Effect of Tentoxin on the Activation and on the Catalytic Reaction of Reconstituted H+-ATPase from Chloroplasts [original]

Effect of Tentoxin on the Activation and on the Catalytic Reaction

of Reconstituted H+-ATPase from Chloroplasts

Petra Fromme

Max-Volmer-Institut für Biophysikalische und Physikalische Chemie,

Technische Universität Berlin, Straße des 17. Juni 135,

D-W-1000 Berlin 12, Bundesrepublik Deutschland

Ingo Dahse

Friedrich-Schiller-Universität Jena, Biologische Fakultät,

Institut für Biochemie und Biophysik, Philosophenweg 12,

D-0-6900 Jena, Bundesrepublik Deutschland

Peter Gräber

Biologisches Institut, Universität Stuttgart, Pfaffenwaldring 57,

D-W-7000 Stuttgart 60. Bundesrepublik Deutschland

Z. Naturforsch. 47c, 239-244 (1992); received September 30, 1991/January 3, 1992

H +-ATPase, CF0F,, Tentoxin, Enzyme Kinetics, Uni-Site Catalysis

The proton-translocating ATPase from chloroplasts, CF0Fj, was isolated, purified and re

constituted into asolectin liposomes. The effect of the energy transfer inhibitor, tentoxin, on

different functions of the enzyme was investigated. Tentoxin does not inhibit the nucleotide

release during energization by a pH/AT jump, i.e. the activation of the enzyme is not influ

enced. ATP synthesis driven by a pH/AT jump and multi-site ATP hydrolysis are completely

inhibited by tentoxin, whereas uni-site ATP hydrolysis is not influenced.

Introduction

Membrane-bound H +-ATPases of the F0F,-type

catalyze ATP synthesis/hydrolysis coupled with a

transmembrane proton transport. These ATPases

have a hydrophilic part, F,, containing the nucleo-

tide-binding sites and a hydrophobic membrane-

integrated part, F0, functioning as a proton chan

nel. The F, part has six nucleotide-binding sites.

Three of them are supposed to have catalytic prop

erties, i.e. they can hydrolyze ATP [1-4]. The phy

totoxin, tentoxin [cyclo(L-leucyl-N-methyl-(Z)-

dehydrophenylalanyl-glycyl-N-methyl-L-alanyl)],

produced by the fungus Alternaria alternata

(= A. tenuis; [5, 6]), binds to a site on the a and/or

ß subunits of the CF, part [7], At low concentra

tions between 0.1 and 1 jim (at a tentoxin: enzyme

ratio of 10:1) tentoxin is a non-competitive inhibi

tor of the enzyme [8]; at high concentrations

Abbreviation: CF0F,, H+-ATPase (ATP synthase) from

chloroplasts.

Reprint requests to Dr. P. Fromme.

Verlag der Zeitschrift für Naturforschung,

D-W -7400 Tübingen

093 9 -5 0 75/92/0300 -023 9 $01.30/0

(10 (i m to 1 m M ) it is able to stimulate ATP hydro

lysis activity of the isolated CF, [9-11], It has been

shown by Junge et al. [12] that inhibition of photo

phosphorylation by tentoxin is accompanied by

the cessation of the phosphorylation-coupled pro

ton efflux through ATPase. This explains the slow

er relaxation of light-induced transmembrane elec

tric potential difference and the enhanced delayed

fluorescence which were observed with tentoxin-

treated chloroplasts [13-15]. The specificity of

tentoxin action prompted us to investigate its

effect on the isolated, reconstituted CF0F,.

Materials and Methods

Isolation and reconstitution o f CF0Fj

CF0F, was isolated from spinach chloroplasts,

purified and reconstituted into asolectin liposomes

by cholate dialysis as described earlier [16-18].

The proteoliposomes finally contained approxi

mately 1 |i m CF0F, and 30 g/1 asolectin, 10 m M

Na-Tricine, pH 8.0, 0.2 m M EDTA, 2.5 m M MgCl2

and 0.25 m M dithiothreitol. Prior to every set of ex

periments the enzyme was reduced in the presence

of dithiothreitol and then activated by a pH /A^

jump as described earlier [19].

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240 P. From me et al. ■ Effect of Tentoxin on H +-ATPase from Chloroplasts

The proteoliposomes were incubated for 1.5 h

with 50 mM dithiothreitol at pH 8.0 at room tem

perature.

Incubation with tentoxin

The reduced proteoliposomes ([CF0F,] = 1 |iM )

were incubated with tentoxin before every set of

experiments: an aqueous solution of tentoxin

(1 m M ) was stored at -20 °C and freshly diluted to

appropriate concentrations. Then 2 |il of this solu

tion were added to the five-fold volume of reduced

proteoliposomes and incubated for the time indi

cated.

Activation o f reduced CF0F,

After preincubation of proteoliposomes with

tentoxin, CFqF, was activated by a pH /A ^ jump

(ApH = 3.2, A'F = 60 mV) as follows: 10 f-il of the

proteoliposome solution were added to 50 |il of the

acidic solution (buffer 1) containing 30 mM

Na-succinate (pH 4.9), 5 mM NaH2P 0 4, 2 mM

MgCl2, 0.5 mM KC1 and 1 jim of freshly added vali-

nomycin. The pH during incubation was 5.0. After

30 s of incubation, 50 |il of an alkaline solution

(buffer 2) consisting of 200 mM Na-Tricine (pH

8.7), 120 mM KC1, 5 mM NaH2P 0 4 and 2 mM

MgCl2 were added. The final pH was 8.2.

The acidic and the alkaline solutions both con

tained the same tentoxin concentration as was

used for the preincubation, because tentoxin effect

is reversible [9], Fifteen seconds after the pH/AT

jump, 75 (il of the reaction sample were used for

the determination of ATP and for the sum of ADP

and ATP by luciferin/luciferase in two separate as

says as described elsewhere [19]. In order to obtain

samples without activation of CF0F, the acidic and

alkaline solution were mixed before addition of

proteoliposomes.

Synthesis o f AT P in a pH IA 'F jump ( A TP yield)

The acid-base transition was carried out as de

scribed for activation except that 200 (aM ADP

were present in the alkaline solution (buffer 3).

Fifteen seconds after the acid-base transition, the

reaction was stopped by the addition of trichloro

acetic acid (final concentration 2% w/v). Ten |il of

this solution was used for the determination of

R ed u ctio n o f C F 0F I in p r o te o lip o s o m e s ATP with luciferin/luciferase as described else

where [19]. The ATP background was determined

by mixing the acidic and alkaline solutions before

the addition of proteoliposomes; fifteen seconds

after this addition the sample was denatured and

ATP content was determined as previously de

scribed. This background was always subtracted.

Uni-site ATP hydrolysis

The enzyme was incubated with tentoxin (1 jiM

CF0F,, 4 hm tentoxin) for 10 min and then activat

ed as described above. Fifteen seconds after the

pH/A1? jump, 40 nM Mg-ATP and 10 mM NH4C1

were added in a solution identical to the reaction

medium. At different reaction times 75 (il samples

were taken to determine the free concentration of

ATP or the sum of ATP and ADP with luciferin/

luciferase.

Multi-site A T P hydrolysis

The enzyme was activated as described above.

Mg-ATP labeled in the y-position with 32P

(30 kBq; final concentration 10 |i m to 1 m M ) was

added fifteen seconds after the pH/AT jump in a

solution identical with the reaction medium but

containing in addition 10 m M NH4C1. At reaction

times between 10 and 30 seconds samples were

taken and denatured with 3% trichloro-acetic acid

(final concentration). The 32P; content of the sam

ples was determined as described elsewhere [19].

Results and Discussion

CFoFj can exist in four different states, oxidized

and reduced and both active or inactive. After re

constitution the enzyme is in the oxidized, inactive

state. The inactive enzyme is unable to catalyze

ATP hydrolysis or ATP synthesis. The isolated, re

constituted enzyme contains 1 ADP and 1 ATP

per CFqFj. The ATP can be released only by dena-

turating the enzyme. The ADP is released during

activation. If the enzyme is activated by a pH/AH'

jump in the presence of phosphate, the ADP is

partly phosphorylated to ATP when the mem

brane is energized (about 1 -2 sec; [20]). Therefore,

the total amount of nucleotides (ADP + ATP) re

leased indicates the amount of the active enzyme.

About 20-30% of the total CF0F, can be activat

ed [19],

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P. Fromme et al. ■ Effect o f Tentoxin on H +-ATPase from Chloroplasts 241

Concentration of tentoxin [ (iM ]

Fig. 1. ATP synthesis by CF0F, (1 (i m ) reconstituted into

proteoliposomes: The ATP yield in a pH/A'P jump is

shown as a function of tentoxin concentration. Preincu

bation medium (5 min) and reaction medium contain the

same concentration of tentoxin.

Fig. 1 shows ATP synthesis by proteoliposomes

in a pH/A1? jump as a function of the tentoxin

concentration. The proteoliposomes were preincu

bated for 5 min with the indicated tentoxin con

centration and then the pH/A41 jump was carried

out (see Materials and Methods). The ATP yield

decreased rapidly and at 8 tentoxin (at a ten

toxin :CF0F] ratio of 8:1), no further ATP syn

thesis was measured. It should be mentioned that

the inhibition of ATP hydrolysis catalyzed by

trypsin-treated CF, occurred at a ratio of ten

toxin : enzyme of 10:1 [10].

Here the question is addressed in which step of

catalysis is tentoxin involved. Does it inhibit the

activation of the enzyme, or one step in the cataly

tic turnover of the enzyme (substrate binding,

catalytic reaction ATP*-»ADPP; on the enzyme,

product release) or the cooperativity between the

sites?

For this purpose the fraction of active CF0F,

was measured as a function of the preincubation

time with tentoxin. The experiment was carried

out as follows: the proteoliposomes were incubat

ed with 2 |im tentoxin for different times. Then,

CF0F, was activated by a pH/A^P jump in the ab

sence of exogenous ADP. In two separate experi

ments free ATP and the sum of free ADP and ATP

was determined. As the control the same experi

ments were carried out without a pH/A41 jump.

The result is shown in Fig. 2:

(A) About 0.2 free nucleotides (ATP + ADP)

per CF0F, were found after pH/AT jump and this

amount does not depend on the incubation time

with tentoxin.

(B) The amount of free ATP per CF0F, after the

pH/A'P jump decreases from 0.06 to 0.01 ATP per

CF0F, during incubation with tentoxin, i.e. the ra

tio between free ADP and free ATP changed al

though the total amount of nucleotides remained

constant.

amount of free nucleotides (ATP + ADP) is 0.05

per CF0F, and does not depend on incubation time

with tentoxin.

(D) Almost no free ATP is detected without

pH/A1? jump.

(E) For comparison the ATP yield in the pres

ence of 100 |j.M ADP was measured with the same

pH/A'F jump as a function of incubation time with

tentoxin (dashed curve, scale on the right side).

The ATP yield decreased continuously with in

creasing incubation time.

These results lead us to the following conclu

sions:

(1) The nucleotide release induced by energiza

tion of the membrane is not influenced by incuba

tion with tentoxin. Since the ADP release from

CF0F, upon energization is related to the activa-

0.24

0.20

| 0,6

< 0.12

T3 0.08

-§ 0.04

0.04

0 , , , ,

-------

0 5 100 5 10

Incubation time with tentoxin [ min ]

Fig. 2. Influence of tentoxin on ATP synthesis and nu

cleotide release during activation. The proteoliposomes

([CF0F,] = 1 jim ) were incubated with 2 p.M tentoxin for

the time indicated. Then the following measurements

were carried out in the presence of 2 |im tentoxin: (A)

The sum of free (ATP + ADP)/CF0F, after a pH/A'F

jump. (B) Free ATP/CF0F, (after a pH/A^ jump). (C)

The sum of free (ATP + ADP)/CF0F, (without pH /A^

jump). (D) Free ATP/CF0F| (without pH/AM' jump). (E)

ATP yield in a pH/A'P jump.

+ ApH ( J )

- ApH

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242 P. Fromme et al. • Effect o f Tentoxin on H -ATPase from Chloroplasts

tion, this result shows that tentoxin does not

change the fraction of the activated enzyme.

(2) A part of the released ADP is re-bound and

phosphorylated to ATP during the pH jump. This

part decreases during incubation with tentoxin in

dicating that the catalytic process is inhibited. The

ATP yield measured in the same type of experi

ment decreases in parallel and supports this con

clusion.

(3) The ATP yield (curve E) was measured in the

presence of 100 |iM ADP and therefore, all accessi

ble ADP-binding sites are involved (multi-site

ATP synthesis). The amount of ATP generated in

the experiment (B) was measured under uni-site

conditions since the concentration of free ADP

was much lower than the enzyme concentration

(uni-site ATP synthesis). Therefore, multi-site and

uni-site ATP synthesis are inhibited by tentoxin.

(4) Tentoxin (2 ^m; tentoxin: CF0F, = 2:1) does

not itself induce a release of tightly bound nucleo

tides (Fig. 2C and D). Therefore, we conclude that

at this low concentration no tentoxin-induced acti

vation of the enzyme occurs.

At high tentoxin concentration (400 j im ) the

ATPase activity of isolated CFj was stimulated

and can be correlated to the release of tightly

bound nucleotides [9, 21]. This stimulatory effect

by high tentoxin concentration can, therefore, be

explained by the release of tightly bound ADP

from the regulatory site activating the CF,.

In the next experiment the effect of tentoxin on

uni-site ATP hydrolysis was measured. CFqF,

was activated by a pH /A^ jump and ATP was

added, so that the initial concentration of ATP

was 50 nM . The concentration of ATP and that of

(ADP + ATP) was measured. Fig. 3 shows the re

sult: ATP was bound to the enzyme and with al

most the same kinetics ADP was released.

From this measurement the rate constant for

ATP binding was determined to be 1 x 106 m_1 s~'.

This is the same number as found in the absence of

tentoxin. In addition, the ADP release is the same

in the presence and absence of tentoxin [19].

Therefore, we have to conclude that neither ATP-

binding, nor ATP hydrolysis on the enzyme nor re

lease of ADP is affected by tentoxin.

In the next experiments the influence of tentoxin

on the rate of ATP hydrolysis was measured at

concentrations of ATP from 10 |i m to 1 m M where

multi-site hydrolysis occurs. First, the rate of ATP

Time [s ]

Fig. 3. Single-site ATP hydrolysis: The proteoliposomes

([CF0F,]= 1 |i m ) were preincubated with 4 tentoxin

for 10 min. Then, the enzyme was activated by a pH/A'P

jump and ATP was added. During the reaction 4 |iM ten

toxin was present. The concentration of free ATP and

the sum of free (ATP + ADP) were measured as a func

tion o f reaction time. The concentration of free ADP

was calculated from the difference between both meas

urements.

hydrolysis was measured without tentoxin. The re

sults are shown in Fig. 4. The rate increases with

increasing ATP concentration. A Lineweaver-

Burk plot of these data gives a Ku = 180 |aM and

Fmax = 58 ATP/(CF0F, * s). For investigation of

Concentration of ATP [ mM ]

Fig. 4. Rate of ATP hydrolysis as a function of the ATP

concentration. CF0F, (1.4 (i m ) was preincubated either

without or with 10 hm tentoxin for 10 min, then the en

zyme was activated by a pH/A'P jump and the reaction

was started by addition of y-[32P]-ATP and 10 m M

NH 4C1 (final concentration).

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P. Fromme et al. ■ Effect o f Tentoxin on H +-A TPase from Chloroplasts 243

the tentoxin effect the enzyme ([CF0F,]= 1.4 ^m)

was preincubated for 10 min with 10 (im tentoxin.

At this concentration ATP synthesis is completely

blocked (Fig. 1). In the presence of tentoxin no

ATP hydrolysis could be detected, i.e. tentoxin

completely inhibits multi-site ATP hydrolysis.

These results can be summarized as follows. (1)

Tentoxin does not change the fraction of activated

CF0F,. (2) In the presence of tentoxin neither mul

ti-site nor uni-site ATP synthesis occurs. (3) Under

uni-site conditions ATP binding, hydrolysis of

ATP to enzyme-bound ADPPj and the release of

ADP are not inhibited by tentoxin. (4) Tentoxin

completely inhibits multi-site ATP hydrolysis.

The differential inhibition of uni-site and multi

site ATP hydrolysis can be explained in two ways.

(1) The Pj release could be inhibited by tentoxin. In

this case all reaction steps before the Pj release can

occur, however, only one turnover of the reaction

is possible since the free enzyme is not regenerated.

Therefore, several turnovers under uni-site condi

tions and multi-site ATP hydrolysis are inhibited.

(2) Uni-site ATP hydrolysis is not inhibited but the

cooperativity between different catalytic sites is

prevented by tentoxin. This suggestion is support

ed by measurements of the differential effect of

acid inactivation on uni-site and multi-site ATP

hydrolysis [22],

Fig. 2 and Fig. 3 show that also uni-site ATP

hydrolysis and uni-site ATP synthesis are differ

ently affected by tentoxin. There are several possi

bilities to interpret this observation.

1) Uni-site ATP synthesis and uni-site ATP hy

drolysis occur at different sites, e.g. the ATP syn

thesis is coupled with proton translocation, the

ATP hydrolysis is not (as for example in CF,-cata

lyzed ATP hydrolysis).

2) Uni-site ATP synthesis and hydrolysis occur

at the same site but tentoxin inhibits the conforma

tional change which is necessary to couple proton

transport through CF0 with the chemical reaction

in CF,. Also in this case, uncoupled uni-site hydro

lysis can occur but synthesis is inhibited.

3) If uni-site ATP synthesis and ATP hydrolysis

occur via the same reaction pathway (microrever

sibility), both directions must be inhibited to the

same extent. The measurements in this part (with

the exception of Fig. 2E) are carried out under

conditions where only a part of the enzymes car

ries out one turnover (“single turnover” condi

tions). If e.g. tentoxin inhibits the conformational

change connected with proton transport (this is

the first step of the ATP synthesis direction), then

ATP synthesis will be completely blocked. How

ever, the first steps of proton transport-coupled

ATP hydrolysis can occur (e.g. ATP binding etc.)

up to that step where coupling to proton transport

must take place. In fact the enzyme is completely

inhibited, however, this does not influence the first

steps. In order to show this, the enzyme must carry

out at least one turnover under uni-site conditions.

As yet, we cannot decide between these possibili

ties.

The dependence of the fraction of activated

CF0F, and the catalytic reaction of C F ^ on

PH/A41 [23] showed that both processes are driven

by the transmembrane electrochemical potential

difference of protons. Our results demonstrate

that ATP synthesis was completely blocked by ten

toxin, whereas the fraction of activated enzymes

was not affected. Therefore, we have to assume

that for activation of CFqFj and the catalytic reac

tion different pathways for protons should exist.

Acknowledgements

We thank Dr. B. Liebermann, Jena, for his gen

erous donation of tentoxin. This work was sup

ported by the Deutsche Forschungsgemeinschaft

(Sfb 312).

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244 P. Fromme et al. ■ Effect o f Tentoxin on H +-ATPase from Chloroplasts

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