Purification and Properties of Vacuolar Membrane Proton ...

T H E JOURNAL OF BIOLOGICAL CHEMISTRY Q 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 33, Issue of November 25, pp. 20068-20073,1989 Printed in U.S.A.

Purification and Properties of Vacuolar Membrane Protontranslocating Inorganic Pyrophosphatase from Mung Bean* (Received for publication, April 17, 1989)

Masayoshi MaeshimaS and Shizuo Yoshida From the Institute of Low Temperature Science, Hokkaido University, Sapporo 060, Japan

Inorganic pyrophosphatase was purified from the vacuolar membrane of mung bean hypocotyl tissue by solubilization with lysophosphatidylcholine and QAEToyopearl chromatography. The molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 73,000 daltons. Among the amino-terminal first 30 amino acids are 25 nonpolar hydrophobic residues. For maximum activity, the purified pyrophosphatase required 1 mM Mg2+and 50 mM K+. The enzyme reaction was stimulated by exogenous phospholipid in the presence of detergent. Excess pyrophosphate as well as excess magnesium inhibited the pyrophosphatase. The enzyme reaction was strongly inhibited byATP, GTP, and CTP at 2 mM, and the inhibition was reversed by increasing the Mg2+concentration. An antibody preparation raised in a rabbit against the purified enzyme inhibited both the reactions of pyrophosphate hydrolysis of the purified preparation and the pyrophosphate-dependent H+ translocation in the tonoplast vesicles. N,N’-Dicyclohexylcarbodiimidebecame bound to the purified pyrophosphatase and inhibited the reaction of pyrophosphate hydrolysis. It is concluded that the 73-kDa protein in vacuolar membrane functions as an H+-translocating inorganic pyrophosphatase.

have been purified from higher plants (5, 8,9) and fungi (10, 11) and characterized at biochemical and molecular levels (12, 13). The tonoplast inorganic pyrophosphatase has not yet been purified, although its enzymological properties in the membrane have been analyzed by some groups (6, 7, 14). We have been conducting a series of experiments to clarify the molecular properties of vacuolar membrane inorganic pyrophosphatase. This paper reports the purification and properties of the tonoplast inorganic pyrophosphatase from mung bean. The purified enzyme was found to be a distinct enzyme molecule from the tonoplast H+-ATPase and tohave unique enzymological and protein chemical properties, differing strikingly from soluble inorganic pyrophosphatases from yeast (15) and membrane-bound inorganic pyrophosphatases from rat liver mitochondria (16) and chromatophores of Rhodospirillum rubrum (17). EXPERIMENTALPROCEDURES

The vacuoles of higher plant cells, acidic organelles, have been shown to be sites of salt and metabolite deposition and of lysis of intracellular components(1,2). The vacuolar membranes have many active transport systems. Recently, attention has been directed to the mechanism and regulation of active transport systems including proton-transport systems in the membrane (3, 4). Studies with isolated vacuoles and tonoplast (plant vacuolar membrane) vesicles have shown the existence of two kinds of proton-translocating enzymes, namely inorganic pyrophosphatase (EC3.6.1.1)and H’-ATPase (5-7). Both proton-translocating inorganic pyrophosphatase and ATPase generate an electrochemical potential difference of protons across the vacuolar membrane, and some active transport systems of the vacuolar membrane work by a mechanism of H+/substrate antiport(3-5). Detailed studies onbothproton-translocating enzymes should promote an understanding of the mechanism of membrane transport processes and thespecific physiological roles of the pyrophosphatase andATPase in plantcells. Vacuolar membrane ATPases

Materials-Seeds of mung bean (Vigna radiata, cv. Wilczek) were inbibed with water and germinated in the dark at26 “C for 3.5 days. Hypocotyl tissue was carefully removed, and the rest of the seedling was discarded. Lysophosphatidylcholine (egg yolk, type I), phosphatidylcholine (soybean, type IV-S), and phosphatidylserine (bovine brain) were purchased from Sigma. ATP, ADP, CTP, UTP, and GTP were from Boehringer Mannheim. QAE-Toyopearl 550Cwas from Tosoh (Japan). Horseradish peroxidase-linked protein A and [“C] DCCD’ were from Amersham Corp., and 4-chloro-1-naphthol was from Bio-Rad. All other products used were of analytical grade. Tonoplast Preparation-Three hundred grams of hypocotyl from etiolated seedlings was homogenized with a Polytron PT30 in a total of 400ml of grinding medium containing 0.25 M sorbitol,5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1.5% polyvinylpyrrolidone, 1% ascorbic acid, and 50 mM Mops/KOH buffer, pH 7.6. The homogenate was filtered and centrifuged at 3,600 X g for 10 min. The supernatant was centrifuged a t 120,000 X g for20 min. The precipitate was suspended in 15 ml of 10 mM potassium phosphate (pH 7.8), 0.3 M sucrose, 1 mM EGTA, and 2 mM DTT and poured into a centrifugation tube. The suspension was overlayered with 10 ml of 5 mM Mops/KOH (pH 7.3), 0.25 M sorbitol, 1 mM EGTA, and 2 mM DTT. After centrifugation at 120,000 X g for 30 min in a Hitachi RP50 rotor, the interface portion was collected and diluted in a half-volume of 5 mM Mops/KOH (pH 7.3), 0.25 M sorbitol, 1 mM EGTA, and 2 mM DTT. Thesuspension was then centrifuged at 130,000 X g for 20 min, and theresulting white pellet(tonoplast vesicles) was suspended in 20 mM Tris acetate (pH 7.5), 20% glycerol, 1 mM DTT, 1 mM EGTA, and 2 mM MgClz (Tris/GDEM) at a final protein concentration of 1.5 mg/ml. Enzyme Purification-To the tonoplastsuspension were added solid KC1 and 5%deoxycholate a t final concentrations of 50 mM and

*This work was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture of Japan (to M. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This articlemusttherefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed.

‘ The abbreviations used are: DCCD, N,N’-dicyclohexylcarbodiimide; Mops, 3-(N-morpholino)propanesulfonicacid; Mes, 2-(N-morpho1ino)ethanesulfonic acid; DTT, dithiothreitol; SDS, sodium dodecyl sulfate; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; Bistris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3diol.

20068

Purification of Vacuolar Membrane Pyrophosphatase 2 mg/mg of protein, respectively, and thesuspension was centrifuged at 150,000 X g for 30 min. The pellet was suspended in Tris/GDEM buffer containing 0.4% lysophosphatidylcholine to bring the volume to 0.5 of the original tonoplast suspension. The suspension was centrifuged at 150,000 X g for 40 min at 8 "C after it hasbeen stirred for 10 min at 25 "C. Thesupernatant (solubilized fraction) was applied to a column (bed volume, 5 ml) of QAE-Toyopearl preequilibrated with Tris/GDEM. The column was washed with 15 ml of Tris/ GDEM buffer containing 50 mM NaC1, then pyrophosphatase was eluted from the column with 10 ml of Tris/GDEM buffer containing 120 mM NaCl. After the peak activity fraction had been diluted with 3 volumes of the buffer without NaCl, QAE-Toyopearl chromatography was again carried out. The enzyme solution was collected and stored a t -80 "C. Antibody Preparation and Immunoblot Analysis-Antibody against pyrophosphatase was raised in a rabbit by injection of purifiedenzyme preparation (0.4 mg). The immunoglobulin G fraction was prepared from the serum by ammonium sulfatefractionation and DEAEcellulose column chromatography as described previously (18). The electrophoretic blotting technique (19) was used to test theantibody specificity. The IgG that reacted to the antigen on a nitrocellulose membrane was detected with horseradish peroxidase-linked protein A using a color development reagent (4-chloro-1-naphthol). Amino Acid Sequence Analysis-Pyrophosphatase was completely purified by SDS-polyacrylamide gel electrophoresis and electrical elution from the gel as described previously (20). The purified preparation (100 pmol) was thoroughly dialyzed against 0.02% SDS and used for amino acid sequence analysis performed with a pulsed liquid phase sequenator (Applied Biosystems, model 477A) and an amino acid analyzer (model 120A). Labeling with [l4C/DCCD-Labeling of the purified pyrophosphatase with [14C]DCCD wasperformed by the method of Uchida et al. (11) with a few modifications. The tonoplast preparation (150 pg) and the purified enzyme (8 pg) suspended in 0.8 ml of 10 mM Tris acetate (pH 7.2) and 1 mM EDTA were incubated with 0.5 pCi of [14C]DCCD(56 mCi/mmol) at 20 "C for 60 min. The reaction was stopped by the addition of 0.2 ml of 50% trichloroacetic acid. After centrifugation, the pellet was rinsed with 0.5 ml of 10%trichloroacetic acid and then with 0.5 ml of 10 mM Tris acetate (pH 7.2) and 1 mM EGTA. The resulting precipitate was subjected to SDS-polyacrylamide gel electrophoresis. The gel was incubated in Enhance (Du Pont-New EnglandNuclear) for 1 h and thenrinsed with cold water. The gel was dried, then theradioactive bands were detected with Fuji x-ray film (RX) after 5 days of storage in the dark a t -80 "C. Analytical Measurements-Two to 10 pl of enzyme was assayed in 0.25 ml of 1 mM sodium pyrophosphate, 1mM MgS04,50 mM KC1,l mM sodium molybdate, 0.02% Triton X-100, 20 pg of phospholipid micelle suspension, and 30 mM Tris/Mes (pH 7.2). After incubation for 10-30 min a t 30 "C, the reaction was terminated, and theamount of Pi released was determined by the modified method of Hoges and Leonard (21). One unit of pyrophosphatase activity was defined as the amount of enzyme which hydrolyzed 1 pmol of PPi/min under the assay conditions. The formation of inside acid pH gradients across the membranes of the tonoplast vesicles was measured as the rateof fluorescence quenching of the permeant amine dye, acridine orange. Tonoplast vesicles (60 pg of protein) suspended in 5 mM Tris/Mes (pH 7.0), 0.25 M sorbitol, and 2 mM DTT were incubated a t 20 "C in 2.2 ml of reaction medium. The reaction medium contained 0.95 mM sodium pyrophosphate, 0.25 M sorbitol, 1 mM MgS04,50 mMKC1, 3 p~ acridine orange, and 25 mM Hepes/Bistris propane (pH 7.2). The changes in fluorescence were monitored with a Shimazu RF-5000 fluorescence spectrophotometer set a t 493 nm for excitation and 540 nm for emission. Protein content was measured by the method of Lowry et al. (22) after precipitation with trichloroacetic acid.Gel electrophoresis on 12%polyacrylamide gel containing 0.1% SDS was carried out by the method of Laemmli (23). RESULTS

Purification of the Tonoplast Pyrophosphatase-The procedure outlined under"Experimental Procedures" for the purification from tonoplast(Table I) was developed from preliminary trials in which the conditions for solubilization and stabilization of the enzyme were established. When the tonoplast suspension was treated with 0.3% deoxycholate in 50 mM KCl, 30% of the tonoplast protein was removed, but the pyrophosphatase was retained. On adding0.4% lysophos-

20069

TABLE I Purification of vacuolar membrane inorganic from mung *DVrODh0SDhta.W " ' " bean Total Total Recovery Specific Fraction units activity protein units/mg

%

mg

Vacuolar membrane 66.060.0 100 vesicles" 65.8 Lysophosphatidylcholine 43.4 21.1 extract 23.3 2.96 35.3 First QAE-Toyopearl eluate 5.36 0.635 8.1 Second QAE-Toyopearl eluate a The vacuolar membrane vesicles were prepared from mung bean hypocotyl tissue and stored a t -80 "C.

100" 80 -

1

-

-

40 -

-

60

30

2

3

1.10 2.06 7.86 8.45 2.7kgof

4

--

20"

FIG.1. SDS-polyacrylamide gel electrophoresis of purified tonoplast pyrophosphatase.Lane 1,tonoplast vesicles (34 pg); lane 2, lysophosphatidylcholine extract (24 pg); lanes 3 and 4, eluates of first and second QAE-Toyopearl chromatography (7 and 5 pg, respectively). The arrowheads indicate the positions of markers (94,67,45, 30, 20.1, and 14.4 kDa), and the figures on the ordinate indicate the scale used to estimate the molecular mass. phatidylcholine, the pyrophosphatase was solubilized. In the subsequent steps of QAE-Toyopearl column chromatography, glycerol (20%) andTriton X-100 (0.1%) were present throughout to maintain solubility and the activity. The tonoplast ATPase in thesolubilized fraction was separated from the pyrophosphatase by QAE-Toyopearl chromatography. As shown in Table I and Fig. 1, the procedure yielded a highly purified preparation inwhich only one band was visible after SDS-polyacrylamide gel electrophoresis. The molecular mass, by comparison with standards, was 73 kDa, and this band could be recognized as a major component of the tonoplast preparation (Fig. 1). The amino-terminal sequence of the isolated pyrophosphatase was as follows. 10

1

NHz-Gly-Ala-Ala-Ile-Leu-Pro-Asp-Leu-Gly-Thr20

Glu-Ile-Leu-Ile-Pro-Val-( )-Ala-Val-Ile-Trp30

Ile-Ala-Phe-Ala-Leu-Phe-Gln-Trp-Leu-

" " "

The 17th residue could not be determined. Nonpolar hydrophobic residues are underlined.

20070

Vacuolar Membrane Pyrophosphatase Purification of

Properties of the Pyrophosphatase-The pH optimum for the enzyme reaction of the purified preparation was 6.5-7.5, and all measurements were done at pH 7.2. The tonoplast pyrophosphatase did not hydrolyze ADP, ATP, GTP, UTP, ITP, NADP, or fructose 6-phosphate. As shown in Fig. 2, the reaction of pyrophosphatase was markedly stimulated by phospholipids and detergents, and 20 pg of phospholipid and 0.02% Triton X-100 were included in the assay medium. At high concentrations, Triton X-100 and deoxycholate inhibited the purified pyrophosphatase even in the presence of a sufficient amount of phospholipid, and the concentrationof both detergents for 50% inhibition was 0.1%. Pyrophosphatase activity depended strictly on the presence of Mg2+ (Fig. 3B) and was stimulated &fold by K+ salts (Table11).The enzyme activity was slightly stimulated by Mn2+ at 1 mM, but Co2+ and Ca2+ had no stimulatory effect on the enzyme activity. As shown in Fig. 3, A and B, pyrophosphatase activity was inhibited by excess M e and PPi. Thus, the relationship between the pyrophosphatase and the substrate concentration was analyzed by keeping Mg2+/PPi in a constant 1:l ratio. As shown in Fig. 3C, the substrateactivity curve was not a typical hyperbolic substrate saturation curve. It was therefore not possible to calculate the K,,, value of the tonoplast pyrophosphatase. The activity was in proportion tothesubstrate concentrationup to 0.6mM, suggesting that the enzyme reaction maybe afirst-order process. The half-maximum activity was obtained at 0.25 mM MgPPi. The standardassay medium contained 50 mM KCl, 1 mM sodium pyrophosphatase, and 1 mM MgS04. The effects of several inhibitors onpyrophosphatase activity from the peak fraction of QAE-Toyopearl chromatography were examined. KN03, azide, and vanadate are known to inhibit the tonoplast, mitochondrial, and plasma membrane ATPases, respectively. KF and molybdate are known as inhibitors of phosphatase. Nitrate (50 mM), molybdate (0.5 mM),and azide (2 mM) had essentially no effect on theenzyme reaction. Vanadate and fluoride a t high concentrations inhibited the reaction, and the concentrations for 50% inhibition were 2 and 5 mM, respectively. As shown in Table 111, the

1

0

2

3

0

Pyrophosphate (mM)

1

2

3

MgS04 (mM)

Pyrophosphate, MgS04 (mM)

FIG.3. Effects of concentrations of pyrophosphate ( A ) and magnesium ( B )on pyrophosphatase activity. A , activity of the purified enzyme was assayed in the medium containing 1 mM MgSO, and the indicated amount of sodium pyrophosphate. The activity is expressed as the percentage of the maximum activity (8.0 units/mg) obtained at 1 mM sodium pyrophosphateand 1 mM MgSO,. B, activity was assayed at 1 mM sodium pyrophosphate. C, pyrophosphatase activity was assayed in the medium containing the indicated amount of MgSO, and sodium pyrophosphate. Mp"/PPi was kept in a constant 1:l ratio.

TABLEI1 Effect of potassium on the activity of vacuolar membrane pyrophosphatase Salt

Concentration

M$+"

mM

Phospholipid oJg/assay) Detergent (%I FIG.2. Effects of phospholipids ( A ) and detergents ( B ) on activity of the purified pyrophosphatase.A, activity was assayed 50

in 0.25 ml of 0.02% Triton X-100 and the indicated amounts of phospholipid micelles. 0 ,bovine brain phosphatidylserine;0,soybean phosphatidylcholine. B, activity was assayed in the presence of 20 pg of soybean phosphatidylcholine. 0, Triton X-100;0, sodium deoxycholate; 0, sodium cholate.

Activity unitslmg

0.038 None +2.03 None 0.01 50 KC1 + 5.74 KC1 50 1.61 NaCl 5.84 50 KN03 + 6.27 K,SO, 25 Activity was assayed in the presence (+) or absence (-) of 1 mM MgSO,.

+ +

Purification of Vacuolar Membrane Pyrophosphatase 1

2

3

20071

4

DCCD (mM)

FIG.6. Effect of DCCD on pyrophosphatase activity. To the reaction mixture was added 50 mM DCCD dissolved in ethanol to the indicated concentration (0).As controls, the same volume of ethanol as DCCD solution was added to thereaction mixture (O), and DCCD solution was added to themixture before determination of the released Pi (A). The initial activity was 6.0 units/mg. FIG.4. Immunoblot analysis of specificityonanti-pyrophosphatase IgG. Lunes 1 and 2, gel stained with Coomassie Brilliant Blue; lunes 3 and 4 , immunoblot with anti-pyrophosphatase IgG and horseradish peroxidase-linked protein A. Lanes 1 and 3, tonoplast preparation (45 pg); lunes 2 and 4 , purified tonoplast pyrophosphatase (5 pg).

IA

I 0

5

1 0 0

Antlserurn (pllassay)

2

IgG

4

e

(JIllassay)

FIG.5. Effect of anti-pyrophosphatase IgG on the activity of purified pyrophosphatase ( A )and PPi-dependent H+ translocation activity in tonoplast vesicles ( B ) . A , the indicated amounts of anti-pyrophosphatase antiserum ( 0 )and control serum (0)were incubated with the purified pyrophosphatase (1.2 pg) a t 20 "C for 5 min, and then the pyrophosphatase activity was measured. The initial activity was 5.2 units/mg of protein without antiserum. B, various amounts of antipyrophosphatase IgG (0,14 mg/ml) and control IgG (0, 14 mg/ml) were added to the mixture containing tonoplast vesicles (60 pg), 0.95 mM sodium pyrophosphate, 1 mM MgS04, 50 mM KCl, 0.25 M sorbitol, 3 p M acridine orange, and 25 mM Hepes/Bistris propane (pH 7.2). After incubation a t 20 "C for 5 min, PPi-dependent H+translocation activity was measured a t 20 "C by following the decrease of the fluorescence of acridine orange. The initial activity was 20% change in fluorescence/min.

FIG.7. Electrophoresis of DCCD-binding polypeptide on SDS-polyacrylamide gel. Tonoplast preparation and purified pyrophosphatase were incubated with 0.5 pCi of [14C]DCCD (56 mCi/ mol) at 20 "C for 60 min and electrophoresed on SDS-polyacrylamide gel as described under "Experimental Procedures," and the DCCDbinding polypeptides were visualized by fluorography. Lunes 1 and 3, tonoplast preparation (45 and 150 pg); lunes 2, 4 , and 5, purified tonoplast pyrophosphatase (4,4, and 8 pg, respectively). Lanes 1 and 2, gel stained with Coomassie Brilliant Blue; lunes 3-5,fluorograph. The closed and open triangles indicate the tonoplast pyrophosphatase and theDCCD-binding subunit of tonoplast ATPase, respectively.

among the tonoplast proteins. As shown in Fig. 5, the antipyrophosphatase antibody inhibited not only the PPi hydrolysis of the purified enzyme(Fig. 5A) but also the proton translocation in tonoplast vesicles (Fig. 5B). DCCD Bincling und Inhibition-DCCD, which is thought to inhibit ATPases by blocking proton conductance (24), was an inhibitor of the tonoplast pyrophosphatase (Fig. 6). Fifty percent inactivation of the pyrophosphatase was measured at addition of ATP, GTP, andCTP decreased the enzyme activ- a concentration of 0.5 mM (71 pmol/mg of protein) DCCD, ity to less than 20% at 2 mM. The inhibition was reversed by which is higher than the concentration that inhibits the increasing the concentration of Mg2+ to the sum of PPi and mitochondrial ATPase (5), the plasma membrane ATPase the nucleotide. (25), and tonoplast ATPase (26). We next examined whether Antibody Experiments-Antibody to thepurified pyrophos- or not DCCD binds to the enzyme. When the tonoplast phatase was raised inarabbitand partially purified by preparation and the purified enzyme were incubated with (NH4),S04fractionation and DEAE-cellulose chromatogra- [14C]DCCD,the pyrophosphatase and three other polypepphy. As shown by the immunoblot analysis in Fig. 4, the anti- tides (36, 24 and 16 kDa) of thetonoplastproteins were pyrophosphatase IgG reacted only with the pyrophosphatase labeled as shown in Fig. 7. The results suggest that the

Vacuolar Membrane Pyrophosphatase Purification of

20072

tonoplast pyrophosphatase may have a region that forms a proton channel initself. Judging from our preliminary experiments, the 16-kDa polypeptide is the DCCD-binding subunit of tonoplast ATPase; the properties of the 36- and 24-kDa polypeptides remain to be characterized. DISCUSSION

This study was done to purify and characterize the vacuolar membrane pyrophosphatase. The purification described here depends on the selective solubilization of proteins of the vacuolar membrane preparation by deoxycholate and lysophosphatidylcholine and theinclusion of glycerol and Triton X-100 in the QAE-Toyopearl chromatography. SDS-polyacrylamide gel electrophoresis gave a single band of molecular mass 73 kDa, which corresponded to a major constituent of the vacuolar membrane fraction (Fig. 1).The molecular mass of the tonoplast pyrophosphatase differs from the membrane inorganic pyrophosphatase from rat liver mitochondria (subunits, 28 and 35 kDa; Ref. 16) and soluble inorganic pyrophosphatase from yeast (32 kDa; Ref. 27), soluble inorganic pyrophosphatase from S. vulgure chloroplast (42 kDa; Ref. 28). The present study revealed two functions of the 73-kDa protein: PPi hydrolysis and proton translocation across the vacuolar membrane. Findings supporting the proton-pumping ability of the pyrophosphatase are: (i) [14C]DCCDlabeled the purified pyrophosphatase (Fig. 7); (ii)the PPi hydrolysis reaction of the purified enzyme was inhibited by DCCD (Fig. 6); and (iii) the anti-pyrophosphatase antibody inhibited the pyrophosphate-dependentproton-pumping activity inthe tonoplast vesicles (Fig. 5). PPi hydrolysis seems to be coupled with proton transport. The low sensitivity of the tonoplast pyrophosphatase to DCCD must be examined further. The region from the amino terminus to the 30th residue of the pyrophosphatase was very hydrophobic because 25 residues in the region were nonpolar hydrophobic amino acids, which indicates that the amino-terminal region may be a part of membrane domain of the enzyme. There was no sequence TABLEIII Effect of nucleotides on the activityof vacuolar membrane pyrophosphatase Activity of the purified enzyme was assayed in the medium containing 1 mM sodium pyrophosphate and indicated amounts of nucleotide and MgSOd. Nucleotide

M P

mM

mM

Activity unitslmg

%

None ATP

1

5.98

1 2 1 2

1 1

3.08 0.661 5.85 5.11

52 11 98 86

3.61 0.806 5.88 5.28

60 14 98 88

4.63 1.20 5.85 5.28

20 98 88

2

100

GTP 1 2 1 2

1 2

homologous to this part in the soluble inorganic pyrophosphatase from yeast (27),which suggests that thehydrophobic region of the tonoplast enzyme is peculiar to the protontranslocating pyrophosphatase. Both PPi and MgZ+ inhibited the tonoplast pyrophosphatase at more than 1.5 mM (Fig. 3, A and B ) . The actual substrate of pyrophosphatase is not simply pyrophosphate but rather aparticularmetal complex such as MgPP:- (29). Divalent cation is required in stoichiometric amounts toform the active substrate. Free PPi and MgzPPi are competitive inhibitors of pyrophosphatase (29). Thus, the enzyme in the reaction medium containing a high amount of PPi may be bound competitively by free PPi. The inhibition of activity by excess M e is thought to be due to thesequestering of PPi as inactive MgzPPi. The inhibition by ATP, GTP, and CTP (Table 111) is a unique property of the tonoplast pyrophosphatase. The extent of inhibition by the nucleotides was reduced by increasing the concentration of M e . The results suggest that thenucleotides and pyrophosphate may compete for Mg2+ in the reaction medium. At present, the molecular mechanisms and physiological significance of stimulation by K' and inhibition by the nucleotides are not clear, but these properties can beused to distinguish the tonoplast pyrophosphatase from other soluble and membrane-bound pyrophosphatases. Natural phospholipid stimulated the pyrophosphatase activity about %fold in the presence of 0.02% Triton X-100 (Fig. 2). The requirement of phospholipid for the activity suggests that tonoplast pyrophosphatase is an integral membrane protein and active in a phospholipid-protein complex. The purified enzyme may need Triton X-100 at a concentration slightly higher than the critical micelle concentration to be reconstituted into the phospholipid bilayer. At high concentrations, however, detergents such as Triton X-100 and deoxycholate may depress the activity by disturbing the interaction between the enzyme protein and phospholipid. Inorganic pyrophosphate is a high energy phosphate compound. The amount of PPi in higher plant cells was determined to be about 20-40 nmol g-', fresh weight, and the concentration of the PPi in the cytoplasm was estimated to be about 0.2 mM (6, 30). Referring to Fig. 3, this PPi concentration is sufficient for the tonoplastpyrophosphatase to express hydrolytic activity at about 40% of the maximum rate. The present work shows that thetonoplast pyrophosphatase exists as a major component of the vacuolar membrane with high enzymatic activity. Further study of the enzyme at molecular and cell biological levels may shed more light on the structure-function relationship and the functional significance of the tonoplast pyrophosphatase. Acknowledgments-We are grateful to Professor T. Asahi, Nagoya University, for his stimulating discussions. We wish to thank K. Sasaki, Center for InstrumentalAnalysis, for her contribution to the amino acid sequence analysis. REFERENCES 1. Wagner, G. J. (1982) inRecentAdvances

CTP 1 2 1 2

77

1 1

2

ITP 1 2

83

1

6.09 4.96

1

6.14 4.90

102

ADP 1 2

103

2. 3. 4.

5. 82

6.

in Phytochemistry (Creasy, L., and Hrazdina, G., eds) Vol. 16, pp. 1-45, Plenum Publishing Corp., New York Nishimura, M.& Beevers, H. (1979) Nature 277,412-413 Anraku, Y. (1987) in Bioenergetics:Structureand Function Of Energy TransducingSystems (Ozawa,T., and Papa, S., eds) PP. 249-262, Springer-Verlag,Berlin Poole, R. J., Sarafian, V. & Blumwald, E. (1987) in Plant Membranes: Structure,Function,Biogenesis (Leaver, C., and Sze, H., eds) pp. 209-222, Alan R. Liss Inc., New York Sze, H. (1985) Annu. Rev. Plant Physiol. 36,175-208 Chanson, A., Fichmann, J., Spear, D. & Taiz, L. (1985) Plant Physiol. 79,159-164

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19. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354 20. Nakagawa, T., Maeshima, M., Muto, H., Kajiura, H., Hattori, H. & Asahi, T. (1987) Eur. J. Biochern. 165, 303-307 21. Hodges, T. K. & Leonard, R. T. (1974) Methods Enzyrnol. 32, 22. Lowry, 0.H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275 23. Laemmli, U. K. (1970) Nature 227, 680-685 24. Solioz, M. (1984) Trends Biochern. Sci. 9, 309-312 25. Kimura, T., Maeshima, M. & Asahi, T. (1988) Plant Cell Physiol. 29,883-888 26. Randall, S. K. & Sze, H. (1986) J. Biol. Chem. 261, 1364-1371 27. Cohen, S. A,, Sterner, R., Keim, P. S. & Heinrikson, R. L. (1978) J. Biol. Chem. 253,889-897 28. Krishnan, V. A. & Gnanam, A. (1988) Arch. Biochem. Biophys. 260,277-284 29. Josse, J. & Wong, S. C. K. (1971) in The Enzymes (Boyer, P. D., ed) 3rd Ed., Vol. 4, pp. 499-527, Academic Press, New York 30. Smyth, D.A. & Black, C. C. (1984) Plant Physiol. 75,862-864