effect of desaspidin on photosynthetic phosphorylationeffect of desas!idin anid...

Effect of Desaspidin on Photosynthetic Phosphorylation" 2

Zippora Gromet-Elhanan3 and Daniel I. Arnon4Department of Cell Physiology, University of California, Berkeley, California

Desaspidini, a phlorobutyrophenone derivative. isused in mledicine as an anthelminltic agent.

CH3 CH,

HO OH CHHO OH

ICH,C3HrCO 7 - COC,H7

0 OH

DE SASPIDIN

Runeberg (20) founid that desaspidin acts as apowerful uncouipler of oxidative phosphorylation.IBaltscheffskv and (le Kiewx-iet (13) ilntro(dticed (lesas-pidin to the stt(lv of photosynthetic phosphory-lation.l'liex sll)(livi(le(l 5 systemls of light-induced p)hos-phorylatioll ililtO 2 grou0ps witl respect to their senl-sitivity to inihibition by desasl)i(lin. In 1 groulp. therate of AE'P formationi was rescluceti l)\ half at a vervlow (10-- : ) conicelntrationi of dlesasl)idlin. whereasin the second grouip a 100 timles greater concentrationof desaspi(lill was reqtuired to give tlle samiie (legree ofillhibitioni.

In the present inlvestigatioin, the inhibitory actionof desaspidin was used to test further the validity ofthe subdivision of photosylnthetic phosphorylation byclhloroplasts inlto 2 distinict types: a type discovered in1954 (3, 6) and(l later designated as cyclic photophos-p)horylation (8). in which ATP formiiationi by illumlli-nate(l chloroplasts proceeds with nio consumption ofeither ani electron donor or an electroni acceptor, anida type discovere(l in 1957 (8 ) ai(i later rename(l nlon-cyclic photophosp)horylation (2). in which ATP for-Matfon is linked stoichiomiietrically with the consumip-tioni of an electron donlor anid ani electroni acceptor.The electron donor was water, andcl its oxidation liber-ated O.2; the electron acceptor was either TPN orferricvanide (8. 9). 'rhe terms cvclic alnd noncvclicwere introduced to denote the couplinig of phosphoryl-atic i to a light-induced closed or open electron flowthat liberates the requisite free energy for the syn-thesis of ATP (2).

1 Dedicated to the memory of David P. Hackett.2 Received June 21, 1965.' Permanent address: Biochemistry Section, WVeizmanni

Inistitute of Science, Rehovoth, Israel. Grateful ac-knowledgment is made of the Charles F. Kettering Foun-dation International Fellowship.

4 This work was .ai(ded by graints fromii the 'NationalTIistitutes of Health, Office of Naval Researchl aind theCharles F. Kettering Foundation.

Sensitivity to desaspidIini inhibition was also use(lin the present investigation to test the vali(lity ofcharacterizing pseudocvclic plhosphorylation as aspecial case of nloncyclic lhotophosp)horylation inwhich an open electron flow is generated betweenwater as the electron donor and 02 as the electronacceptor. This nonicvclic electron flow, fromii w-aterto externial O., gives the appearance of a cyclic elec-troni flow since maiionoetric imieasturiing techniquesgive Ino incdication that ani electron donor and accep-tor are being consumed concomitantly- with ATPformationi. In pseli(locy clic photophosphorylation theconsutniptioin of O., at the terminial end of the elec-tronic pathway is balanced 1b the production of ('.ait the site of electroll donatioln 1bx w-ater (10).

In a(l(lit ion. selnsitivity to (lesasl)i(li illhihitiom)ro\e(l to be uisefiil ill clanrifvingp the type of lhloto-phosphorvlatioll 1)y chloropla. ts (N19) that accom-panies re(lutctionl of ''1PN hv illulniitiate(d chloroplastsill a 11n)1pilysiological non01cvclic svteiii in \Nwhiclh r-e-dluced dichilorophenol ind(lophielnol replaces water asthe electroni (lonor (24 ) ThIis txype of pihotopllo -

phorylationi \w-as pre-iotusly conisi(lere(d (19) to occiir-at the samie site as the photophosphorylation \vhicbaccompanies TPN reduction wshen water is the elec-troni donior (8, 9). A tlifferenzt initerpretation willnow be proposed oni the basis of the liew- results w-ithdesaspidin.

Methods

Brokeni spinach chloroplasts ( P'1 ) \ere l)reparedwithout adde(d ascorbate, as (lescribedI 1v WVhatleyan(l Arnonl (29 ) ChloroplivIl was deteriniiie(d hvthe method of .\rion (1). The reaction was car-ried out in conlical manaomiieter vessels at 200 in aWVarburg water bath with continuious shlakinig (4. 6).The vessels were equilibrated for 10 minutes. prior toillumination, with either argon or air. The vesselswere illuminated from below by a bank of 300- and150-w incaindescenit flood lamps that were so ar-ranged as to give eqiuial illuminationi throughout thebath. In certain experiments monochromatic lightof 714 m,n was suppliedl as dlescribed elsewhere (22).

ATP wras mleasured as (les nribed 1v Nv\vron (11).To 1.5 ml of the reaction mixture, 0.15 mil of 30 %perchloric acidl was added: the resultinlg precipitatewas remove(d by centrifugatioin anid anl aliquot of thesupernatanit fluid was takenl for the ATP assay. Re-(luce(l TPN wvas measure(l1d absorption at 34() 111(26) onl a cenitrifilued aliqutiot (of thl reaction ml'ixtutre

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GROM ET-ELHANAN AND ARNON-DESASPIDIN AND PIIOTOPHOSPHORYLATION0

or directly, without centrifugation. in cuvettes wvith1-mm light path in a Cary spectrophotometer.

Desaspidin was a gift from Drs. B. C. Pressmani(through M. Avroln) and H. Baltscheffsky. Theconcentration of desaspidin used in our experimentswas kept uniformly at 5 X 10- M. Desaspidin stocksolutions were changed once weekly.

Results

Effect of Desaspidin anid CMVU on NoncyclicPhiotophosphorylationi. The photoproduction of 02by isolated chloroplasts is strongly inhibited by suchinhibitors as o-pheinanthrolilne (25) and p-chloro-phenyl-1,1-dimethyl urea (CMIU) (27) which. how-ever, do not inhibit cyclic photophosphorylation (10).These inhibitors, and particularly the substituted ureacompounds that are strongly inhibitory at very lowconcentrations, are, therefore, useful in distinguishingbetween cyclic photophosphorylation where 02 is notproduced and noncyclic photophosphorvlation whichis coupled to the production of 02.

In the present investigation, inhibition by desas-pidin was compared in every instance with inhibitionbv CAM. Table I shows the effect of desaspidin

Table 1. Effect of Desaspidini (iint p-C hiorophenyl-l,j-Din-ieth!l Urea (CMU) on. Noncyclic

PhotophosphorylationThe reaction mixture contained, in a final volume of

3.0 ml, chloroplast fragments conitaining 0.5 mg of chloro-phyll; and the followinig in umoles: Tris, pH 8.0, 80;MgCl, 5; ADP, 10; Ki,HP3204, 10; TPN, 8; and 0.1mg of spinach ferredoxin. The final concentration ofdesaspidin was 5 X 10- M and of CMU, 2 X 10-5 M.The reaction was run for 15 minutes under argon at anillumination of 30,000 lux.

,amoles ATP ,moles TPNH,Treatment formed formed

Control 5.4 7.8Desaspidin 5.2 8.2CMU 0.1 0.6

and CMU on a noncyclic photophosphorylation inwhich water vas the electron donor and TPN theultimate electron acceptor. Desaspidin, at the lowconcentration used (5 X 10-' m), did not inhibitthis type of photophosphorylation, which was, as ex-pected, strongly inhibited by CMU. In 10 differentexperiments, the ATP and TPNH, formed in thepresence of desaspidin ranged, respectively, between83 to 97 and 91 to 108 % of the controls.

Effect of Desaspidin antd CMU otn Cyclic Photo-phosphorvlationt. The effect of these 2 inhibitors wascompletely reversed (table II) in the case of cyclicj,hotopjhosp)lhorylatio1i catalyzed b)y iieliadioine (6, 7)1)henazine nlethosulfate (17) or reduced dichloro-

Table II. Effcct of Desaspidin and p-Chlorophlen-1-1,1-Di,ncthyl tUrea (CMlU) on Cyclic

Photo phosphorvlatioiiExperimental conditions as in table I, except that

TPN and ferredoxin were omitted. Where indicated,the following (in umoles) were added: PMS, 0.1;menadione, 0.3; dichlorophenol indophenol, 1.5. Di-chlorophenol indophenol was added joinltly with 1.5 Amoleascorbate (Z. Gromet-Elhaniaii, in preparation).

,tmoles ATP formedCatalystadded

MenadionePhenazinemethosulfate

Dichlorophenolindophenol

Plus PlusControl desaspidini CMU

8.27 1.63 5.46

2.10

0.09

7.26

7.40

7.34

6.47

phenol indophenol (14. 18, 23). Desaspidin stronglyinhibited this type of photophosphorylation whereasCMU had no significant effect. The cyclic photo-phosphorylation that was catalyzed bv reduced di-chlorophenol indophenol proved to be particularlysensitive to inhibition 1v desaspidin:; the inhibitionhere was always in excess of 95 %.

Ifffect of I)esaspidin mnd CMVUl oni PseudocyclicPhotoph/osphorylatiomi. The previous exl)erimenits'vere carrie(l ouit under argoni. Uln(ler air. as alreadvmentioue(l, certain catalysts of cvclic photophos-phorylation will catalyze a pseudocyclic type of photo-phosphorylatioin which is a varianit of nlonlcyclic photo-phosphorylationi anid depends oni water as the electrondolnor (10). Thus, on the basis of the results giveniin tables I anid II, pseudocyclic photophosphorylationwould be expected to be insensitive to inhibition bydesaspidin and to be strongly inhibited by CMIJ.Table III shows that these results were obtained in2 cases of pseudocyclic photophosphorylation, onecatalyzed by menadione and the other by FMN.

Different results. however, were obtained whenphotophosphorvlation un(ler air was catalyze(d byphenazine metlhosulfate and reduced dichlorophenolindophenol (table IV). In the case of phenazinemethosulfate, ATP formiiation was only mildly in-hibited by CMU. Taken by itself, this result would,uggest that. as in previous inivestigatiolns ( 10), phena-

Table III. Effect of Desas!idin anid p-Chlorophenyl-1,1-DinzethlW Urea (CMU) oni Psenidocyclic

PhotophzosphorylationExperimental conditions as in table I, except that

TPN and ferredoxin were replaced either by 0.3 gmolemenadione or 0.3 umole FMN and the gas phase was air.

-,.moles ATP formedCatalyst Plus Plusadded Control desaspidin CMU

MenadioneFMN

7.627.64

6.907.84

0.090.28

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zine methosulfate catalyzes a cyclic type of photo-phosphorylation, even tinder air. If the photophlos-phorylation catalyzed by phenazine imiethostilfate tin-(ler air is of the same cyclic type as it was tinider ar-gron (table 11), then it should be stronglv inhibitedlby desaspidiin. But table IV shows that, uinder air.(lesaspidin, whether aloone or in combination with1CM U, gave only a mild inhibitioni of the photophos-phorylatioin catalyzed b l)henazine methosulfate.The resistance of phenaziine mletlhosulfate-catalyzedphotophosphorylation under air to inhibitioni by des-aspidin raises the question whether, contrarv to previ-otis interpretations (10(). this photophosphorvlation isin(leed of the samie cyclic type tind(ler air as it is unlderargoni.

Table INT shows that, unlike pheniazine nietho-sulfate, the photophosphorvlatioin catalyzed by di-chlorophenol indophenol displaye(l the same sensi-tivity to inhibition by desaspidin anld the same resist--anice to inhibition by CMU tinder air as tlnder argon(compare table 1I ancd table 11). It appears, there-fore, that uinder these experimental conditions thephotophosphorylation catalyze(d b)y dichlorophenolindopheniol was always of the cyclic type.

In the experiments represelite(l 1y table IV. a

relatively high concentrationi of ascorbate (20umoles/3 mil) was added to (lichloroplhenol indlophenol

Table IV. Effect of I)csaspidin antd p-Chlorophenyl-I11-Diethyl Urea 'CJIML) on Photophosphorylation

Catalyzed by Phen--inc Mctih osulfate (PIS)and Redutced I)ichloropheol.Iol(lophen.ol

(OI'IP)Exp)erimental coniditionis as in table Il, except that 20

yimoles ascorbate was added ini the DPIP series anld thegas phase was air.

Treatnwi iit

ControlI'lus C

desaspidillCMIU an(d desaspidin

,/moles ATP formedIPIMS DPIP

7.77 7.495.98 7.295.07 0.265.44 0.32

to tiuard aoainst its reoxidation b)y air and the ensti-

ing com)plication that a low conicentrationi of oxidized(lichloroplheniol indophenol may give rise to a nonicyclicphotophosphorylation ( 14). The ad(litioni of excess

ascorbate to menadione, FMN or phenazine metho-stilfate under air caused no chanige in the response

of the respective phosphorylations to desaspidin andCMIU.

IEffect of Desaspidinl (11(i CMIU oni NontcyclicPhotophosphorylation with D)ichlorophenol IJdo-phllenol an d Mni 0,. Gromet-Elhananl and Avron (14,15) described a nionicyclic photophosplhorylation xvithwater as the electroni donior anid oxidized dichlloro-phenol indophenol as the electroni acceptor. Oxidizeddichlorophenol indophenol is added in catalytic

Table V. Effect of Dcsaspidi-n and p-Chlorophenyl-1,1-I)i1mcthvl 'rea (CMU') on Noncyclic

Photophosph orylation with OxidizecdI)ichlorophenol Ihdophciol

(I)PIP)Experimental conditions as in table I, except that

TPN and ferredoxin were omitted aind 0.1 gnmole DPIPanId( 4 nig MInO.I [prepared according to Hoclhster and(lQuastel (16)] were added.

Treatmeiit

Controll)esaspidinCMU

JLmoles ATP forme(d

4.804.600.01

amiotiints an(l is mainitained in ali oxidize(d state by anlexcess of MnO_. In the light of the great sensitivityto inhibition by desaspidin exhibited by the cyclicphotop)hosphorylationl catalyzed by redticed dlichloro-phenol indophenol, it became of initerest to comparethe effect of desaspidIini and CllMU oni the nionicyclicphotol)hosl)horylation linked to oxidize(d dlichloro-pheniol indophenol anid MnO_. Table V' shosX tlatthis variant of noncvclic photophosphorylation (wvhichis also accoml)aniedl by photoprodtictioni of ).,) re-sponded to inhibitioin bv desaspidin in the saniie muani-ner as the conventionial inonlcyclic photophosphory-lationi (table I). Tlhe D;PIP catalyzed nolncvclicphotophosphorylation was stronigly inhliibitedl 1y C?\l1Ubut was niot inihibite(d 1bw desaspidin.

Effect of Des(aspidini on1 N'Voncyclic IP10otoplios-phorylation Twith Dichloropheniol Inidophenol-Ascor-b(ate (1s the IElectr-ont Donor Systemi. bosada. et al.(19) observed a photophosphorylation ill a svS teml illwhich TP.N was the tultimilate clectroii accep)tor atindin whlich the electroii dolior was nlot water but re-duced dichlorophenol indophienol, miaintained in theredticed foriii liv ani excess of ascorbate. LosaadL etal. (19') characterized tlle plhosphorylatioii linkedwith this svstem as being of the iionlcclic bacterialtvl) b)ecatiuse it was cotipled to an open electroni flo\xfr-oili an electiron (lonlor othier tilaii water to lpyriliineniucleoti(le as the electroni acceptor anid restiltedl inthe constliipl)tion of 1)oth.

Althotigh there was n1o qtiestioi abotit the noni-cyclic clharacter of the electronl flow fromi redtice(ddichlorophenol ind(lopheniol to TPN in the scheiime ofLosada et al. ( 19), the site of the accompanyllingphosphorylation became open to (loulbt after Trel)staand Eck (23) fotinld anld other investigators coni-firmed that redtuced (lichlorophenol indophelnol (cancatalyze cyclic photophosphorylation (12. 14, 18, 28).Since sensitivity to verv low conicenltrationis of (lesas-l)i(lin appleared to give a clear-ctit dlistinictioni be-tweein cyclic aiid noncyclic photophosphorylatiol.it seemed (lesirable to examine the effect of (lesas-pidin oni the-type of )hotophosphorylationi descri bed1y- Losatda'-t al. ( 19). Figitres 1 ald 2 illtstratcethe effect of desaspidin on AT''1' formation andTP'N redtictioii. '1'PN reductioni was tiiiml)aired

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GROMET-ELHANAN AND ARNON-DEXS.\ASI'lDIN AND PIIOTOPHOSI'HORYLATION

8

I4-

3-

+ Desaspidin

Ixio 5xI05 IxI 5xiAM/or Concentrotlon of Dich/orophenol Indophenol (DPIP)

FIG. 1. Effect of desaspidin on photophosphorylationassociated with noncyclic electron flow from reduceddichlorophenol indophenol to TPN. Experimental con-

ditions as in table I, except that CMU (2 X 10-- m)was always present and 20 ,umoles ascorbate were addedtogether with dichlorophenol indophenol. Open circles,control; full circles, desaspidin.

and even stimulated whereas ATP formation was al-most completely inhibited by desaspidin. The effectof desaspidin was the same over a wide range of con-

centration of dichlorophenol indophenol. It becameclear, therefore, that the photophosphorylation whichaccompanies the noncyclic electron flow from re-

duced dichlorophenol indophenol to TPN and whichis sensitive to inhibition by desaspidini is distinct fromthe desaspidin-insenisitive photophosphorylation which

81

b7

65

4

'A3

2

I I

+ Desaspidin

Control

EFFECT OF DESASPIDIN ON TPNREDUCTION BY ASCORBATE-DPIP

Itl11 'I i I Itti I I I-4

IxIO 5x10 1IxIO 5x I0

Mo/or Concentration of Dich/orophenol Indophenol (DPIP)

FIG. 2. Effect of desaspidin on TPN photoreductionby reduced dichlorophenol indophenol. Experimentalconditions as in figure 1. Open circles, control; fullcircles, desaspidin.

accompanies the noncyclic electron flow from waterto TPN (table I).

The experiments represented by figures 1 and 2were carried otit un(ler argon. Table VI shows thatsimilar results were obtained under air, when CM\fUwas present to block the flow of electrons fromwater. Under these conditions, desaspidin did notinhibit the reduction of TPN by reduced dichloro-phenol indophelnol: in fact, it slightly stimullated it.

Table VI. Effect of Desaspidin oni PhotophosphorylationtCatalved by Dichlorophcnnol Intdophentol lTiUder

Cylic anid Noncyclic ConditiontsIn the noncyclic system the experimental conditions

were as in table II, except that CMU (2 X 10-5 M) was

always present and the following were added in umoles:TPN, 4; ascorbate, 20; DPIP, 0.2; and 0.1 mg of fer-redoxin. In the cyclic system TPN and ferredoxin were

omitted. The gas phase was air.

,umoles ATP ,umoles TPN

Conditions formed reducedCon- + Desa- Con- + Desa-trol spidin trol spidin

Noncyclic 3.27 0.07 3.09 3.66Cyclic 3.18 0.06 ... ...

However, desaspidin strongly inhibited the photophos-phorylation coupled with this noncyclic electron flow.The inhibitory effect of desaspidin was the same hereas on the cyclic photophosphorylation catalyzed byreduced dichlorophenol indophenol (table II).

The validitv of these observations was confirmedby experiments carried out, tnder argon, in mono-

chromatic light at 714 m,*. This illumination doesnot support a flow of electrons from water to TPNbut it provi(les energy for cyclic photophosphorylationan(l for noncyclic photophosphorylation with reduced

Table VII. Effort of Desaspidin on PhzotophosphorylationtCataly_ed by Dichlorophenol Iudophenol at 714 niz,

utnder Cyclic and Noncyclic ConditionsThe reaction mixture contained, in a final volume of

2.0 ml, chloroplast fragments containing 0.5 mg of chloro-phyll, and the following in /imoles: Tris, pH 8.0, 80:MgCl.,, 5; ADP, 10; K,HP3204, 10; TPN, 4; DPIP,0.2; ascorbate, 20; and 0.1 mg of ferredoxin. In thecyclic system TPN, ferredoxin and ascorbate (Z. Gromet-Elhan-n, in preparation) were omitted. Desaspidin (.5X 10-' M) vas added as indicated. The reaction was

run at room temperature for 10 minutes in monochromaticlight at 714 m,u (8.5 pueinsteins per min). The gas phasewas argon.

,umoles ATP ,umoles TPNConditionis formed reduced

Con- + Desa- Con- -± Desa-trol spidin trol spidin

Noncyclic 2.24 0.03 2.22 2.63Cyclic 2.16 0.02

EFFECT OF DESASPIDIN ON DPIP-CATALYZED"NONCYCLIC" ATP FORMATION

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PLANT PHYSIOLO0V

dichloropheinol in1dopheniol as the electron donior(22). Thus, at 714 mnx there is no need to use CMIUto block the electroni flow froml water.

Table VII shows that at 714 mu desaspidini didlnot inhibit the reductionl of TPN bv reduce(d dichloro-phenol indopheniol but it strongly inhibited the asso-

ciated photophosphorylation to the same degree as itinhibite(d the cyclic photophosphorylation catalyzed bydichloropheniol indloplheniol.

Discussion

Our results confirm the findings of Baltscheffskyanid de Kiewiet that low concentrations of desaspidin(ca. 10- M) do not inhibit the classical noncyclicphotop)hosphorylation (8, 9), i.e. the phosphorylationwhiclh is associated with the light-induced, lnoncyclictransfer of electrons fromii water to TPN. Further-more, low concentratiolns of (lesaspidin (lo not inhibitsuiclh variants of noncvclic photophosphorylation as thepsetudocyclic type or the noncvclic photophosphory-lations which are associated xvith artificial electronacceptors, i.e. ferricyani(le or oxidized dichlorophenolindoplhenol writh MnO.,. Low conicenitratioins of (les-aspidin strongly inhibite(d all other photophosphory-lations, whether they were associated with a cvclic

electron flow or with a noncyclic electron flow fromreduce(d dichlorophenol indopheniol to TPN. Thus,sensitivity to inhibition by low conlcentrations of des-aspidin provides a new and seemingly unambiguouscriterioni by which the phosphorylatioin associated withthe plhotooxidation of water is distinguiished from allother types of photophosphorylation, whether theyare associated with a close(d or with anl open type ofelectroni flow.

We conclude, therefore, that, contrary to earlieriilterpretations ( 19, 21), the phosphorylation coupledwith the photooxidationl of water occurs at a sitewhich is not share(d either by cyclic photoplhosphory-lationI or by the phosphorylatioli whiclh is associatedwith the nonicyclic electron flow from reduced di-chlorophenol indophenol to TPN. The latter. on thebasis of its sensitivity to inhibition by desaspidin, ap-

pears to occur at a site comimIllonl to cyclic photophos-phorylation. The bearing of these results onI therelation between phosphorylationl sites atnd varioustyp)es of light-iin(ltuce(l electroln flo\\ are disculssedelsewhere (5,14:l).

'1'u1rning to pseudocyclic photoph)osphorylation, itsinsensitivity to inihibition by low coIncen1trations ofdesasp)i(lin suggests that its ATP formation occuirs atthe same site as in the classical noncyclic photophos-phorylation. This corroborates the interpretationthat pseudocyclic phosphorylation is a variant of non-

cyclic photophosphorylationl in which water is theelectroin donor but external O., replaces TPN as theultimnate electroin acceptor (10).

This interpretation of pseudocyclic photophos-phorylation was borne out by the data wvhen photo-phosphorylation proceeded, under air, in the presence

of menadione or FMN (table III). However, thesame results under air were not obtained in the pres-enice of phenaziine methosulfate or reduced (lichloro-phenol inidophenol. Phenazine methosulfate cata-lvze(d utnder air a phosphorylation that was resistantto desaspidin, CMU. or to both. These observationiscannot be explained satisfactorily at this time. Uni-like phenazine methosulfate, dichlorophenol indo-plhenol (in the presence of excess ascorbate) cata-lvzed, even under air, a phosphorylation wvhiclh oc-cuirred at a cyclic site that was setnsitive to inhibitionb1 desaspidin but not by CMAUI.

The differential inhibition by low concelntration'sof desaspidin lends support to the idea that illumiii-nated chloroplasts form ATP at one site that ispeculiar to noncyclic photophosphorylation and at,at least, one other site which is characteristic ofcyclic photophosphorylationi.

Summary

Sensitivity to inhibitioin by low concentrations (.5X 10- z1t) of a phlorobutyrophenonie derivative,knowni as desaspidin, serves as a uisefuIl criterion fordistinguishing the phosphorylation associated withthe photooxidation of water from all other types ofp)hotophosphorylation.

Desaspidin strongly inhibited cyclic photophos-phorylation catalyzed by menadlione, phenazinemethosulfate or redluced (lichlorophenol indophenol.

The same concenitrationi of desaspidin had nio ef-fect on the noncyclic phosphorylation that is cott-pled with an electron flow fronm water to TPN orfrom water to anI artificial electron acceptor systeml,i.e. oxidized dichlorophenlol plus manganese dioxi(le.

Desaspidin at 5 X 10- Mr was also withotut effecton another variant of noncyclic photophosphory-lation, known as pseudocyclic phosphorylation. inwlhich molectular oxygen replaces TIPN as the tultim-ate electron acceptor.

The phosphorylation which is associated with thenoncyclic electron flov from reduced dichlorophenolindophenol to TPN was also strongly inhibited by 5X 10-7 M desaspidin an(d appears, therefore, to occturat a site which is commiiiion to cyclic photophosphory-lation ani(I niot to the noncyclic photophosphorylationwhich is linlked with photooxidation of water.

Literature Cited

1. ARNON, D. I. 1949. Copper enzymes in isolatedchloroplasts. Polyphenoloxidase in Beta vuiqaris.Plant Physiol. 24: 1-15.

2. ARNON, D. I. 1959. Conversion of light intochemical energy in photosynthesis. Nature 184:10-21.

3. ARNON, D. I., M. B. ALLEN, AND F. R. WHATLEY.1954. Photosynthesis by isolated chloroplasts.Nature 174: 394-96.

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GRO'MET-ELHANAN AND ARNON--DESASPIDIN AND PHOTOPHOSPHORYLATION

4. ARNON, D. I., M. B. ALLEN, AND F. R. WHATLEY.1956. Photosynthesis by isolated chloroplasts.IV. General concept and comparison of 3 photo-chemical reactions. Biochim. Biophys. Acta 20:449-61.

5. ARNON, D. I., H. Y. TSUJIMOTO, AND B. D. Mc-SWN'AIN. 1965. Photosynthetic phosphorylation andelectron transport. Nature 207: 1367-75.

6. ARNON, D. I., F. R. WHATLEY, AND M. B. ALLEN.1954. Photosynthesis by isolated chloroplasts.II. Photosynthetic phosphorylation, the conver-

sioIn of light into phosphate bond energy. J. Am.Chem. Soc. 76: 6324-29.

7. ARNON, D. I., F. R. WHATLEY, AND M. B. ALLEN.1955. Vitamin K as a cofactor of photosyntheticphosphorylation. Biochim. Biophys. Acta 16:607-08.

8. ARNON, D. I., F. R. WHATLEY, AN.D M. B. ALLEN.1958. Assimilatory power in photosynthesis.Science 127: 1026-34.

9. ARNON, D. I., F. R. WHATLEY, AND M. B. ALLEN.1959. Photosynthesis by isolated chloroplasts.VIII. Photosynthetic phosphorylation and thegeneration of assimilatory power. Biochim. Bio-phys. Acta 32: 47-57.

10. ARNON, D. I., M. LOSADA, F. R. WHATLEY, H. Y.TSUJIMOTO, D. 0. HALL, AND A. A. HORTON.1961. Photosynthetic phosphorylation and mole-cular oxygen. Proc. Natl. Acad. Sci. U.S. 47:1314-34.

11. AVRON, M. 1960. Photophosphorylation by Swisschard chloroplasts. Biochim. Biophys. Acta 40:257-72.

12. AVRON, M. 1964. On the site of ATP formationin photophosphorylation. Biochem. Biophys. Res.Commun. 17: 430-32.

13. BALTSCHEFFSKY, H. AND D. Y. DE KIEWIET. 1964.Existence and localization of 2 phosphorylationsites in photophosphorylation of isolated spinachchloroplasts. Acta Chem. Scand. 18: 2406-07.

14. GROMET-ELHANAN, Z. AND M. ANTRON. 1963.Photophosphorylation coupled to the reduction ofindophenol dyes. Biochem. Biophys. Res. Com-mun. 10: 215-20.

15. GROMET-ELHANAN, Z. AND M. AVRON. 1964.The role of indophenol dyes in photoreactions ofchloroplasts. Biochemistry 3: 365-73.

16. HOCHSTER, R. M. AND J. H. QUASTEL. 1952.Manganese dioxide as a terminal hydrogen accep-

tor in the study of respiratory systems. Arch.Biochem. Biophys. 36: 13246.

17. JAGENDORF, A. T. AND M. AvRON. 1958. Cofactorsand rates of photosynthetic phosphorylation byspinach chloroplasts. J. Biol. Chem. 231: 277-90.

18. KEISTER, D. L. 1963. Indophenol dyes as catalystsand uncouplers of photophosphorylation. J. Biol.Chem. 238: PC2590-PC92.

19. LOSADA, M., F. R. WHATLEY, AND D. I. ARNON.1961. Separation of 2 light reactions in noncyclicphotophosphorylation of green plants. Nature190: 606-10.

20. RUNEBERG, L. 1963. The effect of desaspidiin anidrelated plhlorobutyrophenone derivatives from fernextract on oxidative phosphorylation and muscleadenosinetriphosphatases. Societas ScientiarumFennica, Helsinki, Finland. Commentationes Bio-logicae XXVI. 7 Diss. 69 pp.

21. TAGAWA, K., H. Y. TSUJIMOTO, AND D. 1. ARNON.1963. Analysis of photosynthetic reactions by theuse of monochromatic light. Nature 199: 1247-52.

22. TAGAWA, K., H. Y. TSUJIMOTO, AND D. I. ARNON.1963. Separation by monochromatic light ofphotosynthetic phosphorylation from O., evolu-tion. Proc. Natl. Acad. Sci. U.S. 50: 544 49.

23. TREBST, A. AND H. ECK. 1961. Ul1tersuchungenuber die Beteiligung des sauerstoffs in photosyn-thetischen Reaktionen mit Hilfe von Hemmstof-fen. Z. Naturforsch. 16b: 455-61.

24. VERNON, L. P. AND W. S. ZAUGG. 1960. Photo-reductions by fresh and aged chloroplasts: Re-quirement for ascorbate and 2,6-dichlorophenolin-dophenol with aged chloroplasts. J. Biol. Chem.235: 2728-33.

25. WARBURG, 0. 1949. Heniry Metal ProstheticGroups and Enzyme Action. Clarendon Press,Oxford, England. p 213.

26. WARBURG, 0. AND W. CHRISTIAN. 1939. Isolie-rung und kristallisation des Proteins des oxydie-renden Garungsferments. Biochem. Z. 303: 40-68.

27. WESSELS, J. S. C. 1956. The action of some deri-vatives of phenylurethan and of 3-phenyl-1,1-di-methylurea on the Hill reaction. Biochim. Bio-phys. Acta 19: 548-49.

28. WESSELS, J. S. C. 1964. ATP formation accom-

panying photoreduction of NADP+ by ascorbate-indophenol in chloroplast fragments. Biochim.Biophys. Acta 79: 640-42.

29. WHATLEY, F. R. AND D. I. ARNON. 1963. Photo-synthetic phosphorylation in plants. In: Methodsof Enzymology VI. S. P. Colowick and N. 0.

Kaplan, eds. Acadamic Press, New York. p 308--13.

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