micromolar concen-trations of DEPC for 30 min at 370C in the presence of either10 mM ATP or ADP, activity was protected but not returnedto the control level (Fig. 9). Increased inhibition was observedwhen the inhibitor concentration was increased from 0 to 500,uM and this was correlated with a decreasing amount ofprotection against DEPC modification by ATP or ADP.When incubation was performed in the presence of 500 AMDEPC, enzyme activity was inhibited approximately 95% andthe inclusion of either ATP or ADP caused an increase inactivity. However, the enzyme was still inhibited to approxi-mately 65% of the control level.At lower concentrations of inhibitor, activity in the presence

of nucleotide returned to a level closer to that of the controlbut there was less effect of DEPC on subsequent ATPaseactivity at these lower DEPC concentrations. Increasing themagnesium concentration to 10 mM did not increase protec-tion by nucleotides (data not shown), consistent with previousobservations that nucleotide binding to the enzyme can occurin the absence of magnesium (5, 13). In all cases, it appearedas if ATP allowed the inhibited enzyme to recover approxi-mately 30% over inhibited activity and ADP was slightlymore effective as a protectant since activity was increased byapproximately 40%. These results suggest that the modifiedhistidine moiety might not represent a residue (or residues)that directly participates in the binding ofsubstrate to enzyme.These results are somewhat different from those of Morjanaand Scarborough (28) who reported that the addition ofADP,but not ATP, decreased the rate of DEPC inhibition in theH+-ATPase located in the plasma membrane of N. crassa.These authors concluded that the modified histidine probablyplayed a part in the binding of nucleotide to the active site,but they did not rule out longer range conformational changes(28).

Table II. Effect of Hydroxylamine on ATPase Activity in Presenceand Absence of 800 gM DEPC

Enzyme was preincubated for 30 min at room temperature witheither DEPC or ethanol in buffer containing 30 mm BTP/Mes, 3 mMMgSO4, and 50 mm KCI. Various concentrations of NH2OH. HCI (pH6.5 with BTP) were then added and the mixture was incubated for 1h at room temperature. L-Histidine (5 mM) was then added andenzyme was washed two times by centrifugation for 30 min in amicrofuge. Final pellets were suspended in suspension buffer andaliquots were assayed for ATPase activity.

ATPaseHydroxylamine

-DEPC +DEPC

M 4molPi-mg-' h-'

0 27.2 7.7 (28)0.5 40.9 18.7 (46)0.7 37.1 27.3 (67)0.9 38.3 30.6 (80)

a Activities in parentheses represent relative activities based onpercent of control ATPase treated in the same fashion, but in theabsence of DEPC.

I-4

'1

I-

I:

200

150

100

50

00 150 300 500

DEPC Concentration (up'ff)

Figure 9. Effect of ATP and ADP on protecting against inhibition ofsolubilized H+-ATPase by DEPC. Incubation was carried out for 30min at 37°C in a reaction solution containing 30 mm BTP/Mes (pH7.0), 50 mm KCI, and 3 mm MgSO4. After 30 min, aliquots werewithdrawn and then assayed for ATPase activity.

In conclusion, the results of this study indicate that the H+-ATPase associated with the plasma membrane of red beetstorage tissue was sensitive to micromolar concentrations ofthe histidine-specific reagent DEPC. This would confirm theearlier proposal for a role of histidine in the mechanism ofthe plasma membrane H+-ATPase based upon transient statekinetic studies on the enzyme (6). That the characteristics ofDEPC inhibition were similar for the enzyme in the mem-brane-bound and solubilized form would suggest equal acces-sibility and/or sensitivity of histidine residues to this reagentfor both preparations. Recently, DEPC has been used toinvestigate the role of histidine in animal membrane transportproteins and has allowed the identification of essential histi-dine residues associated with a number of transport systemssuch as the band 3 protein from erythrocyte ghosts ( 18), theNa+/H+ exchanger found in renal tissue (15) and the renalH+/organic cation antiport system, where histidine was foundto play a regulatory role in transport ( 17). Preliminary resultsin this laboratory indicate that DEPC may block the transfor-mation of the E P state of the enzyme to the EP state ( 14) asobserved on acid pH dodecylsulfate PAGE (23, 30). Thus,DEPC modification may serve as a useful tool for the furtherstudy of mechanistic aspects of this enzyme. In the future,'4C-DEPC could also be used to probe for the sequencesurrounding the modified histidyl residue on ATPases in theE-P class isolated from various sources in order to determineif this might be a structurally and mechanistically commontheme.

LITERATURE CITED

1. Berger SL (1975) Diethyl pryocarbonate: an esamination of itsproperties in buffered solutions with a new assay technique.Anal Biochem 67: 428-437

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

702 Plant Physiol. Vol. 94, 1990



HISTIDINE RESIDUE IN RED BEET PLASMA MEMBRANE ATPase.

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

4. Briskin DP, Poole RJ (1984) Characteristics of the solubilizedplasma membrane ATPase of red beet. Plant Physiol 76: 26-30

5. Briskin DP (1986) Intermediate reaction states of the red beetplasma membrane ATPase. Arch Biochem Biophys 248: 106-115

6. Briskin DP (1988) Phosphorylation and dephosphorylation re-actions of the red beet plasma membrane ATPase studied inthe transient state. Plant Physiol 88: 84-91

7. Cantley L, Carilli CT, Farley RA, Perlman DM (1982) Locationof binding sites on the (Na,K)-ATPase for fluorescein-5'-iso-thiocyanate and ouabain. Ann NY Acad Sci 402: 289-291

8. Dominici P, Tancini B, Voltattorni CB (1985) Chemical modifi-cation of pig kidney 3,4-dihydroxyphenylalanine decarboxyl-ase with diethyl pyrocarbonate. J Biol Chem 261: 10583-10589

9. Eyzaguirre J (1987) Chemical Modification of Enzymes: ActiveSite Studies. John Wiley & Sons, New York

10. Gacheru SN, Trackman PC, Kagan MM (1988) Evidence for afunctional role for histidine in lysyl oxidase catalysis. J BiolChem 263: 16704-16708

11. Giannini JL, Gildensoph LG, Briskin DP (1987) Selective pro-duction of sealed plasma membrane vesicles from red beetstorage tissues. Arch Biochem Biophys 254: 621-630

12. Giannini JL, Holt JS, Briskin DP (1988) Isolation of sealedplasma membrane vesicles from Phytophthora megasperma f.sp. glycinea. I. Characterization of proton pumping and ATP-ase activity. Arch Biochem Biophys 265: 337-345

13. Gildensoph LH, Briskin DP (1989) Modification of an essentialarginine residue associated with the plasma membrane ATPaseof red beet storage tissue. Arch Biochem Biophys 271: 254-259

14. Gildensoph LH (1989) Chemical modification of essential aminoacid moieties associated with the red beet plasma membraneH+-ATPase. PhD dissertation, University of Illinois, Urbana

15. Grillo FG, Aronson PS (1986) Inactivation of the renal microv-illus membrane Na+-H+ exchanger by histidine-specific re-agents. J Biol Chem 261: 1120-1125

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

17. Hori R, Maegawa H, Kato M, Katsure T, Inui K (1989) Inhibitoryeffect of diethyl pyrocarbonate on the H+/organic cation anti-

port system in rat renal brush-border membranes. J Biol Chem264: 12232-12237

18. Izuhara K, Okubo K, Hamasaki N (1989) Conformational changeof Band 3 protein induced by diethyl pyrocarbonate modifi-cation in human erythrocyte ghosts. Biochemistry 28: 4725-4728

19. Kasamo K (1988) Essential arginyl residues in the plasma mem-brane H+-ATPase from Vigna radiata L. (mung bean) roots.Plant Physiol 87: 126-129

20. Kumagai H, Utagawa T, Yamada H (1975) Studies on tyrosinephenol lyase. J Biol Chem 250: 1661-1667

21. Leonard RT (1984) Membrane associated ATPases and nutrientabsorption by roots. In PB Tinker, A Lauchli, eds, Advancesin Plant Nutrition, Vol 1. Praeger, New York, pp 209-240

22. Levy HM, Leber PD, Ryan RM (1963) Inactivation of myosinby 2,4-dinitrophenol and protection by adenosine triphosphateand other phosphate compounds. J Biol Chem 238: 3654-3659

23. Lichtner RS, Wolf HU (1979) Dodecyl sulfate polyacrylamidegel electrophoresis at low pH values and low temperatures.Biochem J 181: 759-761

24. Lundblad RL, Noyes CM (1984) Chemical Reagents for ProteinModification, Vols I and II. CRC Press, Boca Raton, FL

25. Means GE, Feeney RE (1971) Chemical Modification of Pro-teins. Holden-Day, San Francisco

26. Melchior WB Jr, Fahrney D (1970) Ethoxyformylation of pro-teins. Reaction of ethoxyformic anhydride with a-chymotryp-sin, pepsin, and pancreatic ribonuclease at pH 4. Biochemistry9: 251-258

27. Miles EW (1977) Modification of histidyl residues in protein bydiethyl pyrocarbonate. Methods Enzymol 47: 431-442

28. Morjana NA, Scarborough GA (1989) Evidence for an essentialhistidine in the Neurospora crassa plasma membrane ATPase.Biochim Biophys Acta 985: 19-25

29. Mulhrad A, Hegyi G, HoranyiM (1969) Studies on the propertiesof chemically modified actin. III. Carbethoxylation. BiochimBiophys Acta 181: 184-190

30. Post R, Sen AK (1967) [32P]-labelling of a (Na+, K+)-ATPaseintermediate. Methods Enzymol 10: 762-768

31. Riordan JF, McElvany KD, Borders CL Jr (1977) Arginyl resi-dues: Anion recognition sites in enzymes. Science 195: 884-886

32. Sokol PP, Holohan PD, Ross CR (1988) Arginyl and histidylgroups are essential for organic anion exchange in renal brush-border membrane vesicles. J Biol Chem 263: 7118-7123

33. Xu, K-y (1989) Any of several lysines can react with 5'-isothio-cyanofluorescein to inactivate sodium and potassium ion ac-tivated adenisonetriphosphatase. Biochemistry 28: 5764-5772

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Modification of the Red Beet Plasma Membrane