requirement for plastoquinone a in the hill reaction of ... · requirement for plastoquinone ain...

Plant Physiol. (1966) 4-1, 633-640

Requirement for Plastoquinone A in the Hill Reactionof Isolated Chloroplasts'

P. M. Wood, H. N. Bhagavan', and F. L. CraneDepartment of Biological Sciences, Purdue University, Lafayette, Indiana

ReNvised nmantiscript receive(l October 1, 1965.

SimnlIrv(11'. 1lii order to avoid the l)roblelll of ani uncouplinig effect in stimiiulatioln ofpotential Hill reaction activity durinig extractioln with heptane, Hill reaction activitywas measured with uncouple(d spinach chloroplasts. TI'he reactions were also runi ulnderaerobic coniditionis. Under these conditions a correctioni for a 3- (p-chloroph'enyl) -1.1-dimethylurea insensitive, photostimiiulated O. uptake reaction is necessary, especiallywvith extracted chlori oplasts. The reaction displayed l)roper Hill reaction stoiohionmetrvin untreate(d and restored systems if these corrections were mlade. Plastoquinone Aalways restored some activity to the extracted chloroplasts but never restored activityto the original level. Additional activity could be restored by the crude extract. It ap-pears that there is a clear requirement for p)lastoquinone A in the Hill reaction l)ut therestoratioln of maximuimi activity dlepelnds upon additional factors. The variability ofresponse to plastoquinone A which has been reported in various lalboratories may bedetermined by the degree of extraction of the other mlaterials.

Proposals lhave leen nmade that plastoquinone A(PQA) functions as a redox intermediate in the Hillreaction of chloroplasts and in photosynthesis (1, 2, 5,8, 12, 13, 16). The evidence on which these proposalsare based includes restoration experiments which dem-onstrate that chloroplasts inactivated by solvent ex-tracitioni which removes PQA may be reactivated by,the readdition of purified PQA. Other lines of evi-dence are measurements of UV' difference spectraWvhich show changes upon illumiiniationi resemblingthose brought about by oxidatioln or reduction ofPQA. and measurements of light-induced changes inthe oxidation state of endogenous PQA. Certain re-cent developments have made (desiral)le a re-examina-tion of the requirement for plastoquinlonie A in theHill reaction. Chloroplast extracts were found tocontain 3 other plastoquinones, designlated PO B.PQC, PQD, and at least 3 tocopheryl (quinolles. Thisdiscovery supports the possibility of miore than oneplastoquinone site, as was su-ggested by olther work-ers, and raises the question of the specificity of therequirement for PQA in the Hill reactioni.

The present study deals with the restoration ofHill activity to heptane extracted chloroplasts by theaddition of PQA. We believed that a clearer pictureof the PQA requirement cotuld be obtained if con,di-tiolns wvere arranged so that the Hill reaction was unl-

I This research was supported in part by grant GM-10741 from the National Institute of General 'MedicalSciences and by a David Ross Grant.

2 Present address: St. Joseph Hospital, Lanicaster,Pennsylvaniia.

couipled fromii photophosphorylation, and if assayswere carried out under conditions favoring micaximliunmHill activity. Ferricyanide and NADP were used aselectron acceptors. The Hill reaction is usuallyestimated by measuring the reductioni of an externallyadded electron acceptor or by measuring O., evolu-tion; seldom are both nmeasured on the samne reac-tion miixture. Reports of active O., uptake ly iso-lated chloroplasts uplon illumination, and of photo-oxidations of various substalnces by (letergenit orsolvent-treated chloroplasts led us to feel that a betteranalysis mlight be made if both O., evolution and thereduction of the Hill oxidant were measure(d oln thesame reaction samlple and if attention were given tothe stoichiomietrv. The effect of the inhibitor CMUwas determined onl intact and oni reactivated systems.It is believed that the approach developed 'here willbe of use in evaluating the requiremiient for othersulbstances in the extract.

Methods and Materials

Preparation of Chloroplasts. Spinach was ob-tained froml local markets and the midribs were re-nmoved. For preparation of chloroplasts for extrac-tion about 300 g (wvet wt) of leaves were groundin a WN'aring blendor for 45 seconds in 600 ml ofcold 0.35 AI NaCI adjusted to pH 7.0. The crudestispensioni tlhus obtained was filtered through adouble layer of cheesecloth to remove leaf debris.The crude suspenlsion was centrifuged at 700 X gfor 5 minutes. T'lhe sediment was discarded and thesupernatant fractioni recentrifuged for 8 minutes at

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P'LANTP'I-IYSIOLOGY

1)(() X gIinnudiately, \\xithout fuirtlher washilln,

the sedimiient xwas frozeni in a dry ice-acetonie bathanid ly()philized for a miiaximiiumii of 4 hours. Thelvophilized chloroplasts wx-ere stored ini a desiccatorover calcium chloride at -10° anid w-ere used in allcases \within 4 (lays after their preparation.

I'frcparatiotl of 'lhotosvitti etic Pyridhine Nucicleo-ti(dc Reductasc (PPNR). Partially purified PPN-Rwas used in the experiments in which NADP was

the oxidanit. The PPNR was prepared (in our lab-orator-v) fromii spinach chloroplasts by a procedureessentiall- similar to that described 1w. San Pietroand Lanig ( 14). ''he l)rotaimrine sulfate purificationstel) \as iiot carried out.

recparation of Mlastoqjinbo)ne A (PQA). Puri-fi.ed 1O.\ was prepared in ouir lal)oratory. A crudechloroplast extract was prepared as (lescril)ed byHIenniniger and Cranie (11) and subjected to chromlat-ographly onl a decalso coluninii. The crude PQA prep-aration obtained in this fractionation was furtherplurifie(l 1! thini laver chromatography as described1v D)ille- and Cranie (7).

Otiher Reagenlts. Redistilled heptanie dried over

soditumii sulfate was used in all the extraction-reactiva-tioll experiiiients. All other reagents were obtainedcommniercially- ald were uised without fuirther purifi-cationl.

cllasitrcnent of O. l.7o/utiol. O) evoltition was

mieasuired polarigraphlically on the Oxygraph, an

inistruimiienit miianiufactured by- Gilsoni MIedlical Elec-troniics which uses ani oscillatiing platinum electrode.The reaction cell was enclosed in a glass jacketthrotugh \which water at conistanlt temlperature (250)-.was ral)idlv circulate(l.

of Ferriecani'de Rednctioni and 0,, Evoli-tioni. The reactionl miiixture was prepared in a glassstopl)ere(d ttube. a fresh miiixtture beilng prepared be-fore each assay. It contained the following: 75uLmole Tris (hydroxvmethyl aminomiethanee)-HCbuffer, pH 7.0: 3.0 ttnole potassiumn ferriceainide;chloroplasts; watel to 4.5 ml. A-kdditions to thisbasal reaction mllixtuire are indicated whllere aippropri-ate. The chloroplasts were added last, and the com-l)lete miiixtuire was wvell miiixed by inversion of thetulbe. As the rate of reactioni sometimfnes varied (le-p)eldil-g oln the lenlgtlh of timiie the chloroplasts wtereexl)osedl to the reaction mlixtuire, the lengtll of timiieafter the additionl of the chlloroplasts prior to illumii-iiiatiol w as kept constant. The enitire l)rocess vas

carried out in dini light, in wvlhiclh no significanit re-

action occtirre(l. After proper mixing. 1.; ml of thereaction mixture w-as pi)etted into the Oxygraphcell xhile the remainder -as kept as a clark control.T'lhe light reaction was carried out in thle Oxvgraphcell, which xwas mnaintained at 250. After an initial(lark period, the cell xwas illuminated by a 100 wv

tuingsten lamp and ( ), evolution wxas folloxved hvnmeasurintlg> tlhe increase of O. concentrationi in solui-tion. \fter the liglht xwas tuirned off, the light an(l

dlrVk re;it1ion mixtulrc;e x-relre prateini7cdl x-ith TCA

aiid the rcesidual ferriexanide \xxas (let ermiinied 1b meas-

uriii- the absorption at 420 imi/u.-Issay of N.AJJ)J' 1- Rediction (1)1(d O., LvolhitioMn.

A basal reactioni imiixture xx-as prepared in a glassstopperedl tube containuing the folloxxin-g: 50 uiiiolesTris-H[Cl buffer 1)1 /7.0-; 2 mig NAD[); PPNRZ [4units as definie(d bx Sani Pietro and Lail- (1chloroplasts ith a chlorophyll contelt ralnging frtirol0.0-5 to 0.150 mg xx;water to 3.() 1lz. Trlhe proce(ldurfor illutminationI xas idenitical to that described foirthe ferricyainide assav. After illuliniation. 1.0 nil

aliquots of both lihlt anld dlark mixture.s xxere eichpipetted direcilx inito 2.0 ml xi sod(itim carboliate.''lhc samlples xere centrifuged in a clinlical centrifilugeto precipitate the elloroplasts. tle silperiliatantsx\vcrctraiisferredl to ctvettes. anid the al)sorb-aluce at 34(

VL \xas iiieastire(l. 'I'lhe ad(lditioml of N;tLC( )8. prexvemitclthe gradual reoxi(lation of N AI)IPH observed ill it.salbseiice.

11ctho/d of k.vlrhatio11. 1, vophilized chloroplastsxx-re extracte(l \ ith 20 ml of ice cold heptanie 1)er5 lug of chloroplast chlorophyll. 'T'he extraction xascarrie(l otut in aL gIass ho01mog-enizer \ ith a g-entlehomogenization \ ith a loose fitting teflon pestle forlength of timlle ran-ginig fromli to 15 miniutes. )ur-

iing the extractionl the holmiogeniizer \xas iniserte(l ill a

bucket of ice. After extraction, tlle extract \asremoved by filtration through a siiitered glass funlnlel.T'he solv-ent remaining in the chloroplasts xxwas re-

inoxed 1v directing- a cturrenlt of liitroeil olito tllechloroplasts.

Readdition (oOinones (11( 1 .extraet. th'lle cx-

tracted chloroplasts \xxcre scral)e(l fromii the funnl,el,divid(led into thle de.sired number of samples an(l placediln imldlividual tubes. The extract \as evapl)oraite(l todrviless anid redli>ssolxed ini a-. smiiall volulnme if betl'mllso that the extralct could lie added to its originial lexini oine of thlie samples iii a volulme of l. Ouillolesor extract \xxere a(l(led ini 1 nil hleptanie anid tIle sam

ples \ ere iixecl xx-itl a. glass stirrimlg rod. ()u1ioloexxas added in amiiotunts aimingii- at a final ratio ofchlorophyll:quiloie of 10 :1. Extract xvas a(lde(lto a level equal to that ini tlhe ori-imald chloroplasts..\fter piroler inix'ig, tlhe heptaclle xxas rellioveci in

vactiii, and( the chlo,roplasts xwere res1slteipcled il \xaterto a colncenitratioil of chlloroplhyll of about 1 IIIg

per ml.An1alvsis ofl .rtr-acf: Qitalitative. Qutalitative amid

qunaititative aiialvsis of the extracts miiade use of theteclhniqlue of tlimi laver chromiatography. Thili layerplates 0.25 mmii1z thick of IMN-Kieselgel GHR xvere

sedI ('Machevey. Nagel. aild Company). For anialx-sis of PQA. a 60:40 miiixture of bezene:heptane Nvasuse(d as the developinig solx-eiit. For analysis of POCr PODQ)I cllorofoi ll x-x as used as the solxenct.

OtiiiioiC spots erc detected li\ sprainil- itlh

re(duiced m neth vlenc lilute xwhich t1 i(poi (ixidationll the

qtuilicole forme(d a blue spot. A solutioni of re(duice(dmiiethvlivie blute xxwas pirepare(l 1y sxvirlin.g I zziic

(lust and 2 nil 0.r1 met lcue blt me wx ith l ccil

ciutrate(d silfU rie ci I.\\IWhen ceolor1c>.s. the :S,ltltilion

o34

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WVOOD ET AL.-PLASTOQUINONE A IN THE HILL REACTION

wvas filtered through glass wool to remilove zilnc dust,and was used as a spray before significant 'blue colorreappeared.

A thin layer plate was divided into 4 bands, ap-proximiiately eqtual in width. Samples vere appliedabhout 3 cim from one end of the 'bands. The leftmllost portion was used for a plate control. Theextract was applied in duplicate to the middle sec-tions. Test spots for spraying were placed in thelast section. After developnment of the plate, it wasremoved from the tank, allowed to dry, and the testsection of the pilate was sprayed while the other sec-tions were covered. After location of t'he quinone,a corresponding band is scraped from the plate fromeach of the other 3 sections and placed in a testtube. Each sample is eluted with 5 ml ethanol byalternate mixing and standing. After proper elutionthe mixture is filtered through filter paper. Theabsorbance of the filtrate at 255 m,u is determinedbefore an'd a'fter reduction with potassium borohy-dride, and the concentration of PQA can be calcu-lated if the change in millimolar extinction, oxidizedminus reduced, at 255 mju is taken as 15.

Detcrinination of Chlorophyll. In the extractionexperiments it was convenient to hiave a rapid methodfor determining the relative chlorophyll concentra-tion of the samples. Chlorophyll in vivo has 1 ab-sorption maximum at 675 ml,u. It was found thatthe absorption at 675 depended in a linear manneron the concentration of chloroplasts. Since all thesamples in any 'particular extraction study were fromiithe samiie original chloroplast preparation, and sinceall had received similar solvent treatment, this methodwas adopted as valid for determining the relativeamoiunt's of chilorophyll in each sample.

Absolute chlorophyll concentrations were deter-mined by the method of Arnon (3).

Characterization of the Intact Systemn. Prelim-inary experiments aimed at a characterization ofthe Hill reaction under our conditions were carriedotut using intact or unextracted systeims. Freshlyprepared chloroplasts showed comparatively low ratesof Hill activity as measured by 0, evolution at pH7.0 in the absence of uncouplers (NH4CI). The samechloroplasts catalyzed much 'better rates of 02. evolu-tion at pH 7.8 in the presence of phosphate acceptors,or at pH 7.0 in the presence of uncouplers. Chloro-plasts lyophilized for 4 hours shiowed good rates ofHill activiity in the absence of uncouplers. Table Ishows the results of an experiment in which both 0^evolution and photophosphorylation were measuredunder conditions identical to those used in otherassays except for the presence of the phosphate ac-ceptor sys;teim.

The effect of ammonium chloride iat pH 7.0 and7.8 and the effect of lyophilization was determined.The chloroplasts were able to catalyze a Hill reactioncoupled to photophosphorylation at pH 7.8 and thecoupling was released by the addition of ammoniunmchloride. At pH 7.0 a very low rate of phosphory-lation occurred and the rate of O. evolution waslimited by the low rate of photophosphorylation. Theadditiion of ammonium ions eliminated photophos-phorylation and greatly stimulated O., evolution, aresult which is consistent with anl uncoupling effect.Lyophilized chiloroplasts in the absence of anmmoniumwere able 'to catalyze good rates of O.2 evolution, withverv little concurrent ATP formaation. Lyophiliza-tion has accomplished uncouplinig, or has activatedla pathway for ferricyanide reduction anid O., evoltu-

Table I. 0 Evolutiont and ATP Formation, by Fresh and Lyophil&sed Chloroplastsin the Presence and Absentce of NH4Cl

Basal reaction mixture: 25 umoles Tris-HCl buffer, pH as indicated; 2.0 ,umoles potassium ferricyanide; 1.0 ,umoleMgCl,; 1.0 umole KH,PO4-K,HPO4 adjusted to the pH inidicated and enriched with 32p so that the specific activitywas 21600 cpm/,umole for the pH 7.0 and 21850 cpm/,mole for the pH 7.8 P; chloroplasts; H,O to 2.0 ml. The reactiontime was 5.0 minutes.

Conditions

Reaction mixturescontaining freshchlioroplasts witla chlorophyllcontenit of 0.064 lilgpH. 7.8

Freslhchlioroplasts(as before)1H 7.0

Reaction mixturescontaining lyophilizedchloroplasts witha chlorophyll contentof 0.075 mgpH 7.0

Additions tobasal mixture

None1 /umole ADPI gimole ADP + 10

,uniole NH1CI

None1 ,Imuole ADP1 jumole ADP + 10

/Amole NH4Cl

1 4Amole ADP1 ,rmole ADP + 10

/,mole NH4Cl

02 evolved(umoles/mg chl/hr )

1013

ATP formed(,umoles/mg chl/hr)

020

17

76

26

19

0

0

2

23

635

1



PLANT PHYSIOLOGY

tion niot coul)led to photol)hosp)hic)rvl.ationi.One of the I)rol)lems enicounltere(l in the mieasure-

eent of (., evolutioli by the 14ill reactioni is thatof light stimuilated C)., uptake reactionls of the chloro-plast suspenlsion (9). The rates of 0., uptake withwhole chloroplasts were ustually smnall (ca. 5 % ofthe rate of 0), evolntion). but occasionally freshprel)arations showed a nmarked ).. tiptake. (O. uptakeof 10 to 20 % of the rate of ()., evolution was colm-monly' observed with extracte(l chloroplasts.

The O. ulptake reactioni of whole chloro)lasts wasfirst nioticed when a study was made of the effectof the inhibitor, CAMU. Concentrations of CAIUsufficienit to enitirely inihibit O., evolution did tnotinhibit ferricyanide reductioni. By increasinig theconicenitration of CMU, ferricyanide redluctioni couldlbe comipletely eliminiated, thus exposinig an O., llptakereaction. Table II illus;trates 1)oth extreme andi tvpi-cal exanmlples of this .situiation. The relationi of thisreaction to the 0., evolution reaction is niot clear.That the (., ul)take occurs independently of the [-illreaction is ind(licated by the fact that 0., uptake occutrsin the absence of the Ilill oxi(lanit. alind that 0., uip-take in the Cabsence of Hill oxicdants is niot inhibitedby CAlU (table 11). Observationis of O, lll)takereactioins simiiilar to tho.se in the first l)art of table Ithave been reported lby de Kouichkovsky (6). wvhoattributes the reactioni to a Afehler reaction. where(., itself serves as oxidant for a Hill reactioni. C)i

the other hand, Fork (9), has observed this (., up-take and feels that it is based oni eni(logenious oxi(lallt.h'l'e lack of effect of CMRL is niot conlsistenit wNrith aM ehler reaction.

It might also be reasonedl that C)., uptake occursonly w-heln the Hill reactioni is blocked either by CMA tiii-llihbition or by niot supplying a Hill oxidant. Itwould niot be possible to dleci(le this maatter if 1oth(., uptake an(l )., evolution were always linear asthey istlally are. \Vith occasional l)rel)arations (.uptake ceases before 0.r evolution. TIn this case tlherCslhould( be an apparent increase in the rate of 0., evo-Ittioni as the rate of C)., uptake declines. Figure 1showN-s that this is the case. It is apparent that theOX, evolution obtained by miieasulr-emiienit of the niet ()changes in the mlixture may nlot represent the absolutevalue for the O., evolved as a restilt of Ifill reactionactivity. WVe feel that a better estimiiate can be o0-taimied if the value for ()., up)take in the presence ofenouglh CM_U to totally inhibit the MHill reaction isa(l(le(l to the value for (O) ev-oltutioln in the al)senceof inhiitor. Tlshis correctioni was routiniely appliedin stoichiometrv Inieasnilren)lents with ilntact chl)r(-)-l)lasts.

Experiments vere coni(lucte(l to measure tlle stoi-chiomnetrv of the reaction tull(ler onr cond(litionis. TI'hestoichio'nietry olbtainie(d wN-ith intact chloroplasts ap-l)roache(l 4:1 for miioles of electrons transferred tomoles of 0. evolved wheni the correction \\was alpp)lie(lfor CM F' insen.sitive *. release.

Table II. 0, Uptake Reaction of [['iolc Chfioropla.stsThe ferricyani(le l)asal mixture containe(l: 25 /umoles Trris-HCl buffer, pH 7.0; 1.0 uniole ferricyai(le; chlorroplalsts:

H,O to 1.5 ml. The NAD)P basal mixture contaie(l: 25 ginoles Tris-HCl huffer, oH 7.0; 1.0 mIg NAI)I' t-; PPNR(2 units) ; chloroplasts; H.,O to 2.0 ml.

Systemii

Ferricyanide mixture+ NH4ClFerricyanidle mixture+ NH4Cl + CMIU(4 X 10 ' M\)Ferricvanide imlixtuire+ NH 4C1 + CMU

(4 X 10k' M)

Ferricvanide mixtureiniu.s ferricyanide

Ferricvanide mixtureiliillEtS ferricyanide,CNML (4 X 10- -- ) add(le(d

Ferricyanide IlliXtUre+ NH4CIFerricyani(le mixture

- NH 4Cl + CML (4 X 10 xi)

NADP + mixture+ NH C1NAI)P + mnixture A

N H4Cl + (4 X 10 \I)

O.,evolved

,umoles/mgchl /hr

30()

12 *

18

-18

71

5

Ferricyanidereduced

ylmloles/niigchl /hr

NADP-reduce(l

,uilR)les/lllu-chl /hlr

258

8

0

0

352

0

16875

6

* Negative values indicate O.. uptake.

636

Chloroplastpreplaration

I

(Chlorophyllcontent ofthe reaction.imiixtutres wvas0.036 nig)

II(Clhlorophyllcontent ofthe reactionmixtures -was0.028 m-g)



WVOOD ,T AL.-PLASTOQUINONE A IN THE HILL REACTION

0.08

to0I

E

w

LL

x

0wzw

0

0.06j.

0.04

0.02F

0.00

-0.02

- (YA4

B

1.0 2.0 3.0 4.0TIME (Minutes)

FIG. 1. 0, uptake by whole chloroplasts in presenceof CMU. Curve A. 0, evolution by chloroplasts asobserved in the presence of ammonium chloride with fer-ricyanide as oxidant. Curve B as for curve A but inpresence of CMU.

To comiplete the oharacterization of the inltactsysteml the effect of CMU was studied. Table IIIreports the effect of CMU onl the Hill reaction withferricyanide as electron acceptor. The reactions wereconducted 'in the presence of ammonitum chloride.CMU is seen to be effective at low concentrationsin 'inhibiting the reaction.

Reactivationt Experi;inents. Heptane extractiolnof lyophilized chloroplasts results in partial or conm-plete loss of the ability of dhiloroplasts to catalyzethe Hill reactioni. If the crude extract is concen-trated and added back to the extracted chloroplastsat a level eqtlivaleiit to'that in the original chloroplasts

Table III. Effect of CAMU on Ferricvanide Reductionand on O., Evoluitiont

Reduction mixture as shown for table IV with CMUas shown. Chloroplasts containing 0.032 Itig chlorophyllused. Reaction time was 4.0 miniutes.

CMU

None3.5 X1.8 X3.5 X3.5 X3.5 X

Ferricyanidlere(luced

,umoles/mngchll/hr

3833131701294044

10 Nr10 ;I106ei I1 0-s-,110-4 M

O., evolvedl,gmoles/mgchl/hr

725124140

- 4*

Nl*, Negative valu,e indicates O., u-ptake.

the photoreductase activity of the chloroplast is par-tiallly restored. In table IVT data is presented fromextractions of different preparations of chloroplasts.An interesting fealture of extracted chloroplasts isthe enhaniced 02 uptake reactions. This is shownby cases where there is 02 uptake and ferricyanidereldtiction is zero, or in cases when the apparent 0Oevolution is zero and significant anmotunts of ferricy-anide are still reduced.

Table V presents tlhe results of an experimenttesting, the effect of various amounts of PQA addedto approximately equal amounts of extracted chl,oro-plasts. The extract was later analyzed and theamount of PQA actually remloved was calcuflated foreach sample. O2 evolution is shown with ferricy-anide and with NADP+ as the Hill oxidant, and forcomparison th,e rate obtained with lyophilized chloro-plasts is also presented. The effectiveness of lowlevels of PQA should be noted. The best restorationoccurred with levels of PQA slightly higher than thatremoved. A different experiment in which the ex-t;ract also was added back is reported in table VI.The ability of the extract to restore activity is appar-ently better than that of pure PQA. That effect of

Table IV. Hill Reactiont wzith Ferricyanide ofter Extractionantd ofter the Readditiont of Extract

Reaction mixture as describe(d in Methods. Reactioni time was 5.0 minutes.

mg chlReaction mixture

0.0300.0370.0350.0760.0470.0570.0290.026

* Negative value for 0, indicates 0., uptake.

Preparation

1

2

3

4

Sample

ExtractedReactivatedExtractedReactivatedExtractedReactivatedExtractedReactivated

Ferricyanidereduced

,umoles/mngchl /1hr0

59459650

1032168

O., evolvedumoles/mgchl/hr

-6.

6

0139

20213

_ .T .

637



PLANT PHYSIOLOGY

the extract is due simply to its having the correctlevel of PQA is n1ot likely since the data of table Vind(1icates that excess PQA does not haxve a greatlyinlhibitory effect. In aniother exl)eriment this possi-bilitv was checked l)v a(lding pure PQA to a saturat-

ing level l)lus extract to half its original level. Thestipplementarv extract did not inhibit but stimulatedthe rate over that obtainied with satturatinig PQAalone.

Somethiing slhouild be sai(l about the percentatge

Table V. Effcctivcniess of I 'ariou,s Amounts of Purified IQA in the Reactiviationtof Extracted Chlor-opl(asts

Extractioni and readditionis as described inNMethods. CMIU wvas a(lded in 0.02 ml ethanol. The reaction mixturesare described under Methods. The reaction time was 3 miniutes.

'I'reatmenit

UinextractedUIlextracted + CMU(4 X 1(0-4 )ExtractedFxtractedExtractedlExtracte(lExtractedExtracted + CMU(4 X 10-4 \I )

illg chlReactionimiixture

0.05

0.050.080.110.120.100.12

0.12

mg PQARemoved

0

00.070.090.100.080.10

0.10

nug PQAAdded

0

00.000.010.060.120.24

0.24

O., Evolvedferricyanidleas oxidanit,umoles/nmg

chl /hr

5

06

1010(1 312

O., EvolvedNADP+ asoxidant

)u1nl01es/Ingchl /hr

26

-3"

679X

-4

Negative valuies for O., indicate O., uptake.

Table VI. Comn parison of the Ability of t(e Extract and of Pullrified PQA4to Reactizvatc Extracte( C(i loroplo(sts

Reaction mixtures as (lescribe(I under Methods. The reaction time was 3.0 minutes.

Uniextracted1Extracte(lExtractedExtractedExtracted 4- Extract

n.ig chl.Rteactionmixture

0.0950.1200.1700.1300.155

iiig PQAReiemoved

0.120.180.140.17

Ing PQAAdded

Nonec0.12(0.24().16

O., Evolvedferricyanideas oxidanitugiioles/miig

clhl/hr

32481013

O., EvolveclN AI)P+as oxidantAjmiioles/milg

clhl/hr

1536612

Thle extract was ad(led to a level equal to that in the utnextractedI chloroplasts. Upon anlalysis the amolunt of extractadl(de(d was found to conltaini 0.16 mg PQA.

Table VI f. I)etermnination of the Stoiiomeoltry of Re(ativationCalculation of stoichiomiietry as (describe(d in text. The reactioln mlixtuires are (lescribe(l tn(ler Methods. The

reaction timile is 5.0 iinutes.

Uerricyaiiide as oxidant

Chlilorophlyllper reactiisiinxture (mg)

0.0)310.0330.0310.0420.0390.042

Ferricyanidere(litce(l

(uninoles/ingchl/hr)

19

106

( )., evolv1ed1(tlnolesX/lim,i'chl/hr)

3 :

11

I I)

NAD.1) as oxidant

N .\l )Pre(luceld

(/.nl(ole/Ingchl/hr')

61327

O., evolved( ,uniole/inlg

chl/hr)

-7.4-4.6

1.7

- Negative values indicate 0, uptake.

\dditions tothe extractedcl loroplasts

NoniePQAExtractNonePQAExtract

6)38



WOOD E1T AL.-PLASTOoIJNONE1 A IN TIIE HILL REACT'ION

of restoration of the activity of the original chloro-plasts achieved with the reactivated chloroplasts.There are several reports in the literatuire which showrestoration of 100 % or more of the original activityof the unextracted chloroplasts by adding pure PQA(4,10, 13). A general feature of all the extractionexperiments carried out in this study is that thedegree of restoration did not exceed 70 %, even witlhthe whole extract, land the degree of restoration ob-tained with pure PQA alone did not excee-d about30 %. For this reason the rate obtained bv ad(iingback the extract may be a better measutre of thep)otential activity after solvent treatment.

Several experiments were carried out in whiclhattention was given to the stoichionmetry of the reac-tivate(l Hill reaction with ferricyanide and NADTP+as oxidants. Data from a representative experimentis reported in table VII. In calculating the ratios,the values for the amount of photoreduction anid 0Tevolution restored may be obtained by sultractingthe value for these quantities as shown bv the ex-tracted chiloroplasts from the value as shown by thereac'tivated system. We feel thait these (liffereinces,if due to a reactivation of the Hill reaction, slhouildshow Hill reaction stoichiometry, anid should be ab-solute values. The effect of CMU on the reactivatedchloroplasts was checked, and it was found that lowlevels (4 X 10-' At) eliminated the reaction.

Discussion

The data for the stoioh.iomiietrv and for theeffect of CMU are favorable to the conclusion thatthe stimulation of ferricyanide and NADP reductionani(l of 0 evolution is due to a stimulation of theHill reactioni. Qualitatively this confirmiis the reportsof earlier workers.

Two additional facts emiierge from this stutdy)which appear to be a result of the use of a nonphos-phorylating system operating tunder collntions wheremaximumll raites of Hill activity may be ob)tainled. Thefirst is that solvent treatments similar to those uisedby other workers result in irreversible loss of somleof the 'original activity, i.e., 100 % restoration wvasnever obtained. Secondly, as may be seen froml talblesVI and VII, pu,re PQA never gave as miuch restora-tion as (lid the extraot. In fact, the anm,otunt of res-toration with PQA was ussually about half that ob-tained with the crude extract.

'T'he simplest interpretation of these resuilts is that-the extract contains a substance other thaii POAwhich function-s in the Hill reaction. The natuireof this substance is being investigated. A study ofthe components of the extract has showln that thereduced form of PQA 45 is one of the major factorsin the additional activity of the extract (18) in res-toration of the activities reported here.

The evidence presented indicates that addition ofplastoquinone A alone to heptane extraoted chloro-

plasts does nlot restore Hill reaction to the originialuncoupled level. Other factors such as the reducedplastoquinone A are involved in developing maximumiiactivity. Sutch factors can acciount for some of thlevarial-ility observed in restoration with purified PQA.

Literature Cited

1. AMESZ, J. 1964. Spectrophotometric evidence forthe participation of a quinone in photosynthesis ofintact blue-green algae. Biochem. Biophys. Acta70: 257.

2. ARNON, D. I. AND A. A. HORTON. 1963. Site ofaction of plastoquinione in the electron transportchain of photosynthesis. Acta. Chem.. Scand. 17:135.

3. ARNON, D. E. 1949. Copper enzymiies in isolatedchloroplasts. Plant Physiol. 24: 1.

4. BISHOP, N. I. 1961. The possible role of plasto-quinone (Q-254) in the electron transport systemof photosynthesis. In: Quinones in ElectronTransport. G. E. W. Wolstenholme and C. M.O'Connor, eds. Little, Brown and Company,Boston. p 385.

5. CRANE, F. L., B. EHRLICH AND L. P. KEGEL. 1960.Plastoquinone reduction in illuminated chloro-plasts. Biochem. Biophys. Res. Commun. 3: 37.

6. DE KoUCHKOVSKY, Y. AND J. M. BRIANTAIS. 1963.Light induced oxygen reactions in isolated chloro-plasts. In: Photosynthetic Mechanisms of GreenPlants. National Academy of Sciences, NationalResearch Council, Washington, D. C. p 362.

7. DILLEY, R. A. AND F. L. CRANE. 1964. Light de-pendent conversions of endogenous-tocopherol-quinone and plastoquinone D in Spinacia oleraciachloroplasts. Plant Physiol. 39: 33.

8. DUYSENs, L. M. N. 1963. Studies on primary re-actions and hydrogen or electron transport inphotosynthesis by means of absorption and floures-cence difference spectrophotometry of intact cells.In: Photosynthetic Mechanisms of Green Plants.National Academy of Sciences, National ResearchCouncil, Washington, D. C. p. 1.

9. FORK, D. C. 1963. Action spectra for 0., evolu-tion by chloroplasts with and without added sub-strate, for regeneration of 0., evolving ability byfar-red, and for 02, uptake. Plant Physiol. 38:323.

10. HENNINGER, M. D. AND F. L. CRANE. 1963. Res-toration of photo-reductase activity in acetone ex-tracted chloroplasts by plastocquinones and toco-pherylquinones. Biochemistry 2: 1168.

1 1. HENNINGER, M. D. AND F. L. CRANE. 1964. Iso-lation of plastoquinones C and D from spinachchloroplasts. Plant Physiol. 39: 598.

12. KLINGENBEWG, M., A. MULLER, P. SCHMIDT-MENDE,H. T. WITT. 1962. Changes in absorption dur-inig photosynthesis in the ultra-violet spectrum.Nature 194: 379.

13. KROGMAN, D. W. AND E. OLIVERO. 1962. Thespecificity of plastoquinone as a cofactor for photo-phosphorylation. J. Biol. Chem. 237: 3292.

14. SAN PIETRO, A. AND H. M. LANG. 1958. Photo-synthetic pyridine nucleotide reductase. I. Par-tial purification and properties of the enzvme fromspinach. J. Biol. Chem. 231: 211.

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PLANTr PIIiN S1OfLOG,Y

15. T1AkGAWA, K., H. TSUJIJMOTO, AND) 1). I. ARNON.1963. Analysis of plhotosynithetic reactions by theuse of monochromatic light. Nature 199: 1247.

16. TREBST, A., H. ECK ANI) S. \WAGNER. 1963. Ef-fects of quinones and oxygen in the electron trans-port system of chloroplasts. In: PhotosyntheticMechaniisms of Green Plants. 'National Academyof Sciences, National Researchi Counicil, \VNashing-ton, D. C. p 174.

17. \VFINON, L. P. AN)) \\. S. ZAUGG. 1960. Plhoto-reductions by fresh and aged chloroplasts: Re-quiremenit for ascorbate and 2,6-Dichlorophenol-indopheniol with aged chloroplasts. J. Biol. Cheim.235: 2728.

18. WOOD, P. M. AND) F. L. CRANE. 1965. A require-ment for reduced plastoquinone A in the Hill re-

actionl of extracted chloroplasts. Biochemii. Bio-phys. Res. Commun. 20: 274-78.

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requirement for plastoquinone a in the hill reaction of ... · requirement for plastoquinone ain...

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