ScL R'eTI_)，' ]{aR21.Za't}Va U:nivo

Vol. 24， No， 2， pp， 81-89

December 1979

l味付I[)fGalae~ül.üp1di'l IChloJrupla日11:8 Lcdthin

m 必 P'.re泊四~ce u1sf LYE\{]!~edt TI1 ii ll1i

HOSHJiNA

I和l[!ψ-zi(:al liu'Jt!:♂よCtlj/){i

(REC巴lV巴dSe'pt己1百ber29， 1975)

ßUJI~í11s- æ:Ltl，;t 'Th日 bindingof iYElo!ecithin (J.y<，opbc叩 b'3.tiGylchuline) to chIoropla;~t

tn.eIn1JnU1eS and {esu~ti日g 1'::;lE.，asf: rn日ナ:melipids weri2 inve"tigatωt stt ]ovi.r YeJ.icJ，s

of lyso!edthi汀 to dllcfL'opbyl.! j !_::/:SOJ(::I:乙ithiE 世 Jncorp(!:r~孔巴ιinto th色

chlor・opï日~st rn訓.nbrane2 ~Nitho\.:.rt 1083 of :rrJc111br2U'1I2 COA江.1pon召nt3， High ratios of

1';7801にιithin c.blOf:[)phylJ. 日xtractE:d 50~Gü l;jS of rn(H1ogal研工士山2.yland digalw.:tJsyl

diglyc引:ide:s iecithin (ph心sph日UdylchoHn::¥ 8r~d. ~.O-15 ，，:，1 pho:~~'phaticlyl 立lYClt:: !'Tll ，

snHo"=luinovusyl rEglyc日dde~n(l (_~.hJüroplJ.yn :fron1 chloroniasl2.. VII"i;en soyi):: 3D 1日cithin

1l，)O;，コ肌巴'S. "N占reack1ed to lysoI6:itJ:in~treated chllOtOpl asts， lysoleじithi!1 Sこoic11.io-

E1etricaHy aplaルed.by lecithin， ~rhe distrib仁tiorrof hcid mむ!ie.. 亡~:Je3 in the laterai plan(~

01 ムゾlakc，id~11ernl>raJ1C:S is uiscussecL

J'，ltl"OOUCti.OI1

and Hosb.ina a:nd N ::"hida that W{J日n

'¥Alhich i:-::" 1ive:l -knu¥，!(/H fOI・ it~f

。fel~~ctr(，γ1 transport'Nas stimnlated 乱，~-;;; a

at 10'1'¥.1 r，rJ.tJ.O-S of lvs仁Jβc:ithin

勺， l1nd lTlh:l.b.jt7o:ci ，)f e~ectron 2r.Ilso 'i}¥7aS ob:serγ日 at

that EiIl reaction ir:.hibited

acil1s 197;'， ;

19ti'l ;l:'1xlfGrd ，，'門dE日 belつ工198tα ヨ五;E'f.rar'

lTle;s.C1Irnorphisrn d.es(_'.:"Hふとd Hel配nn':;河川"1dSirnons

to rrler~lb'C'anefs，.引Jch ct3 rny"ピlinf~ じent ci

e

』

門

JnM 毛孔

下

lrιー泊目、

ハLI

--3d

t

I

， J97:3; Lε-ib-D，'IJitz-B.日占企巴110れどl

。rsoÌ'ublHzélidon つ th\~ :rnernc'lrane cOirilp;{)nentB1 -su.ch as

ann

ホ s!，.bl:...:;:-e¥/lations u.sed ~'lr{~: LP(.':J l;rsolec~ぽü::'~ ，::'Yf]-:):'Jhosph[!.tidyl c.hoHrH:::); LPC'/cbl， ratio f)Iμg

lysoh~cithi:;l 'P，eι ，ug c111uIophyll Eo足司l11utln.十;;1lysolec~.tbin adde(~ to chkn:o:Jl.?，st Susp，'2~ns:ijon.

31

82 Satoshi HOSHINA

There hav日beenmany pape内 inthe literature (Helenius and 19'75 ; Tanford

and on t11尽 usεof in the re:seardLof membr豆~ne structure and

fUl1ctiom;， A typεof these studies vlIth

mode of assodation of membnme proteins vvith

interaction with diffεr団 1tchemlc司11ydefined

bOUl1d th(ヨstructureof the

inter:action of and thylakoid

used for

the exact

01

lipid molecules on the influence

Then~ are a fevl 町 portsabont the

have b日en

reaction centers 01'

Okamoto

the instructive results oI

si:udies on the interaction of linolenic acid and chloroplast rnernbranes， shm刊 d

that bindin2: of linolenk acid to

add between tbLC membrane and aqueous Inedium

also reported that linolenic add-indu正:ed

cause of the chan:zes in caDDcitl' of

The purpose of the present

into

structure were th君 main

to bh下dfree fauv add (Okamむ(0et

is to de凶e剖I肘l

released frorn the

and to elucidate the interactiofl betvveen In

the present paper describes the interaction be(ブ~7fveen andョcithinin

mernbranes as the inhibition of thεHilI reaction VJas

reactivai:ed 1Jv thεaddition of lecithin，

lWat町 ialsand ThlIethods

fl合om leaヮe~3 at

0，3 IvI NaCl and 50 rnM Tris-HCl

The isolated chloropla.sts vvere

in the same medium to obtain 0.2-0.4 mg per ml， and at

0-4CC before use， Chim・ophyllconcentrations were n:easurec: the method of Aでnon

Treai仰 nt

were incubated 'with lysolecithin at about 25'C for

to the effect

Vvhen sovbean lecithin

8-10 min as the incubation tim色

of on the Hm reaction

'iposomes were added to the

continued for anotehr 8-10 min at about

rεstoration of the Hill reaction in

6 min

chloroplasts， 1:he illcubation 'vvas

lecithin on

RVas COIrtPIeteWIthin

50 mM Tris-I-ICl

Retlacement 01 chloroplast lipids by lecithin 83

and 0岨6% ethanoL After the incubation oI the with or v，アith

and th♂ suspenSlOns were cooled placing on ice and

at x g for 1 hr at 4"C.

vw，re extracted from the 8upernatant or the chlofoform-methanol-'Nater n1ロムiλtur喝eaccorてdi

1机九ν吋y礼ithDEA生ムE-celluloS!悶色むolumn1 s;hca

G thin--Iaver as d泊こribe仁1 For lipiゴthe !ocatlons ofi.:he were rev日aledby iodine vatJor a'1d each

¥/:aS carぽ rernoved off the silica gel vvith 3， razαr blacte after the iodine

was evaporatea were detennined colむrimetricallyin tbe pres必nceof the siiica

by tbe phenol-sulfuric aod uf anC: Batt

liNf~re rff.eaStlfe(l in thie pres合nc♀ ofthe silica只elby the coIorimetric

me::hod of Farker and P日t.erson

So!ven版 01 were without r行distiHatiolL Lvsol台cithin(tyP(~ 1，

colnrnn

lecithin

tyx:川Il-S)Vlゥγe from

"7VI，:lS us仔dvvithoul: purific.ation， althougb it was

lecithin was

foHowed bv DEAE-亡elluIosecolunm

in thi:::: ¥NョySb，o'iNed :a spot ;.3iHco!

lecithin llpos()llD.es i¥J"lere as

Et昨日t]la自

口 thin-layerchrl'-

descnb合d

HosrjnaヲmdNishid2! describ"od that eff日ctof 1)みoIecithinon the

ot on th日 ratio 0;[ to

rath.cr than on its勺oncentrationa}one¥

boun，d to were determined as a Iunction of th行口-ttjり 0-2 /-4~

f¥t 10vv ratios of

was

At raLios lower than l.O~LG LPCichl予 the8xnount of

日1づlOunt心f うじith:b，] btlt at ratios

le88. }'tt 1.0-1.5 LPCichi， thどconcentTationof free

u; Vei"Y close tothe critic日 lTace}lec()D.centration of

et Pl. ca ve.n:~us

line園 Thi"indicatec， that the increasein binding at feltJOS t}!I

Satoshi 1-:1: 0:ヨHINA8'1

)U仏」

O 4 6，

:3

2

20EbbμJtω¥叫仏」

}U仏」勺C30心

Fig. L Binding oI lysolecithin with chloroplast membnmes as d. functio口af lysoIecithin addε士 Chloroplasts(:302μ，g chl/mI) werei.21cu.bated v，rith variol1s concentrations cf Iysolecithin. The suspensions wej"色 centrifugedand the s1Jpernatant was assay巴cl determine the conc巴D.tration of lysokci山1ユ Theai110unts of bo!md Iysolecithin which was given dS the ratio of J1101e lysolecithi日 permole chlorophyll， vvers calcu!ated from the diffsr巴nceof the concentrations of free and added lysolecithin. In the ョbS>211C巴 ofchloropJasts， lysolecithin addecl was totaUyγecoverεd th巴supematant.

W日間 released fron~

At 1.1 the release of

was detected. Th日

H1creasεwith

50-60 of mono

were detected 巴 th色

and about 10 % detected ir; the

and the release of

be not could mt日e

h

ι1ι

various amounts 01

1j+/3S observed to be

and

to

de<:en:nined.

2 ShOViS that

13

membranes

ratio of

a;nd

The extraction oI

¥'laS rel四 3ed at 5.4

obtained from

increasedwith

of canounts

were no:e

tfom

and

that exl:raded

τhe合xtractionof Iecithin as weli as

shovvs

memhranes.

勺

i Table

Rゅlaceme持 01chlorotlast lipids ゐ1よとcithin 85

100 守ヨ~.1i 的

c ~l

唖F'!.島

色ぬ園'cl

.岳 50町~-。同月

ロ

qtもz凶3 a

E匂L》

O

品

rii寸il

mM

円

U

E

D

同

G

削W

円

μ

6

(LPC/chi， 11'¥1l5lIg，h'け

Fig.2. R.el巳as己ofchlorophyll， monogaiactosyl and digalacto勾rIdiglycerides by th担 aclditionof lyso]日cithin. Exp日runentalconditions are clescrib日din Fig. 1. Th巴 susp日nsionof control chloToplasts and the supernatant of ]ysolecithin-間以edchloroplasts w吃reassay吋 tod抗告nnin巴 th巴 2，rnountsof chlorophylJ. and galactolipids目 [nthεsuspension of contwl chloroplasts， contents of monむgalactosyland digalacto8yl diglycerid巴S，and chkn・ophyll'?;leI日 82.6) 39.4 n.rnoles/rnl 在日d 30.2 μg/cn11 n:~spectively. IV1G-DC;'j monogalactosyl c1iglyc日Tide; DGDG， digalactosyl diglyceride.

'¥vas 50~60 % GI the v:::Llue in the untreat伐 i

It was οbtain日正l:romthr日e' sεparate

relε 伝 Vlere82-98 ~~，ら of theτrahle o"f bound

'The clata s}J.c)t'vvn :ln Table 1 1N'(:1S ;obtained fror1i1l. the

summ(~r. It may be noriced tbat th'l日 arnounts01' lecithin ancl

this 日川内runentaZi;ぅ lov/cr vJIih dat'-l

1966; Hoshina et 197ら Lichtenth211例市在日dP乱rk，)ID6;:;;

f(~3nIti) ohl仁川in日 ifnxn 弓 v1ith nonnal 心 in'Ninteγ and

.1l.Yiaterials also ，~~h仰町ed 2 SUrlUar trぞndto th市 seJ.ectiv巴，sxtract1onof

日日clCI1l0rOpl1yllvvas

the 日1行as位。fphosphatidyl inositol可7i，Ta巴

lipids. Total

and th日 arはountof

as calcuIated from Tablε"， 1.

th<:l.t lotal an.10tU1ts 01:

while

T'he

iHustr日tのぐ1in Table L

As the reactivation of the Hili n~act i.on Jn

o1]ser~\j'(:;d のn the additiun吋 !ec:ithin

it V"J~.:j"S that the

lecithiれ Table ~2

明rer合 recon:~;t. J.t"Lrt(! ュ可r1ith

were :scdded 1:0 t11昭

the amouム::1:8of !ysoledthin in i:he supernatant vver・e

ω 日an l\'~cithü.1 adde(cl， /¥t工b.e

reelrCti"v:ati.on

86 Satoshi HOSHINA

Table 1 Lysolecithin-induced release of glycerolipids from chloroplast membranes

chl LPC 乱!J:GDG DGDG SQDG PC PG PI

μg!ml nmoles!ml

suspension of control chloroplasts 27.5 Trace 77.9 32.6 12.2 2.3 1.8 2.0

supematant of lysolecithin-treated chloroplasts 4.0 160.9 40.4 18.7 1.9 1.3 0.3 0.5

Experimental procedures are described in Fig. 2 except that chloroplasts (27.5μg chl/ml) were incubated with lysolecithin (5.9 LPC!chl). The concentration of lysolecithin was 237.5 nmoles!ml. MGDG， monogalactosyl diglyceride; DGDG， digalactosyl diglyceride; SQDG， suJfoquinovosyl diglyceride; PC， lecithin (phosphatidyl choline); PG， phosphatidyl glycerol; PI， phosphatidyl inositol.

1979b)， the amount of bound lysolecithin was reduced to about half of the value without

lecithin， and the chloroplasts took up lecithin instead of lysolecithin. When 417

nmoles/ml of lecithin was added， the amount of bound lysolecithin further decreased，

and the arnount of lecithin taken up by chloroplasts almost corresponded to the amount

of lysolecithin released. Table 2 also shows that chlorophyll released by lysolecithin is

again taken up by the reconstituted chloroplast membranes. However， the amounts of

galactolipids in the supernatant were not changed by the addition of lecithin (data

were not shown).

Table 2 Replacement of lysole氾ithinbound to chloroplast membran四 bysoybean lecithin

No

可

i

ワM

句、

υ

lncubation with Lipids in supe口latant Calculation

LPC PC chl LPC PC binding binding release of of LPC of PC LPC by PC

nmoles!ml μg!ml nmoles!ml moles/mole chl

157 1.6 15 Trace 5.54

157 107 1.1 75 82 3.20 0.98 2.34 157 426 0.4 112 323 1.76 3.95 3.78

No 1: Chloroplasts (23.0μg chl/ml) were incubated with lysolecithin. No 2 and 3: Chloroplasts (23.0μg chl/ml) were incubated with lysolecithin， followed by incubation with soybean lecithin. After the incubation， the suspensions were centrifuged and the contents of lipids in the supematant were determined. The amounts of lysolecithin released by lecithin were calculated from the difference in the amounts of bound lysolecithin in the presence and in the absence of lecithin. The amount of lysolecithin added was 4.7 LPC!chl. When lecithin was incubated with lysolecithin in the absence of chloroplasts， both molecules were totally recovered in the supematant. PC， lecithin.

Discussion

The present study suggests the following sequence of events as increasing amounts

ltゆlace11'ze河tcり(chlorotlasi litids by lecithi抑 87

of lysolecithin are added to men'lbranes.

Stage 1: At ratios lower tha11 1.0 molecule was incorporated

into ch!oroolasts "Nithout 108s of m印 lbra.neCOffi1;J'Onents

decrease of chloroplast volurne was observed (Hoshina Hnd

Howe'ler， th台

The

may cause in

'In uncoupling of photophosphorylation 1979a; Hoshina and Nishida，

Stage 2: At 1.0-1.5

caused the extraction ()f and

membranes 'W'ithout release of chlorophylL 心ithinalso

caused the inhibh:iol1 of electron transport activities 01 bothγstems I and n (Hoshi口氏 1979a;Hoshina and

3: At ratios than 1.5 into

membranes further increased with ~oss of glycerolipids and

in disinteぽrationof membranes into

。rstTands

h'Nas showl!1 that caused the selective e:'Ctractiun of

221、able1). The releat，e of phosphatidyl ino:".札。1Vlas int日 mediai:ebetween

and 白 l:mtth校 release of

than 25 9~~J iN8.E often o~克己rved in the other

inosite.1 ro叫 tothe gr01Jp of whieh are

more easHy ex.tractecl‘1'he 01' lipid solubilization may many different

exp!anationsおご tothose Helenius and Sirnons 1.

rnay havεdifferent poweγs for different ふ 2.The ot

solubilization may ref1ect the differenc吃 of distribution in different the

membran尽s. 3. '1'h日目 mayre~3u]t Irom interactions betwc:en and

other n日emlJranecornpOJ切れts(I1vJ.In!y 'Vvhich would influence the of

extrぷ心tw口e

Itm正Iybe

chelTucal なnd

that explanation 1 ca1 be

of

ch日rgedgrのtlpぽ日nd

or

exteI机 aii half of the mem brane

in the inncr half

J弘llenet al.

simiIar in gn.1Jl.a and 8tr心Il~a

totzJ basis. It m.Ry

L.

IてJt'naybe ，f'ft":3Sりて1ablethat

more di:ffi心ulttoextγact

beca L18;2 1;JIG

suC'.h ::1S

19G6 ; Lichtenthaler and Park，

in tJnε

loe~丘町d

1972企

were

eithet on a pwtεin or a

to lmd郎、standthe

In s心的.11:'

88 S:Htosl:i I-IOSHD':<IA

or labεUed reagents， m配mbranεareShO¥Nl1 1:0 bεclassified into

tVlJO groups， One of these 1S caUed fluid matrix able to diffuse

of t11.e . The other group 13 imrnobile bound

et 1973; Trヨi，ubleand 1973 ; V'.f anen et

The latter are so-called

Lee'iこhand assuムmedthat

acids murht 10e the candidates for thε f1uid

7，9 % oi total ledthin固

actio目、iJil記re

that t11e circular dichn.Jlsm of 13

the ω:ldition of

品トvn

I

vd mvu

1

1

eyL

d

nru 、ISTι

バU

6

0

市山

ト

Hh

唱吋μー

{L

色

町

d

m凶

Rlh

t

qu ・-QU

OAu f-

fI'OlH

in1eraction of rnembτane componen;:s obs，ヨrvr::din vitro indicates

distribution of membTane compむnentsin native

It

membranes are

that the amounts of

to those

'，vas extracted

the amounts of lecithin incorporated 勺realulOst

membrane can be other

t11日sereconst1tuted membranes may b¥ラuseIulin

membranes.

of

Ack710wle紅側7山一一一一一 thankProf. K Nishida for his helpful comments and suggestions

durh1g the work目 Thanl，saJso are due to Mr. N. Tan'lai 8nd Th. 1(， Ryoyama fOT their valuable

disιussions， and to IVlr. ~叫 Kawamoto for drawing th邑figur但s.

Replace押ze匁t01 chloroPlast lipids ~ッ lecithin 89

Refe:rences

ALLEN， CT， P. GOOD， T. TROSPER and R. B. PARK (1972) Bioche押z.Biophys. Res. Commun. 48， 907-913.

， O. F!lRAYAMA and P. GOOD (1966) Bioche制 istry0/ Chloro.戸lasts. vol. 1，巴d.T.W. Goodwin.

Academic Pr号ssInc.， London and New York， p. 195-200.

ANDERSON， J，M. (1975) Biochim. Biolうわs.Acta 416， 191-235

ARNON， D.L (1宮49)Plant Physiol. 24， 1-15“

BLlGH， E目G.and羽T，J.DYER (1臼59)Ca侭.]. Bioche沖Z.Physiol. 37，911-917

BOARDMAN， N.K. (1970) Aηn. Rev. Plant Physiol. 21， 115-140目

DEAMER， D.W. (1973)]. Biol. Chem. 248， 5477-5485.

GENT， E.LG.， N.A. GREGSON， D.B. GAMMACK and J，H. RAPER (1964) Nature 204， 553-555

HALL， D.o. (1972) Nature， New Biol. 235， 125-126.

HEISE， K.P. and G. ]ACOBi (1973) Z. Natuゆrsch281:， 120-127.

HELENIUS， A. and K. SIMONS (1975) Biochim. Biojぅ，hys.lIcta 415， 29-7玖

HlRAYAMA， O. and T. NOMOTOBORI (1978) Biochim. Biophys. Acta 502， 11-16.

HOSHlNA， S. (1979a) S.α. Rep. J{anazawa Univ. 24， 45-53.

(197gb) Plant & Lell Phy引iol.20， 1107-1116.

， T. KAjI and K. NISHlDA (1975) Plant c島CellPhysiol. 16， 465-474.

一一一一← andK. NISHlDA (1975) Plant & Cell 1コ'hysiol. 日，4'15-484目

JOST， P.c.， O.H. GRIFFITH， R.A. CAPALDI and G.γANDERKO心1(1ヲ73)Proc. Nat. Acad. Sci. 70， 480-484.

KLOTZ， I.J¥1. (1946) Arch. Biochem. 9， 10吉一117.

LEECH， R.M. and D，J. MURPHY (1976) The Intaa Chlorop的お.品J.Barber. Elsevier ScL Publishing

Comp.， An正lsterdam，p. 365-401.

LElBOVITZ-BENGERSHON， Z.， L KOBlLER and S. GATT (1972)よBiol.Che加. 247， 6840-6847.

LlCHTENTHALER， H.K. and R.B. PARK (1963) Nature 198， 1070-1072.

MENKE，羽T.，A. RADUNZ， G.H. SCHMlD， F. KOENIG and R.-D. HlRTZ (1976) Z. Naturforsch. 31<:， 436

444

l'.iEUMANN， W. and E. HABERMANN (1957) Nature 180， 1284.

OKAMOTO， T. and S. KATOH (1971) Plant & Cell Physiol. 18， 539-550

， S. KATOH and S目 MURAKAMI(1977) Plant & Lell Physiol. 18， 551-560.

PARKER， F. and N.F. PETERSON (1965)]. L争idRes. 6， 455-460.

RADUNZ， A. (19'72) Z. Natu1プ'msch.27b， 822-826

一一一一一一一 (1976)Z. Naturforsch. 31c， 589-593.

ROUGHAN， P.G. and R.D. BATT (1968) Ar，ωl. Biochem. 22， 74-88.

SHlPLEY， G.G.， 1.P. GREEN and B.N. NICHOLS (1973) BiochimよEβ20βめ札 Acta311， 531-544.

T ANFORD， C. and J，A. REYNOLDS (1976) Biochim. Biophyso Acta ，1[;7， 133-170

THORNBER， J.P. (1自75)Ann. Rell. Plant Phy，iol. 26， 127-158.

TRAUBLE， H. and P. OVERATH (1973) Biochi悦 Biophys.Acta 307， 491-512.

WARREN， G.B.， M.D. HOUSLAY， J，C. METCALFE and N，J.M. BIRDSALL (1975) Nature 255ラ 684-687.

WlNTERMAl判， J，F.G.M. (1960) Biochi隅 BiolうわS.Acta 4ふ49-54.