J. Biol. Macromol.3 (2) 69-74 (2003)

ArticlesATP-binding proteins of spinach chloroplast membranes
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

The chloroplast membrane proteins from spinach were solubilized by various
detergents, and adsorbed to ATP-agarose gels.

Triton X-100 was most useful forsolubilization, and most proteins were adsorbed to the ATP-agarose gel that had
matrix attachment of N-6 of ATP.

About 10 chloroplast membrane proteins werebound to ATP-agarose gels, which included H+-ATPase β subunit.

Addition ofATP disturbed the adsorption of the proteins to the ATP-agarose gel, and theadsorbed proteins were eluted by ATP from the ATP-agarose gel.
Key words: ATP-agarose; Detergent; ATPase; Chloroplast; Spinach

Mg2+

works

asakeyfactoronphotosynthesis in plant chloroplasts.

adsorbed the solubilized proteins toATP-agarose gels.

We have a final purpose to make theMg2+transportchloroplasts.

proteinsclearin1)

In the preceding paper ,Materials and methods
Materials

we characterized ATPase activities ofspinach

chloroplast

thylakoid

andenvelope membranes, but however, weDetergents and ATP-agarose gelswere purchased from Sigma, and 5’-ATP,
2Na was obtained from Yamasa.

could not identify “Mg2+-dependent”
ATPase.

As another approach for

Solubilization of chloroplast membranes

obtaining

ATP-bindingandby detergents

transport proteins of

Spinach2+purifying the Mg

proteins

chloroplast

membranes

chloroplast

membranes,

weherewere obtained as described previously1).

solubilized

chloroplast

membrane

Membranes were incubated for 1 h at

proteins by various detergents, and4°C in solubilization medium (0.2 M

Abbreviation: PVDF, polyvinylidene difluoride.

K. Kishimoto et al.

70

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

2 mM EDTA, 5 mM mercaptoethanol)

centrifuged at 100×g for 2 min. This

containing

gel-washing procedure was repeated

1%(v/w)

detergentasindicated. After incubation, the mixture

twice.

was centrifuged at 60,000×g for 90 min.

Theandtheproteinssuspension

adsorbedtowastheThe supernatant was again centrifuged atATP-agarose gel were eluted with the60,000 × g for 1 h.

SDS sample buffer (10% glycerol, 64

The supernatant

was applied to an ATP-agarose gel.

mM Tris-HCl (pH6.8), 87 mM SDS, 0.64
M mercaptoethanol), and analyzed with

Binding of proteins to ATP-agarose gel
Three kinds of ATP-agarose gels were

SDS-polyacrylamide gel electrophoresis(PAGE).

used (Table 1) in a 1.5 ml of sampling
tube.

The

solubilized

chloroplast

N-Terminal

membranes were added to the gel thatanalysis

was equilibrated with an equilibration

Theamino

proteins

acidweresequenceseparated

bymedium (40 mM Tricine-KOH (pH 7.5),SDS-PAGE and then blotted to PVDF

5 mM MgCl2, 100 mM NaCl). Aftermembrane.

incubation at 4℃ for 5-15 min, the tubes

with Coomassie brilliant blue R-250.

were centrifuged at 100×g for 2 min.

The protein bands were cut and applied

After the supernatant was removed, 100

to a Shimadzu PPSQ21 sequencer.

The proteins were stainedµl of the equilibration medium wasTable 1.

ATP-agarose gels used.

MatrixMatrixMatrixattachment

spacer (atoms)

activation

N-6

11C-8

9ribosehydroxyls

11Matrix

cyanogencross-linked 4%

bromidebeaded agarose

cyanogencross-linked 4%

bromidebeaded agarose

cyanogencross-linked 4%

bromidebeaded agarose

ATP(nmol)/gel(µl)

1.3

3.1

2.9

ATP-binding Proteins of Chloroplast Membranes

X-100, octylglucoside, CHAPS, andResults and Discussion
Bindingof

71

chloroplast

membranecholate.

The proteins adsorbed to theproteins to ATP-agarose gel

ATP-agarose gels were eluted with the

The chloroplast membrane proteins wereSDS sample buffer, and analyzed withsolubilized by detergents such as TritonSDS- PAGE(Fig. 1).

Similar(B)1235(D)

123(A)

123454but

97,40066,20045,00031,00021,50014,400(C)1234545Fig. 1. Adsorption of chloroplast membrane proteins to ATP-agarose gel. Thechloroplast membrane proteins were solubilized with 1% Triton X-100 (A),octylglucoside (B), CHAPS (C), and cholate (D), and the solubilized proteins wereadsorbed to the ATP-agarose gels (Table 1). The proteins adsorbed to the gel wereeluted with the SDS sample buffer, and applied to SDS-PAGE. Lane 1, molecularweight standards: Phosphorylase b, 97,400; serum albumin, 66,200; ovalbumin,45,000; carbonic anhydrase, 31,000; trypsin inhibitor, 21,500; lysozyme, 14,400.
Lane 2, the solubilized protein fraction. Lanes 3-5, the protein fractions adsorbed to
the ATP-agarose gels that had matrix attachment of N-6 (lane 3), C-8 (lane 4), andribose hydroxyls (lane 5) of ATP.

K. Kishimoto et al.

72

different patterns of protein bands wereobserved:

123

many protein bands wereobserved with solubilization with Triton
X-100 and octylglucoside.

Among 3kinds of ATP-agarose gels tested, the gelthat had matrix attachment of N-6 of ATP(the N-6 gel) adsorbed many kinds ofproteins. Then, the chloroplast membraneproteins were solubilized by Triton X-100,and adsorbed to the N-6 gel.

ATP wasadded during or after incubation of thesolubilized proteins with the N-6 gel.
Addition of ATP during incubation of thesolubilized proteins with the ATP-agarose
gel disturbed the adsorption of theproteins to the ATP-agarose gel (Fig. 2).
When 50-fold molar amount of ATP･Mg
was added to the ATP-agarose gel afterFig.2. ATP disturbs the adsorption of
the proteins to ATP-agarose gel. The
chloroplast membrane proteins weresolubilized with 1% Triton X-100, andthe solubilized proteins were incubatedwith the ATP-agarose gel that had matrix
attachment of N-6 of ATP in the absence(lane 2) and presence (lane 3) of 3.5 mM
ATP.
Lane 1, molecular weightstandards (see Fig. 1).

the solubilized proteins were adsorbed to
the gel, the adsorbed proteins were eluted
from the gel to the supernatant (Fig. 3).
These results indicated that the proteins
adsorbed to the ATP-binding gel did
adsorb to the ATP moiety.

binding proteins
The chloroplast membrane proteinssolubilized by Triton X-100 and adsorbed
to the ATP-agarose (N-6) gel wereapplied to N-terminal sequence analysis.
proteins

SDS-PAGEandwereobserved

8

protein bands were applied to N-terminal
amino acid sequence analysis (Fig. 4).
Only when the solubilized proteins wereadsorbed to another ATP-agarose gel that

N-Terminal amino acid sequence of ATPThe

membrane, and usually

separatedblottedtobyPVDF

had matrix attachment of ribose hydroxyl
groups of ATP, 40 kDa protein band wasobserved. This protein was also applied
to N-terminal sequence analysis. Figure
4B shows N-terminal sequences of theproteins.

Unfortunately,N-terminal

ends of 6 proteins were blocked, but
N-terminal amino acid residues of theATP-binding Proteins of Chloroplast Membranes

(A)

M

12

3

45(B)

12373

4M

Fig. 3. The proteins adsorbed to the ATP agarose gel are eluted by ATP. Equal(3.5 mM, lane 1), 5-fold (17.5 mM, lane 2), 10-fold (35 mM, lane 3), and 50-fold (175mM, lane 4) molar amount of ATP were added to the ATP-agarose (N-6) gel, to which
the proteins solubilized with 1% Triton X-100 were adsorbed. After incubation at
4℃ for 15 min, the gel suspensions were centrifuged at 1,000×g for 3 min, and theproteins that remained to be adsorbed to the gel (A) and were eluted to the supernatant(B) were applied to SDS-PAGE. Lane M, molecular weight standards (see Fig. 1);
lane 5 in (A), the proteins adsorbed to the ATP-agarose gel without addition of ATP.

otherproteins

weresequenced.

identified, maybe due to the blocking ofComputer database search with thetheir N-terminal ends.

FASTA programindicated

proteins

homologous

Amino acidrevealedthat

thesequence analysis of the internal peptides

havesameorof the proteins whose N-terminal ends

aminoacidN-terminal

are blocked are now in progress.

sequences.
The 51 kDa protein was identified tobe H+-ATPase β subunit.

Conclusion

Chloroplast

About 10 chloroplast membrane

H+-ATPase (CF1) is composed of 5proteins from spinach were bound tosubunits, and the β subunit binds ATP2).

ATP-agarose

These results indicate validity of this

H+-ATPase β subunit.

methodproteins and their amounts were different

forobtaining

ATP-binding

proteins, but now no other ATPase wasgels,which

included

The adsorbed

depending upon the detergents used forK. Kishimoto et al.

74(A)

(B)
Proteinband
94k

N-Terminal
sequence
blocked77k

blocked55k

blocked

51k

MRINP

37k

blocked28k

blocked27k

blocked

Identified
protein

H+-ATPase β subunit

26k

GNDAW
Chlorophyll binding protein
AYGEAYNVFGKPY(K)XN
Oxygen-evolving
enhancer protein 2
40k* AKKSVGDLTSAD
Phosphoglycerate kinase*Obtained with the ATP-agarose gel that had matrix attachment ofribose hydroxyls of ATPFig. 4. N-Terminal amino acid sequences of the proteins adsorbed to theATP-agarose gel. The proteins adsorbed to the ATP-agarose gel that had matrix
attachment of N-6 (otherwise indicated) or ribose hydroxyls of ATP were separated by
SDS-PAGE (A), and applied to N-terminal amino acid sequence analysis (B).
Computer database search with the FASTA program revealed that the proteinsindicated as “identified” have same or homologous N-terminal amino acid sequences.
solubilization and the ATP-agarose gelsthat had different matrix attachment ofATP. Triton X-100 was most useful forsolubilization, and most proteins wereadsorbed to the ATP-agarose gel that had
matrix attachment of N-6 of ATP.
In the preceding paper1), we
solubilized ATPase activities of spinach
chloroplast membranes by detergents,
but however, we could not identify
Mg2+-dependent ATPase.
Studies on
ATPase activities of the preparations
purified here by the ATP-agarose gel are
now in progress.

Acknowledgments
This study was supported in part by Grantin-Aid for Scientific Research (C) (15580096)
from the Ministry of Education, Culture,Sports, Science & Technology of Japan.
References
1)

K. Kishimoto, S. Ishijima, and M.
Ohnishi:

J. Biol. Macromol., 3, 61-68(2003).
2) A. Blair, L. Ngo, J. Park, I.T. Paulsen, andM.H. Saier:

Microbiology, 142, 17-32(1996).

Communicated by Hayakawa Kiyoshi.