J. Biol. Macromol.3 (2) 61 -68 (2003)

ArticlesCharacterization of membrane ATPase activities of spinach chloroplasts
Keiko Kishimoto, Sumio Ishijima and Masatake Ohnishi
Graduate School of Agriculture, The Prefecture University of Kyoto(Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan)
Received June 3, 2003; Accepted July 11, 2003
Mg2+ works as a key factor on photosynthesis in plant chloroplasts. We have a
final purpose to make the Mg2+ transport proteins clear in chloroplasts. The
existence of Mg2+-dependent ATPase has been suggested in 1973, but has not been
elucidated yet.

ATPases of spinach chloroplast membranes were solubilized bydetergents, and their activities were characterized.

Octylglucoside was the mostvaluable detergent for solubilization of thylakoid ATPase; however, taurocholate
was more suitable for solubilization of envelope ATPase.
envelope ATPases had the optimum pH of around pH 7.5.

Both thylakoid andBoth ATPases required

Mg2+ for activity, but our results indicated that the ATPases used Mg･ATP chelates
as a substrate, and free Mg2+ was inhibitory.

Thus, we could not identify“Mg2+-dependent” ATPase in thylakoid and envelope membranes, and otherapproaches are necessary for purifying Mg2+ transport proteins and making themclear.
Key words: ATPase; Mg2+; Detergent; Thylakoid; Envelope; Chloroplast; Spinach

The plasma membrane of plant cells

energy production and CO2 fixation in theand plant organellar membranes areplant chloroplast is ion concentrations.

involved

regulation,

In the chloroplast stroma, concentrationstransepithelial transport, and regulation ofof the physiological important ions are on

the membrane potential.

the order of 150 mM K+, 50 mM Cl− ,involumeOne of thefactors that have decisive effects on theand 5 mM Mg2+ 1).

Abbreviations:
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonicacid; DCCD, N,N’-dicyclohexylcarbodiimide.

K. Kishimoto et al.

62

During photosynthesis of the plant

Mg2+

transport

proteins

clear

in

chloroplast, light-driven H+ gradients arechloroplasts.

generated between the cytosolic site (~pH

ATPase enzymes in spinach chloroplasts,

7), the stroma (~pH 8), and the thylakoid

which act as important adjustment of thelumen (<pH 6). Ion transport proteinsion concentrations.

We here characterizepresent in the chloroplast inner envelope
membrane

and

in

the

thylakoid

membrane appear to be involved in thedevelopment

andregulationofH+

gradients and membrane potentials acrossMaterials and methods
Preparation
membranesofchloroplast4) ,9)

these membranes2). Due to the pH

Spinach leaves were obtained from

optimum (~pH 8) of the key enzymes of

local markets. Leaves were ground withthe photosynthetic carbon reduction cycle,

grind medium (50 mM Hepes-KOH (pHthe rate of light-dependent CO2 fixation

7.5), 0.33 M sorbitol, 2 mM EDTA, 1

in

the

mM MgCl2, 1 mM MnCl2, 5 mM

maintenance of a high stromal pH . The

ascorbate) twice for 10 s in a Waring

uphill H+ flux across the chloroplast

blender. The slurry was passed throughenvelope is probably driven by afourchloroplasts

depends

on3)+

layersofMiracloth

beforeH -ATPase and indirectly coupled to thecentrifugation at 5,000 × g for 15 s.

uptake of potassium (K ＋ channel) from

Pellets were resuspended in the grind

the cytosol 1),4). Also, the concentration

medium and layered onto 30% Percoll

of

2+

Mg

increases

due

to

the

solution in 50 mM Hepes-KOH (pH 8.0)

light-dependent release of Mg2+ fromandthylakoid membranes5).

centrifugation at 700×g for 15 min, the

0.33

M

sorbitol.

After

As ion transport systems in the

intact chloroplasts in the pellets werechloroplast, the existence of Ca ２＋ /H ＋

resuspended in the same solution andantiporter6) and K ＋ -channel7) is known.

centrifuged at 1,000×g for 10 min.

2+

However, the existence of Mg

Thetransport

intactchloroplasts

wereproteins is not yet clear, though thesuspended in hyperosmotic medium (0.6

existence of Mg2+-dependent ATPase has

M sucrose, 10 mM Tricine-KOH (pH 7.5),

8)been suggested in 1973 . Mg

2+

works

2 mM EDTA, 5 mM 2-mercaptoethanol)

as a key factor on the photosynthesis.

andrupturedbyexposure

totwo

We have a final purpose to make thefreeze/thaw cycles (freezing at -20°C forATPase Activities of Chloroplast Membranes

ATP hydrolysis was determined bya minimum of 1.5 h followed by thawing
at room temperature).

The medium63

the release of Pi.

Standard reaction

containing ruptured chloroplasts was thenmixtures (25 µl total volume) contained

diluted by adding 2 volumes of medium

40 mM Tricine-KOH (pH 8.0), 10 mM

containing no sucrose.

After gentle

MgCl2, 10 mM ATP, and 2.5 µg protein.

shaking for 5 min at 4°C, the medium

Release of phosphate from ATP waswas centrifuged at 15,000×g for 20 min

determined by a colorimetric procedure

to pellet membranes. The pellets wereusing ammonium molybdate10).

suspended in medium that had 0.2 M
sucrose.
For separation of thylakoid andResults

envelope membranes, the membrane

Solubilization of chloroplast membrane

fraction suspended in 0.2 M sucrose was

ATPases by detergentscentrifuged at 6,000 × g for 30 min topelletthylakoid

membranes.

A variety of ionic, nonionic andThezwitterionic detergents were tested forsupernatant was centrifuged again attheir ability to solubilize chloroplast

40,000×g for 1 h to pellet chloroplast

membrane ATPases. Membranes wereenvelope membranes. The yellow pellets

incubated for 1 h at 4 ℃ in medium

were resuspended in the medium that had

containing a detergent with gently stirring.

0.2 M sucrose.

Thesuspensions

obtained

were

centrifuged, and the ATPase activity in
Solubilizationofmembranesbydetergentsthe supernatant was assayed.
The effect of detergents on the

Membranes were incubated for 1 h atsolubilization of thylakoid membrane

4°C in solubilization medium containing

ATPase activity is shown in Table 1.

detergent as indicated. After incubation,

Highest ATPase activity was obtained

the mixture was centrifuged at 60,000×g

withfor 90 min. The supernatant was again

detergent, and dodecylmaltoside.

centrifuged at 60,000×g for 1 h. The

ionic and zwitterionic detergents tested

supernatant was recovered and assayedwere less effective in comparison withfor ATPase activity.

the non-ionic detergents. These results

octylglucoside,anon-ionic
The

indicated that octylglucoside was mostMeasurement of ATPase activity

useful tosolubilize

ATPasefromK. Kishimoto et al.

64

Table 1.
Effect of detergents on
solubilization of ATPase activity fromthylakoid fraction. Thylakoid fractions(2.5 mg/ml protein) were incubated inpresence of various detergents andcentrifuged as described under Materials
and methods. The final concentration ofdetergent was 1.0% (v/w) except for
Nonidet P40 and Tween 20.
Thesupernatants were assayed for ATPase
activity.
ATPase activity(nmol Pi/hr)
Brij 350Digitonin0
Dodecylmaltoside
1.01
Nonidet P40 (0.50%)
0.22
Octylglucoside
1.70
Triton X-100
0.03
Detergent

Tween 20 (0.50%)

0.46

CholateDeoxycholate
Taurocholate
CHAPS

0.08
0.12
0.08
0.38

thylakoid membranes.
Table 2 shows the effect of detergentsonthesolubilizationofenvelope

Table 2. Effect of detergents on
solubilization of ATPase activity
from envelop membrane fraction.
Envelope membrane fractions (3.3
mg/ml protein) were incubated inpresence of various detergents andcentrifuged as described under
Materials and methods. The final
concentration of detergent was 1.3%(v/w). The supernatants were assayedfor ATPase activity.

Detergent

ATPase activity(nmol Pi/hr)

Brij-35
Digitonin
Dodecylmaltoside

0.07
0
0.28

Octylglucoside

0.19

TritonX-100

0.25

Tween 20
CholateDeoxycholate

0
0.45
0.20

Taurocholate

0.48

CHAPS

0.19

centrifugation.

The

non-ionic

zwitterionicdetergents

and

such

as

dodecylmaltoside, Triton X-100, andmembrane ATPase activity. In contrast

CHAPS were less effective.

tothylakoid

results indicated that taurocholate wasmembrane ATPase, the ionic detergents

most useful to solubilize ATPase fromwere effective. Highest ATPase activity

envelope membranes.

thesolubilization

of

These

was obtained with taurocholate andcholate. With treatment of taurocholate,Comparison of thylakoid and envelope

41%

ATPase activities

of

the

ATPaseactivitywasrecovered in the supernatant after

The effects of pH on the thylakoid

35

25

30

20

25

ATPase activity

30

15
10
5

(μmol Pi/hr/mg protein)

ATPase activity(μmol Pi/hr/mg protein)

ATPase Activities of Chloroplast Membranes

65

20
15
10
5

0
5

6

7

8
9
pH

10

11

12
0
0

Fig. 1.
pH dependence of thechloroplast ATPase activity.
The
ATPase activity of envelope membrane(▲) and thylakoid (●) fraction wasmeasured at various pH.

and envelope ATPase activities were

5

10

15

20

2+

addition of [Mg ] (mM)

Fig. 2.
Effect of Mg2+ on thechloroplast ATPase activity.
The
ATPase activity of envelope membrane(▲) and thylakoid (●) fraction wasmeasured except that the concentration of
MgCl2 was varied.

examined (Fig. 1). The optimum pH ofboth activities was estimated to be aroundpH 7.5.

The effects of Mg2+ added were
examined in the presence of the fixed

The ATPase associated with theconcentration of ATP (10 mM). Highest

chloroplast envelope is typically referred

activity of the thylakoid and envelope

to as a “Mg2+-dependent” enzyme8).

ATPase was obtained at equimolar (10Previous studies11),12) have demonstratedmM) concentration of Mg2+ and ATP (Fig.

that the Mg2+ dependency of various

2). This is consistent with the substrate

ATPases is not cue to the presence of abeing Mg ･ ATP.

catalytic site on the enzyme for Mg2+ but,higher concentration of Mg2+ (allowing

rather, because the Mg･ATP complex isfor the presence of free Mg2+ in thethe actual substrate for the enzyme. The

reaction solution), inhibition of thecharacterization of the ATPase activity

enzyme activity was noted. Thus, freeassociated with the chloroplast thylakoid

Mg2+ actually inhibited the thylakoid andand envelope, therefore, focused on thisenvelope ATPases.

Mg2+ dependency.

In the presence of

K. Kishimoto et al.

100
90
80
70
60
50
40
30
20
10
0

140
120
ATPase activity (%)

ATPase activity (%)

66

100
80
60
40
20

0

15
30
45
60
incubation time (min)Fig. 3.
Effect of DCCD on the
ATPase activity of thylakoid fraction.
After incubation with DCCD at 37°C,
the ATPase activity of thylakoid fraction
was assayed.
The concentration of
DCCD was 0 (♦), 10 (□), 50 (▲) µM.

0
0

20
40
incubation time (min)60

Fig. 4. Effect of DCCD on the ATPase
activity of envelope fraction. Afterincubation with DCCD at 25°C, theATPase activity of envelope fraction was
assayed. The concentration of DCCD
was 0 (♦), 50(▲), 100(●), and 500 (■)µM.

Effect of DCCD on the ATPase activity
DCCD is an inhibitor of thylakoid(and mitochondrial) H＋-ATPase13). The

Discussion

effects of DCCD on the thylakoid andA better understanding of the enzymaticenvelope ATPase activity were examined.

and transport functions of the chloroplast

Both ATPase activities were thermolabile

thylakoidand lost rapidly during incubation, but

requires a detailed knowledge of theirFig. 3 indicated that DCCD inhibited theconstituent

thylakoid ATPase.

Incubation with 10

purification of their proteins, especiallyand 50 µM DCCD for 15 min induced

envelope proteins, is hampered by several

73% and 84% inhibition, respectively.

factors: (a) envelope membranes are only

In contrast, no inhibition of the envelope

a minor membrane structure within the

ATPase activity was observed withplant cell; they do not represent more

50-500 µM DCCD (Fig. 4).

than 1-2% of the total chloroplastandenvelope

proteins.

membranes
However,proteins; (b) compared to most plant cell
membranes, envelope membranes have avery high lipid to protein ratio (1.2-1.5

ATPase Activities of Chloroplast Membranes

67

mg lipids/mg protein), and their complete

enzyme in review articles. However, wesolubilization requires large amounts ofcould not identify the Mg2+-dependent

detergents that might be deleterious to

ATPase, but our results indicated that the

enzymaticaenzyme uses Mg ･ ATP chelates as acompromise between solubilization andsubstrate, and Mg2+-dependent ATPase isactivity should be found, and thea minor component, if any.

detergent carefully selectedtransport

activities.

Therefore,should

be

the

Mg2+

necessary

The choice of a detergent for themechanism in thylakoid and envelope.

solubilization of a membrane is rather

Other approaches are necessary for

empirical.

purifying the Mg2+ transport proteins and

The experiments describedabove have indicated that only a few ofmaking them clear.

detergents are useful for studies ofthylakoid and envelope ATPases.

Two

detergents were found to be superior tothe others for the studies of the thylakoid
ATPase,i.

e.

dodecylmaltoside.

octylglucosideInandcontrast,

Acknowledgments
This study was supported in part by
Grant- in-Aid for Scientific Research (C)(15580096)

from

the

Ministryoftaurocholate and cholate were found to be

Education, Culture, Sports, Science &superior for the envelope ATPase.

Technology of Japan.

It is well known that illumination ofchloroplasts causes stromal alkalinization
and a release of Mg2+ from thylakoid
membranes.

In thylakoid, H+-ATPase

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