Phosphoryl Group Exchange between ATP and ADP Catalyzed ... - NCBI

5 downloads 27 Views 659KB Size Report
cation (Glynn, 1985). The formation of E-P from ATP leads to the release of ADP and, later, Pi in a uni-bi-ordered reaction. In a reversible cycle, as is the case ...
Plant Physiol. (1997) 114: 1397-1403

Phosphoryl Group Exchange between ATP and ADP Catalyzed by H+-ATPase from Oat Roots' Custavo Helguera and Luis Beaugé* División de Biofísica, Instituto de lnvestigación Médica "Mercedes y Martín Ferreyra," Casilla de Correo 389, 5000 Córdoba, Argentina ATPases, an ATP synthesis resulting from the interaction of ADP with some form of E-P has been well characterized (Andersen, 1989; Beaugé and Glynn, 1979). Likewise, this has also been observed with the synthesis of ATP, starting with the interaction of the enzyme with Pi, or ATP-Pi exchange, in the PM H+-ATPase from yeast (Malpartida and Serrano, 1981a, 1981b; de Meis et al., 1987). ATP-Pi exchange requires the interaction of ADP with the phosphoenzyme; however, we are not aware of any report of a phosphoryl group exchange between ATP and ADP catalyzed by any plant H+-ATPase, the existence of which has been suggested by Briskin and Poole (1983). In the present work we describe and characterize, for the first time to our knowledge, an ATP-ADP exchange reaction catalyzed by the PM H+-ATPase from higher plants.

ATP-ADP exchange was estimated i n the presence of plasma membrane H+-ATPase of oat (Avena sativa) roots partially purified with Triton X-1 O0 by measuring ['4C]ATP formation from [14CIADP. Most studies were done at 0°C. At p H 6.0 the exchange showed: (a) Mg2+ requirement with a biphasic response giving maximal activity at 152 p~ and (b) insensitivity t o ionic strength, [Na+], and [K+l. ATP and ADP dependence were analyzed with a model in which nucleotide-enzyme interactions are at rapid-random equilibrium, whereas E,ATP E,P-ADP transitions occur in steady state. The results indicated competition between ADP and ATP for the catalytic site, whereas ATP interaction with the ADP site was extremely weak. At 0°C the exchange showed a 3-fold p H increase, from p H 5.5 t o 9.0. At an alkaline p H the reaction was not affected by sodium azide and carbonyl cyanide p-trifluometoxyphenylhydrazone, had a biphasic response t o Mgz+(maximal at 51 3 pm), and was insensitive to ionic strength. At 20°C ATP-ADP exchange was p H insensitive. At both temperatures ATP hydrolysis displayed a bell-shaped response, with a maximum around p H 6.0 t o 6.5. Because no adenylate kinase activity was detected under any condition, these results demonstrate the existence of an ATP-ADP exchange reaction catalyzed by the plant H+-ATPase.

MATERIALS A N D METHODS Source of Enzyme

Seeds of oat (Avena sativa L. Seagram var) were obtained commercially. Oat roots were grown hydroponically for 7 d in deionized water at 20°C in the dark. For the preparation of the enzyme, the method of Vara and Serrano (1982) was followed with some modifications. A11 of the operations were carried out at 4°C. Oat roots (100 g) were cut and homogenized in 150 mL of a medium containing SUC30% (w I w), 250 IXIM Tris-HC1, pH 8.5, 25 mM EDTA, 2.5 m M DTT, and 8.1 mg mL-l PMSF. The homogenate was passed through four layers of cheesecloth and the filtrate was centrifuged for 25 min at 6,OOOg. The pellet was discarded and the supernatant, containing the microsomal fraction, was centrifuged at 85,OOOg for 30 min. The pellet (microsomal fraction) was resuspended in GTED 20 (20% glycerol, 10 mM Tris-HC1 [pH 7.6 at room temperature], 1 mM EDTA-Tris, and 1 mM dithiothreitol) and applied to a discontinuous SUCgradient (3346%, w / w ) with TED (10 mM of Tris-HC1 [pH 7.6 at room temperature], 1 mM of EDTA-Tris, and 1 mM of DTT). After centrifugation for 120 min at 85,00Og, the interface fraction enriched in PM was removed, washed according to Hodges and Leonard (1974), resuspended in GTED 20, and stored at -20°C at 4 mg total protein mL-'. For Triton X-100 treatment 1.5 mg mL-' of the PM fraction was incubated for 10 min at 0°C with 3.7 mg mL-' of the detergent in 0.5 M (final concen-

The PM H+-ATPase from higher plants belongs to the family of (E-P)ATPases that catalyze cation transport across membranes (Serrano, 1990). These enzymes are also involved in Na+, K+, and Ca2+ transport (Goffeau and Slayman, 1981; Serrano, 1990) and share a basic part of the reaction cycle involving the formation and subsequent hydrolysis of an acid-stable acyl-phosphate intermediate, E-P. The cycles also include the shifting between two conformational states: (a) E,, which represents the enzyme with a high affinity for the substrate and the cytoplasmic cation to be transported; and (b) E,, which has a low affinity for the substrate and the intracellular-transported cations. In coupled systems such as the Na+,K+-ATPase system, the E, state has a high affinity for the extracellular-transported cation (Glynn, 1985). The formation of E-P from ATP leads to the release of ADP and, later, Pi in a uni-bi-ordered reaction. In a reversible cycle, as is the case with a11 studied This work was supported by grants from Consejo Nacional de Investigaciones Científicas y Técnicas, Consejo de Investigaciones Científicas y Technológicas de la Provincia de Córdoba (3511/95), and Volkswagen-Stiftung (I/72 122). This work is part of the fulfillment for G.H. for a doctoral degree. * Corresponding author; e-mail [email protected];fax

Abbreviations: AP,A, PM, plasma membrane.

54-51-695163. 1397

P',P5-di(adenosine-5')pentaphosphate;

1398

Plant Physiol. Vol. 114, 1997

Helguera and Beaugé

tration) KCI (Serrano, 1988). After centrifugation at 85,OOOg for 45 min in the cold, the pellet was resuspended in GTED 20 at a final protein concentration of 4 to 6 mg mL-'. Aliquots of 100 PL of this enzyme suspension were stored at -20°C until use. Biochemical Determinations

ATP hydrolysis was determined by following the [32P]Pi released from [y-32P]ATP of known specific activity by isobutanol benzene extraction (de Meis and Carvalho, 1974). The total amount of hydrolysis never exceeded 2 to 4%; this means that even at 0°C there is still a substantial overall cycle of ATP hydrolysis. Therefore, in each experimental condition, ATPase activity was simultaneously determined and the rate of exchange was corrected for ATP hydrolysis according to Beaugé and Glynn (1979). The ATP-ADP exchange rate was determined from the [14C]ATP formed from [14C]ADP (Beaugé and Campos, 1986). The nucleotides were separated on Dowex 1 X 8 4 0 0 resin columns. The total eluent volumes passed were 7 mL of 10 mM HCl, 30 mL of 20 mM HCI-NH4Cl, and 15 mL of 250 mM HC1. In a11 of the cases the exchange fraction never exceeded 10% of the expected equilibrium fraction. To reduce the fractional ATP breakdown most experiments were performed at OOC; however, some studies on the pH effects were also carried out at 20°C. Solutions contained 50 mM Mes-Tris, p H 6.0, or Tris-HC1, pH 9.0, 0.5 mM EGTA, 0.16 mM ammonium molybdate, 1 mM EDAC (1-ethyl-3-[3dimethylamino-propyl] carbodiimide), and 5 mg mL-I oligomycin. ATP, ADP, Mg2+ concentrations, and other modifications are indicated in the figure legends. Before starting the reaction by the addition of the nucleotides, the enzyme was preincubated 12 min in the reaction medium without the nucleotides. Except when ATP and ADP dependente were investigated, the concentrations routinely used were 3 mM ATP and 2 mM ADP. Total protein concentration ranged from 0.05 to 0.5 mg mL-l in a reaction medium of 100 pL.

RESULTS Mgz+ Dependence

The presence of Mg2' ions is an absolute requirement for the activity of a11 transport ATPases, including the plant PM H'-ATPase (Brooker and Slayman, 1983; Serrano, 1984). On the other hand, in the Na+,K+-ATPase, the optimal free Mg2+ i s much higher for the overall ATP hydrolysis (about 1 mM) than for the ATP-ADP exchange (about 20 p ~ (data ) calculated from Beaugé and Glynn, 1979; Beaugé and Campos, 1986). Therefore, we started by exploring the Mg2+ dependence of both reactions in the plant H+-ATPase. These results are illustrated in Figure 1, A and B. For the H+-ATPase activity (Fig. 1A) the Mg2+ dependente follows a Michaelian function with a K , of 120 f 5 p ~ Figure . 1B shows that at p H 6.0 the response of the exchange reaction to [Mg"] is biphasic, with an optimum at 150 p ~ On . the other hand, the 21% inhibition observed at 4.2 mM Mg2+ is less marked than that seen with the Na+,K+-ATPase (Beaugé and Glynn, 1979).

a I

1 B

L i

Protein Determination

Total protein concentration was determined by the method of Bradford (1976) and modified by Read and Northcote (1981) using BSA as a standard.

l

i

Solutions

A11 solutions were made with Milli-Q water (Millipore) and the reagents were of an analytical grade. Vanadium-free ATP and ADP was from Boehringer Mannheim. Ionized Mg2+ concentrations were estimated with the Maxchelator Program (Hopkins Marine Station, Pacific Grove, CA) and dissociation constants were corrected for temperature, ionic strength, and pH. [Y-~'PJATPwas enzymatically labeled by the method of Glynn and Chappell(1964), as modified by de Meis (1972). [14C]ADPwas from DuPont-New England Nuclear and [32P]Piwas purchased from the Comisión Nacional de Energia Atómica (Buenos Aires, Argentina).

O

1

2

3

4

5

tMg2'1 (mM) Figure 1. H'-ATPase activity (A) and ATP-ADP exchange (6) at different [Mg'+] in H+-ATPasefrom oat root PM treated with Triton X-I 00. H+-ATPaseactivity was calculated from the [32Plphosphate released from [y-32P]ATP. The exchange rate was estimated on the basis of the [14C]ATP formed from ['4C]ADP. The H+-ATPaseactivity (A) is stimulated by Mgz+ following a Michaelian function with a K,,, of 120 2 3 p~ Mg2+. On the other hand, a biphasic response can be observed for the ATP-ADP exchange (B) with the peak stimulation at 150 PM Mg2+; higher [Mg2']s are slightly inhibitory. Each point is

the mean

? SE

of triplicate determinations. See text for details.

1399

ATP-ADP Exchange by Plant H+-ATPase

The Mgz+ dependence of the ATP-ADP exchange rate was also explored at pH 9.0 (see below). At this stage we wish to point out that the response to [Mg"] was also biphasic, but with a maximum at 510 p~ Mg2+.The highest [Mg"] investigated, 3.4 mM, produced a 27% inhibition, which is not very different from that seen at p H 6.0 (not shown). The possible effects of the Na+ and K t ions on H+ATPase and ATP-ADP exchange activities were also studied at 0°C at concentrations between O and 100 mM. On the whole, and except for an inhibition of H+-ATPase activity by [K+] above 40 mM, no other significant effect was observed (not shown).

4 ,

1

O

We next studied the dependence of this ATP-ADP exchange reaction on the concentrations of ATP and ADP. Two series of experiments were done, a11 at O"C, pH 6.0, and 152 p~ free Mg2+. In the first experiment (Fig. 2A), the concentration of ATP was varied between 0.05 and 3 mM at two fixed concentrations of ADP, 0.2 and 2 mM. In the second experiment (Fig. 2B), the concentration of ADP was varied between 0.05 and 2 mM at two fixed ATP concentrations, 0.3 and 3 mM. The points in the figures correspond to the mean t- SE of triplicate determinations. The ATP and ADP dependence were analyzed on the basis of a simplified kinetic model (Fig. 3) with the following assumptions: (a) ATP can compete with ADP for E,P, but it does not affect E,P dephosphorylation; (b) ADP can compete with ATP at a single catalytic site, but it does not phosphorylate E,; (c) ATP and ADP unions are in rapid equilibrium; (d) the E,.ATP e+ E,P.ADP transitions are at steady state; and (e) ATP hydrolysis is not included in this scheme; however, exchange estimations were always corrected for the simultaneous ATPase activity. The model, solved with the KingAltmann approach as modified by Cha (Segel, 1975)' gave the following equation: k,k-lDT[Et]

+ T) + k,T(K', + D)

2

3

[ATP] mM

Dependence of ATP and ADP Concentrations

k-,D(KIT

0.2mMADP 2mMADP

1

V =

I

I

(1)

where Et is the total enzyme; k , is the forward rate constant for the E,.ATP-E,P.ADP transition (the rate constant for enzyme phosphorylation); k - , is the backward rate constant for the E,.ATP-E,P.ADP transition (the rate constant for ATP synthesis); T = pmol ATP; D = pmol ADP; Ks, is the dissociation constant for the E,.ATP complex; Ks, is the dissociation constant for the E,P.ADP complex; K', = (Ks, [ l + Ki,/[ADP]]); K ' , = ( K s , [l + KiT/[ATP]]); Ki, is the dissociation constant for the E1.ADP complex (inhibition constant for ADP); and Ki, is the dissociation constant for the E1P.ATP complex (inhibition constant for ATP). The curves through the points (in Fig. 2, A and B) are the simultaneous best fit of a11 of the data to Equation 1 done with the Scop Program (Simulation Resources, Inc., Berrien Springs, MI) using the 2 criterion. As this figure illustrates, a good fit was observed. Note that the actual fitting parameters are six because the apparent affinity constants are fully determined by the dissociation and inhibition

4 ,

I

I

I

1

O ?

O

O

3mMATP

I

I

I

1

2

3

[ADP] mM Effect of variable [ATP] at two fixed [ADPI (A) and of variable [ADP] at two fixed [ATP] (6) on the ATP-ADP exchange catalyzed by Hf-ATPase from oat root PM treated with Triton X-1 O0 at pH 6.0. In A the [ATP] varied from 50 p~ to 3 mM at 0.2 and 2 mM ADP. In B [ADP] varied from 50 p~ to 3 mM at 0.3 and 3 mM ATP. The incubation times were 5 to 30 min, according to the optimal equilibrium fraction calculated for each condition. The experimental points correspond to the mean i SE of triplicate determinations. The lines are the best simultaneous fit of all the data points from both figures to Equation 1 . See text for details. Figure 2.

constants. The number of data points taken was 78; this means 13 points per parameter, a more than reasonable ratio for an adequate fitting. The values of the different constants are given in Table I. Their implications will be addressed in "Discussion." Effect of Proton Concentration

Previous works on the pH profile of the H+-ATPase activity, assayed at 20°C and in the absence of ADP, showed that it is bell-shaped with a maximal rate at 6.5 (Serrano, 1990; Roberts et al., 1991). The left side of Figure 4 describes experiments at 0°C analyzing the pH effects on ATPase (A) and ATP-ADP exchange (B) reactions in the presence of 3 mM ATP and 2 mM ADP. The H'-ATPase activity displayed an approximately bell-shaped behavior with a maximum at pH 6.0 and decreasing to zero at pH

1400

Helguera a n d

Beaugé

Plant Physiol. Vol.

50

‘\ Ki,

E,P T \

40

I

1

I

alyzed by PM H+-ATPase from higher plants. Shaded regions belong to rapid equilibrium segments, and the rest belongs to the steadystate segment. The details of the model are given in the text.

9.0. On the other hand, the exchange reaction remained practically unaffected from pH 5.5 to 7.0, whereas from that value it sharply increased to reach a 3-fold factor at pH 9.0. In the absence of any bound ligand, the conformation equilibrium of ATPases is strongly temperature dependent. In Na+,K+-ATPase at 0°C about 40% is E,; that percentage is increased to 90% at 20°C and to 99% at 37°C (Repke and Schon, 1992). It is not unlikely that in the plant H+-ATPase high temperatures also favor the E, state. Given the possi~~

I

I

I

A

-

30 -

C -

800

_-

600

20

-

__

400

10

-

__

200

1v O

I

I

T.

+=?B

6 3

Figure 3. Simplified model for the ATP-ADP exchange reaction cat-

I

I

1 1 4, 1997

o -

I

I

O

I

D 60 45 30 15 O

Figure 4. p H effect on ATPase activity (A-C) and ATP-ADP exchange (B-D) catalyzed by the H+-ATPase from oat root PM treated with Triton X-l 00. The experiments were performed at 0°C (A and B) and 20°C (C and D) between p H 5.5 and 9.0 in media of the following composition: 3 mM ATP; 50 mM Mes-Tris, p H 5.5 and 6.0; 50 mM Tris-HCI, p H 7.0, 8.0, and 9.0; 0.5 mM EGTA; 1 mM EDAC; 0.16 mM ammonium molybdate; 5 mg mL-’ oligomycin; and 8 mM MgSO,. The incubation time was between 1 O and 30 min a i the different pHs and temperatures. Note that a i O and 20°C the H+-ATPase activity displays bell-shaped behavior with a maximum a i p H 6.0 and nil at p H 9.0 (A-C), whereas the exchange rate a i O” increases 3-fold from p H 7.0 to 9.0 (B), and at 20°C the p H shows no stimulation (D). Each point is the mean +- SE of triplicate determinations. See text for detai Is.

Table 1. Values for ATP- and ADP-enzyme dissociation and inhibition constants and forward and backward rate constants for the transition

16

E ,.ATP-E,P.ADP

The values were obtained from the simultaneous fit of the data of Figure 2 to Equation 1. Note that due to the extremely weak interaction of ATP with E, P the true and apparent dissociation constants for the E, P.ADP complex have the same values. Type of Constant”

..-

h

0Control fg@ + AP,A

LT

Value

Unidirectional rate

3.6 i 0.7 min-’ 6.1 i 1.2 min-’

kl

k-

1

True dissociation Ks,KST True inhibition Ki, Ki,Apparent dissociation %o., G

3

Kh.2 KI”

792 5 158 p~ 564 i 133 .LLM

4

2993 2 599 p M 2 0.8 x 109 p~

x 10’

792 2 158 p~ 792 2 158 p~

601 2 120 p M 941 2 188 UM

a The meaning of the symbols is in text except: Kb,,,, apparent dissociation constant for the E,P.ADP complex ai 0.3 mM ATP; Kb3, apparent dissociation constant for the E,P.ADP complex ai 3 mM ATP; K;o,2, apparent dissociation constant for the E, .ATP complex a i 0.2 mM ADP; and K;2, apparent dissociation constant for the E, .ATP complex at 2 mM ADP.

O pH 6.0

pH 9.0

Figure 5. [14C] labeling of ATP in the presence of cold ATP and [14C]ADP catalyzed by H+-ATPase preparation from oat root PMs treated with Triton X-I00 at p H 6.0 and 9.0 at 0°C and in the presence and absence of AP,A. The reaction was determined in 3 mM ATP, 2 mM [l4C1ADP, 50 mM Mes-Tris, p H 6.0, or 50 mM Tris-CI, p H 9.0, 0.5 mM EGTA, 1 mM EDAC, 0.1 6 mM ammonium molybdate, 5 nig mL-’ oligomycin, and 1 or 4 mM MgSO, at p H 6.0 and 9.0, respectively, and in the presence/absence of 0.2 mM AP,A. The incubation time was 25 min at p H 6.0 and 15 min ai p H 9.0. Observe that the inhibitor has no effect on the [l4C1ATP labeling. Each bar is the mean 5 SE of triplicate determinations. See text for details.

ATP-ADP Exchange by Plant H+-ATPase

A

T

T

0AMP @@B ATP il

0AMP

'

T

@%ATP %%I

-+ATP

-ATP

- AP,A

+ATP

-ATP

+ AP,A

Figure 6. [l4C1AMP production and [l4C1ATP production or synthe-

sis catalyzed by H+-ATPase from oat root PM treated with Triton X-1 O 0 at pH 6.0 (A) and 9.0 (B) in the presence and absence of AP,A and ATP. The experiments were carried out at O"C, pH 6.0 and 9.0 in the following media: O and 3 mM ATP, 2 mM ADP, 50 mM Mes-Tris, pH 6.0, or 50 mM Tris-HCI, pH 9.0, 0.5 mM ECTA, 1 mM EDAC, 0.16 mM ammonium molybdate, 5 mg mL-' oligomycin, and 1 or 4 mM MgSO, at pH 6.0 and 9.0, respectively, and in the presence/absence of 0.2 mM AP,A. The incubation time was 25 min at pH 6.0 and 15 min at pH 9.0. Note: (a) there is no net synthesis of ['4CIAMP in any condition; (b) there is no net synthesis of [14C]ATP in the absence of cold ATP; and (c) the kinase inhibitor has no effect o n the [14C] labeling of ATP in the presence of 3 mM ATP. Each bar is the mean +SE of triplicate determinations. For details, see text.

bility that this shift also applies to the E,P-E,P equilibrium, if the pH effect is related to an increase in E,P, we might observe that at high temperatures: (a) ATP-ADP exchange increases less than ATP hydrolysis, and (b) the effect of alkalinization is modified. Accordingly, we explored this point at 20°C. These results are shown in Figure 4, C and D. As expected, the ATPase activity also displayed a bellshaped behavior, with the peak stimulation at pH 6.0; at that point the rate of hydrolysis was about 20-fold higher than that observed at 0°C. On the other hand, ATP-ADP exchange, which increased 8-fold over the maximum at O'C, was completely insensitive to pH.

Specificity of the Exchange Assays

A major problem with the determination of [I4C]ATP production in this preparation was the simultaneous pres-

1401

ente of an adenylate kinase activity, particularly efficient at alkaline pH. This enzyme catalyzes the reaction of two ADP molecules, leading to the synthesis of one molecule of ATP and the release of one AMP. Severa1experiments were done to investigate this point using 200 PM of the specific adenylate kinase inhibitor AP,A; note that at 1 FM this compound produces 98% inhibition of the rabbit muscle enzyme (Lienhard and Secemski, 1973). Figure 5 shows that there is no effect of AP,A on the rates of ATP-ADP exchange either at pH 6.0 or 9.0. Figure 6 summarizes the results at pH 6.0 (A) and pH 9.0 (B) on the appearance of [14C]ATP, or its net synthesis, and on the production of [14C]AMP when the enzyme preparation was incubated with 2 mM [14C]ADPin the presence and absence of 3 mM ATP. It can be shown that: (a) with [14C]ADPand ATP, the appearance of [14C]ATPis the same without and with the inhibitor (it concurs with Fig. 5 ) . In addition there is no production of [14C]AMPin any case; and (b) with [14C]ADP alone there is no net synthesis of either ATP or AMP in the absence or presence of AP,A. The facts that (a) AP,A has no effects at concentrations expected to be more than saturating, and (b) that ADP alone does not induce net synthesis of ATP, clearly indicate that in our preparation there is no detectable adenylate kinase activity that could bias the ATP-ADP exchange results. We also ruled out any effect due to contamination by mitochondrial H'-ATPase. At pH 6.0 and 9.0 the ATPase activity and the ATP-ADP exchange rate were not affected by sodium azide and FCCP (carbonyl cyanide p-trifluometoxyphenyl-hydrazone) (data not shown).

DISCUSSION

The work presented here shows that the plant HtATPase enzyme can catalyze a sizable phosphoryl group exchange between ATP and ADP. The existence of this reaction is predicted from the overall cycle suggested earlier for this enzyme (Briskin, 1990) and outlined in Scheme I, but up to now it had never been experimentally verified. The two main results we present have to do with the nucleotide requirements and interactions and the effects of pH. The model in Figure 3, which concentrates on the phosphoryl group-exchange part of the ATPase cycle, can adequately account for the nucleotide dependence of the exchange process. It could be argued that this approach is an oversimplification, particularly since it does have the two simultaneous ATP-binding sites postulated for this enzyme (Roberts et al., 1991, 1995). In our view the simplification is justified because leaving out the overall ATPase cycle, it is unlikely that the regulatory site (or role) of ATP will come into play. Some of the conclusions that can be drawn from the fitting parameters (see Table I) are indeed very interesting. On the one hand, there is a similar true affinity of the catalytic site for both nucleotides (about 40% smaller for ADP); this means that ADP can effectively compete with ATP for that site. On the other hand, the affinity for ATP of the ADP-binding site in E,P is so low that in practice ATP does not influence ADP binding and dephos-

Helguera and Beaugé

1402

ATP

ACKNOWLEDGMENTS

ElATP

E1

Plant Physiol. Vol. 1 14, 1997

EIP.ADP

i

The authors thanks Myriam Siravegna for her skillful technical assistance and Drs. Graciela Berberidn and Gretel Roberts for their critica1 reading of the manuscript. Received February 24, 1997; accepted April 28, 1997. Copyright Clearance Center: 0032-0889/97/ 114/1397/07.

Pi Scheme 1.

phorylation. These two characteristics have also been observed in the Na+,K+-ATPase (Beaugé and Glynn, 1979; Klodos and Norby, 1988). The fact that high p H completely inhibits ATP hydrolysis while it enhances ATP-ADP exchange (at OOC) or does not affect this reaction (at 20°C) indicates that, whatever step is involved in ATPase inhibition, it cannot be due to stabilization of any given complex. The reason is that stabilization would stop not only ATP hydrolysis, it would halt the exchange reaction as well; i.e. the stable state would behave as a sink. Good examples can be found with the Na+,K+ATPase. In that enzyme extracellular Na+, acting with intermediate affinity, stabilizes E,P, whereas extracellular K+, following dephosphorylation at low ATP concentrations, does so with E,(K); in both cases ATP hydrolysis and ATP-ADP exchange are inhibited (Beaugé and Glynn, 1979; Beaugé and Campos, 1986). On the other hand, N-ethylmaleimide and oligomycin block the E,P-E,P transition and inhibit ATP hydrolysis, but increase ATP-ADP exchange (for references, see Beaugé and Glynn, 1979). To account for our observations, alkalinization must retard or block some forward transition(s) following E,P (inhibition of ATPase activity), while accelerating some backward steps starting at E,P without removing enzyme units from the reaction pathway. In yeast H+-ATPase, at 35"C, alkalinization inhibits ATP hydrolysis while enhancing, by about 4-fold, ATP-Pi exchange (de Meis et al., 1987). ATP-ADP and ATP-Pi exchanges share the initial partial reactions of the cycle (see Scheme I); ATP-ADP exchange ends at E,P, whereas ATP-Pi exchange goes as far as E,. This indicates that high pH halts the overall cycle at the E,-E, transition, and it is consistent with our observations provided some backward rate(s) starting a t E,P, which are limiting a t 0°C but not at 20°C, are accelerated when pH is increased. Other comparisons with the ATP-Pi exchange data are difficult because of (a) the large ADP concentration differences, and (b) the very low rates of ATP formation from Pi (0.21 nmol min-l mg-' at 35°C) compared with those obtained in our ATP-ADP exchange experiments (12 and 45 nmol min-' mg-' at O and 20°C, respectively). Incidentally, in the Na+,K+-ATPase it has been proposed that alkalinization slows the forward E,P-E,P transition in the phosphorylation step (Forbush and Klodos, 1991). The pH effects on the plant H+-ATPase cannot be the same because a reduction in the phosphorylation reaction should lead to an inhibition, not an activation, of the ATP-ADP exchange. A complete understanding of the mechanism will require the knowledge of a11 of the intermediates and unidirectional rate constants involved.

LITERATURE CITED

Andersen J (1989) Monomer-oligomer equilibrium of sarcoplasmic reticulum Ca-ATPase and the role of subunit interaction in the Ca'+ pump mechanism. Biochim Biophys Acta 988: 47-72 Beaugé L, Campos MA (1986)Effects of mono and divalent cations on total and partial reactions catalyzed by pig kidney Na+,K+ATPase. J Physiol 375: 1-25 Beaugé L, Glynn MI (1979) Sodium ions, acting at high affinity extracellular sites, inhibit sodium ATPase activity of the sodium pump by slowing dephosphorylation. J Physiol 289: 17-31 Bradford M (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principie of protein-dye binding. Anal Biochem 72: 243-254 Briskin D (1990) The pIasma membrane H+-ATPase from higher plant cells: biochemistry and transport function. Biochim Biophys Acta 1019: 95-109 Briskin D, Poole RL (1983) Plasma membrane ATPase of red beet forms a phosphorylated intermediate. Plant Physiol 71: 507-512 Brooker RJ, Slayman C (1983) Effects of Mg2+ ions on the plasma membrane H+-ATPase of Neurosporu crassa: inhibition by N-ethylmaleimide and trypsin. J Biol Chem 258: 8827-8832 de Meis L (1972) Phosphorylation of the membrane proteins of the sarcoplasmic reticulum. Inhibition by Na+ and K+. Biochemistry 11: 2460-2465 de Meis L, Blanpain JP, Goffeau A (1987) Pi ATP exchange in the absence of proton gradient by the H+-ATPase from yeast plasma membranes. FEBS Lett 212 323-327 de Meis L, Carvalho MGC (1974) Role of Ca2+ concentration gradient in the adenosine 5'-triphosphate-inorganicphosphate exchange catalyzed by sarcoplasmic reticulum. Biochemistry 13: 5032-5038 Forbush B, Klodos I(1991) Rate-limiting steps in Na+ translocation by the Na+/Kt pump. Society of General Physiologists Series 16: 210-225 Glynn IM (1985) The Enzymes of Biological Membranes, 2nd Ed, Vol 3. AN Martonosi, ed, Plenum Press, New York, pp 35-114 Glynn IM, Chappell IM (1964) A simple method for the preparation of 32Pi-labeled adenosine triphosphate of high specific activity. Biochem J 9 0 147-149 Goffeau A, Slayman C (1981) The proton-translocating ATPase of the funga1 plasma membrane. Biochim Biophys Acta 639: 197-223 Hodges TK, Leonard RT (1974) Purification of plasma membranebound adenosine triphosphatase from plant roots. Methods Enzymol32: 392-406 Klodos I, Norby JG (1988) Does ATE' affect the interconversion and the dephosphorylation of the phosphoenzymes of the Na+,K'-pump? In JC Skou, JG Norby, AB Maunsbach, M Esmann, eds, The Na+,K+-Pump Part A: Molecular Aspects. Alan R. Liss, New York, pp 321-326 Lienhard GE, Secemski I I, (1973) P1,P5-Di(adenosine-5')pentaphosphate, a potent multisubstrate inhibitor of adenylate kinase. J Biol Chem 248: 1121-1123 Malpartida F, Serrano R (1981a) Proton translocation catalyzed by the purified yeast plasma membrane ATPase reconstituted in liposomes. FEBS Lett 131: 351-354 Malpartida F, Serrano R (1981b) Reconstitution of the proton translocating adenosine triphosphatase of yeast plasma membrane. J Biol Chem 256: 4175-4177 Read S, Northcote D (1981) Minimization of variation in the response to different proteins of the Coomasie blue G dye binding assay for protein. Anal Biochem 116: 53-64

-

ATP-ADP Exchange by Plant H+-ATPase Repke KRH, Schon R (1992) Role of protein conformation changes and transphosphorylations in the function of Na+,K+-ATPase: an attempt at an integration into the Na+,K+ pump mechanism. Biol Rev 67: 31-78 Roberts G, Berberián G, Beaugé L (1991) Some properties of the H'-ATPase activity present in root plasmalemma of Avena sativa L.: two different enzymes or one enzyme with two ATP sites? Biochim Biophys Acta 1064: 131-138 Roberts G, Berberián G , Beaugé L (1995) Evidence for two catalytic sites in the functional unit of H+-ATPase from higher plants. Plant Physiol 108: 813-819 Segel IH (1975) Enzyme Kinetics. John Wiley & Sons, New York

1403

Serrano R (1984) Plasma membrane ATPase of fungi and plants as a nove1 type of proton pump. Curr Top Cell Regul23: 87-126 Serrano R (1988) H+-ATPase from plasma membrane of Saccharomyces ceveuisiaeand Avena sativa roots: purification and reconstitution. Methods Enzymol 157: 533-542 Serrano R (1990) Plasma membrane ATPase, l n C Larsson, IM Moller, eds, The Plant Plasma Membrane. Springer-Verlag, Berlin, pp 127-153 Vara F, Serrano R (1982) Partia1 purification and properties of the proton-translocating ATPase of plant plasma membrane. J Biol Chem 257: 12826-12830