Plant & Cell Physiol., 8, 269-281 (1967) - CiteSeerX

Plant & Cell Physiol., 8, 269-281 (1967)

PHOTOCHEMICAL ACTIVITIES OF SONICATED LAMELLAEOF SPINACH CHLOROPLASTS WITH SPECIAL

REFERENCE TO THE ACTION OF CRS(CYTOCHROME C REDUCING SUBSTANCE)

YOSHIHIKO FUJITA AND FUMIO MURANO

Institute of Applied Microbiology, University of Tokyo,Tokyo

(Received December 22, 1966)

The photooxidation of reduced DPIP with NADP and O2 as finalelectron acceptors was studied with sonicated lamellae of spinachchloroplasts, with special reference to the possible role of CRS, a newlydiscovered factor of photochemical system I. A correlation betweeninactivation of NADP-photoreduction and dissolution of CRS was observedon treatment of the lamellae with various organic solvents. The treat-ment also suppressed the O2-linked photooxidation of reduced DPIP,although the suppression was not so marked as in the former reaction.The suppressed photooxidation of reduced DPIP (with O2 and NADPas electron acceptors) was partially restored on addition of dyes ofnegative redox potential, such as methyl viologen. The experimentalresults indicate that a factor participates in the reactions of photo-chemical system I, probably functioning as an electron carrier couplingthe photooxidation of P700 with the reduction of ferredoxin or O2.The finding that the above mentioned activity of the factor is destroyedby treatments which are effective in solubilizing CRS from the chloro-plasts suggests the identity of the factor with CRS.

The lamellae of Anabaena cylindrica, from which water-soluble materialhad been exhaustively washed off, were still found to contain a redox substance,which was reduced by the action of the photochemical system I (longer wave-length photochemical system in photosynthesis) and reoxidized by ferricyto-chrome c {1,2). In the washed lamellae of other photosynthetic organisms,spinach, Chlamydomonas, Porphyridium and Anacystis, we also found thesubstance with the same function and the same physicochemical propertiesso far examined (S). The substance, which we have tentatively named"cytochrome c reducing substance" (CRS), is rather tightly bound to the

Abbreviations: ATP, adenosine triphosphate; CRS, cytochrome c reducing sub-stnace; DEAE-, diethylaminoethyl; DPIP, 2, 6-dichlorophenol indophenol; DQ, diquat;FMN, flavin mononucleotide; EDTA, ethylenediaminetetraacetic acid; MV, methylviologen; NADP, nicotinamide adenine dinucleotide phosphate.

1 Present address: Nomura Research Institute of Technology and Economics,Kamakura, Kanagawa.

269

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270 Y. FUJITA AND F. MURANO Vol. 8 (1967)

lamellar structure, and it can be solubilized only after treatment with dilutesolution of polar solvents such as acetone (2). The content of CRS in thelamellae is as high as 1/10 of chlorophyll a on a molar basis, if it is two-electroncarrying substance (2). We inferred that it may form an electron pool inthe reaction of photochemical system I.

However, no direct evidence has thus far been obtained for the participationof CRS in the oxidation and reduction in the photosynthetic electron flow invivo. As an approach to this problem, we examined the possible correlationbetween solubilization (or removal) of CRS from the chloroplasts and the lossof activities of photochemical system I; NADP-photoreduction with theascorbate-DPIP couple and O2-linked photooxidation of reduced DPIP in sonicatedspinach chloroplasts. The results reported here indicate that the reductionof ferredoxin is linked to the photooxidation of P 700, through intermediationof an electron carrying factor, probably CRS.

MATERIALS AND METHODS

Preparation of sonicated lamellae

Spinach whole chloroplasts were prepared with sucrose- Tris medium (0.4 Msucrose, 0.001 M NaCl, and 0.001 M sodium ascorbate in 0.1 M Tris buffer, pH7.6) and washed once with the same medium. Washed chloroplasts weresonicated in Tris-NaCl medium (0.35 M NaCl and 0.001 M EDTA in 0.1 M Trisbuffer, pH 7.6) for 10 to 15min, and the broken lamellae were collected bycentrifugation at 27,000Xg for 2hr and washed once with the same medium.

Reagents

Ferredoxin was prepared from spinach leaves according to the method ofTAGAWA and ARNON (cf. -4). Ferredoxin-NADP reductase was prepared bythe method of SHIN and ARNON (cf. -4). The NADP-reductase preparationwas shown to be free from plastocyanin by the spectroscopic examination.Plastocyanin was isolated from the supernatants of chloroplast-sonicate ac-cording to the method of KATOH et al. (5) with a slight modification. Plasto-cyanin was eluted from the DEAE-cellulose column with 0 . 1 5 M to 0.2 M Trisbuffer (pH 7.6). NADP, FMN, DPIP and MV were obtained from TokyoKasei Co. DQ (California Chemicals) was kindly supplied from Prof. J. MYERS,

The University of Texas.

Assay for activities of photochemical reactions

For NADP-photoreduction, [basal reaction mixture contained sonicatedlamellae of 30 f*g chlorophylls, l^mole NADP, 20A<moles sodium ascorbate,2.0^moles DPIP, 0.02 ftmo\e ferredoxin, 40/^moIes Tris buffer (pH. 7.6), andsufficient amounts of ferredoxin-NADP reductase in 1 ml. The reaction wasrun in flasks having glass and rubber stoppers, after replacement of the airin gas-atmosphere with purified N2. The light-intensity was 20,000 lux(incandescent light), and the temperature, 25°. At time intervals (3min),




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CHLOROPLAST REACTION AND CRS 271

alquots were removed anaerobically by the aid of a syringe, and the reactionwas stopped by tipping the sample into an alkaline ammonium sulfate solution.After dilution with saturated ammonium sulfate solution, the reduced NADPwas measured photometrically with the colorless supernatants. The reactionwas linear with time at least for the first lOmin. Without illumination, nodetectable reduction of NADP was observed. The reaction rate was estimatedfrom the linear part of the time-course of each experiment.

For O2-linked photooxidation of reduced DPIP, basal reaction mixturecontained sonicated lamellae of 60 to 90 pg chlorophyll, 4.8/^moles DPIP, 48/^moles sodium ascorbate, 4.8^moles KCN, 96 ^moles Tris buffer (pH 7.6) and0.24 //mole FMN (or other dyes as stated) in 2.4 ml. KOH (20%, 0.1 ml) wasplaced in the center well of the flask. The reaction was followed manometricallyat 25° under illumination with 20,000 lux incandescent light. Although thereaction rates in the dark were far smaller than those in light, the valuespresented were corrected by subtracting those in the dark.

Assay of CRS-content in extracts

The amount of CRS solubilized from the lamellae on treatment with organicsolvents can be measured by two methods. The first is the calculation from thekinetic data of photochemical oxidation and reduction of horse-heart cytochromec by the sonicated lamellae; ferrocytochrome c is oxidized in the light andreduced to the original reduction state in the subsequent dark period underanaerobic conditions, depending on the presence of CRS in the reaction mixture.The amount of CRS in the reaction mixture can be estimated from the amountof cytochrome c oxidized in the light (or reduced in the dark) and the velocityconstants of the light oxidation and the dark reduction (see 2).

The second method is based on the reactivity of CRS to O2. CRS cansupport Cvdependent photophosphorylation in spinach broken chloroplasts (6).The reaction rates are proportional to the amount of added CRS at concen-trations less than 10"6 M. Because of limitations in available experimentalfacilities, we used the latter method in the present experiment. Relative rateof photophosphorylation was also used as an index for CRS-content of thesample. The reaction rate at saturating concentration of CRS, 10~6 M (90-100/tmoles ATP formed/mg chl/hr), was at the same level as that obtained inFMN-supported photophosphorylation. The reaction was measured by usingradioactive phosphate according to the method of AVRON (7).

RESULTS

Characterization of NADP-potoreduction and Orlinked photooxidation ofreduced DPIP by sonicated lamellae

Besides ferredoxin and NADP-reductase, the NADP-photoreduction requiredDPIP of high concentration (Table I). The addition of plastocyanin did notsignificantly stimulate the reaction, when DPIP was given at a high concen-tration (2 x 10"s M). The NADP-photoreduction with ascorbate-DPIP in sonicated




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272 Y. FUJITA AND F. MURANO Vol. 81(1967)

TABLE I

NADP-photoreduction by sonicated lamellae of spinach"

Reaction system*

Complete 132—ferredoxin and 0NADP-reductase—Ascorbate 0-DPIP 18Complete + 136plastocyanin

° Experimental procedures, see text.' Complete system contained, besides sonicated lamellae, 0.03/'mole fer-

redoxin, sufficient amount of NADP-reductase, 1.5 /(moles NADP, 30/<moles Naascorbate, and 3/*moles DPIP in 1.5 ml.

chloroplasts {8) and in detergent-treated chloroplasts (9) has been reported torequire plastocyanin as electron carrier. It was found that on lowering theconcentration of DPIP (10"4M or less) the plastocyanin requirement becamesignificant. On the other hand, the photooxidation of horse-heart cytochromec in the presence of the lamellar preparation (without addition of DPIP) strictlyrequired the addition of plastocyanin indicating the absence of plastocyaninin our lamellar preparation (3).

The O2-linked photooxidation was also dependent on the addition of DPIP(Expt. 1 in Table II). Plastocyanin was not required for this reaction (Expt.1 in Table II). This property was not altered by the sonication-time (Expt.2 in Table II.)

The photooxidation of horse-heart cytochrome c has been reported to bestrongly stimulated by added dyes of negative redox potential (2,10). Asimilar stimulating effect of the dye was also observed in the photooxidationof plastocyanin (Expt. 3 in Table II). However, the DPIP-photooxidation wasnot so strongly stimulated by such dyes. This indicates the difference inmechanism of stimulating in these reactions. We have proposed an explanationthat, in the cytochrome c photooxidation, reduced CRS is rapidly reoxidizedby cytochrome c, thus lowering the efficiency of the photooxidation (2). Theadded dye suppresses this back flow of electrons by stimulating the flow toO2. The same mechanism will work in the photooxidation of plastocyanin.In DPIP-photooxidation, such back flow of electrons is probably not significant,and the dye simply stimulates the over-all reaction by forming a rapid electronflow to O2.

For both reactions, DPIP was an essential factor. Therefore, we assumethat these reactions share a common path of electrons via DPIP, only differingin electron accepting system. Under the present experimental conditions,however, the reaction rate of NADP-photoreduction, even in the presence ofexcess amount of ferredoxin and NADP-reductase, was always smaller than




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TABLE II

02-linked photooxidation by sonicated lamellae of spinach"

Reaction system

Expt. 1Complete-DPIP-FMNComplete +plastocyanin

Expt. 2 (complete reaction medium)Broken chloroplasts2min sonicated5 min sonicated20 min sonicated

Expt. 3DPIP-ascorbate, with MV (5X10" 6 M)DPIP-ascorbate, without MVPlastocyanin-ascorbate,with MV (5X1O"6M)Plastocyanin-ascorbate,without MV

O2-uptake{ft equi/mg chl/hr)

3163

306328

399426399408

242238238

56

° Experimental procedures, see text. In experiment 1, complete systemcontained 4.8 pinoles DPIP, 48 /«moles Na ascorbate, 0.12 fimole PMN and sonicatedlamellae of 71 i«g chlorophylls in 2.4 ml. In experiment 2, chloroplasts werebroken by suspending in dilute buffer (Tris, 10~3M) before sonication. Brokenchloroplasts were suspended in Tris-NaCl medium. At time intervals, aliquotsof sonicates were removed, centrifuged and resuspended in fresh medium. MV(5X10~ 5 M) , instead of FMN, was added to the reaction mixture. In experiment3, basal reaction mixture for plastocyanin photooxidation contained 0.024 /^moleplastocyanin, 48 jumoles Na ascorbate, 48 j"moles Tris buffer (pH 7.6) and sonicatedlamellae of equal amount to that in DPIP-photooxidation (60 pg chlorophylls)in 2.4 ml.

that of (Vlinked photooxidation of reduced DPIP. However, a reproduciblevalue for the relative ratio of the two reaction rates was obtained for eachlamellar preparation (Table III).

Effects of treatment with various organic solvents on DPIP-photooxidationFirst, the effects of treatment with various organic solvents on the photo-

chemical activities of lamellae were examined. For treatment with polarsolvents, the sonicated lamellae were added to cold solvent (final concentrationof solvent, 60%) and held for 5 min at —20°. After removal of the solventby centrifugation followed by evacuation, the treated lamellae were incubatedanaerobically in Tris-NaCl medium for 20 hr in the cold. As a control, non-




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274 Y. FUJITA AND F . MURANO Vol. 8 (1967)

TABLE III

Correlation in relative rates of NADP-photoreduction and On-linkedphotooxidation of DPIP on limiting lamellar amounts'1

Lamellae added{fig chl/ml)

45231545 (60%acetone-treated)

NADP-reduction

(/* equi/ml/hr)

3.722.481.580.19

Relativerate

1.501.000.640.08

O2-uptake•p

(fi equi/ml/hr)

15.410.47.18.7

ielative

1.481.000.680.84

" Experimental procedures, see text. Concentration of acetone-treatedlamellae was expressed as chlorophyll concentration of the original lamellae.In O2-linked photooxidation of reduced DPIP, MV (5X10~5M) was added aselectron carrier to O2.

TABLE IVEffects of treatment with polar solvents on photochemical activities of

sonicated lamellae and on CRS-solubilizationa

Treatment of Chlorophylls NADP-reduction Oz-uptake CRSlamellae extracted (%) (̂ equi/mg chl/hr) extracted6

Non-treatedAcetone-treatedMethanol-treatedEthanol-treated

—10.1

1.3

3.6

1100

0

5

462352

388

412

1.02.3

1.6

1.7

" Experimental procedures, see text. In Ch-linked photooxidation, FMN(5X10~5M) was added as electron carrier to O2. Activities were expressed onbasis of chlorophyll in the lamellae before treatment.

6 Expressed by the relative rates of photophosphorylation stimulated bythe extracts of treated lamellae. The supernatants of the cold-extracts ofnon-treated lamellae also stimulated the photophosphorylation; the stimulationseemed to be mainly attributed to active substances other than CRS, whichhad been contaminated in washed lamellae. The stimulation was ratherunreproducible.

treated lamellae were also incubated in the same way. The incubated lamellaewere centrifuged and resuspended in the fresh medium. The supernatantswere used for the assay of CRS. For treatment with non-polar solvents, thelamellae were first lyophilized. The lyophilized lamellae were suspended inthe solvent and incubated for lOmin at 0°. The treated lamellae wereincubated in the buffer and centrifuged as described above, and the supernatant




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TABLE V

Effect of treatment with non-polar solvents on photochemical activitiesof lamellae and CRS-solubilization"

Treatment of Chlorophylls NADP-reduction C-2-uptake CRSlamellae extracted (%) (p equi/mg chl/hr) extracted

Lyopmlized,non-treatedPetroleum ether-treated•re-Hexane-treated•re-Heptane-treatedPetroleum ether-,acetone-treated

0.1

0.10.15.2

121

134

121128

0

370

354

302366270

1.0

1.1

0.70.82.3

° Experimental procedures, see text and Table IV. In Ch-linked photooxida-tion, FMN (5X10~5M) was added as electron carrier to O2.

\.O

<\ B

/c

£ •

/ D

-

-i—V,O 50 100

ACETONE CONCENTRATION (%)

Fig. 1. Effects of treatment at various acetone-concentrations on activitiesof sonicated lamellae. Relative activities are plotted against concentrationsof acetone in the treatment. Curve A, chlorophyll content of treatedlamellae; Curve B, activity for NADP-photoreduction; Curve C, O2-linkedphotooxidation of DPIP in the presence of FMN (5X10" 5 M) ; Curve D, indexfor CRS solubilized (photophosphorylation increase caused by the addition ofsolubilized CRS to broken chloroplasts, see METHOD). Experimental pro-cedures, see Table IV and text.

was used for CRS-determination.The results are summarized in Tables IV and V. Although the chlorophyll

in the lamellae was not significantly extracted with the polar solvents, thereoccurred a marked suppression of NADP-photoreduction caused by the treatment




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(Table IV). However, the O2-linked photooxidation of reduced DPIP in thepresence of FMN was not strongly affected. The treatment also causedsolubilization of CRS from the lamellae as detected by subsequent extractionwith the buffer. The treatment with non-polar solvents, on the other hand,did not cause any significant inactivation of the two photochemical reactionsunder investigation or solubilization of CRS (Table V). When the lamellaetreated with petroleum ether were further treated with acetone (the lowestline in Table V), the results obtained were the same as in the simple acetone-extraction, namely, there was a selective inactivation of NADP-photoreductionaccompanied by marked solubilization of CRS.

The effects of treatment at various concentrations of acetone are shownin Fig. 1. Significant extraction of chlorophyll occurred only on treatmentwith acetone at concentrations higher than 40% (Curve A), whereas theinactivation of NADP-photoreduction and the solubilization of CRS weremarked even on treatment with 20% acetone (Curves B and D). The Cvlinkedphotooxidation of reduced DPIP in the presence of FMN was not so stronglyaffected at all the acetone concentrations examined (Curve C).

As the treated lamellae retain the activity for DPIP-photooxidation, thetreatment with polar solvent must have acted in blocking between photochemicalsystem I and the NADP-reducing system, or in stimulating the NADPH2-photooxidation linked to the contaminating O2 or some electron acceptingsubstances contained in the lamellae. The latter possibility, however, can beexcluded by the fact that the rate of NADP-photoreduction by non-treatedlamellae was not affected by the addition of treated lamellae even in an excessamount. The most plausible explanation for the observed correlation betweeninactivation of NADP-photoreduction and solubilization of CRS on treatmentwith organic solvents is that CRS is an essential factor for the photoreductionof NADP (or ferredoxin) by the photochemical system. I. However, the back-addition of the extracts or the addition of purified CRS preparation did notrestore the activities of the lamellae treated with organic solvents (Table VI).Further, the inactivation of NADP-photoreduction is an immediate results ofthe acetone-treatment, the inhibition being observed without subsequentextraction of CRS from the treated lamellae. The lost activity of NADP-photoreduction was partially restored by the addition of MV in the reactionmixture (Expt. 2 in Table VI). However, the addition of MV completelyinhibited the NADP-photoreduction in non-treated lamellae in the gas-atmosphereof purified N2. The inhibition was still significant even when the trace of O2in the reaction mixture was removed so far as possible by the use of H2-paliadium black couple or by preceding DPIP-photooxidation (Expt. 2 in TableVI). If we consider this circumstance and assume that the same event isoccurring in the treated lamellae, then the real grade of acceleration by MVmust have been larger than the observed value shown in the table.

Acetone-treatment and Oz-linked photooxidation of reduced DPIP

As shown in Tables III and IV and Fig. 1, the (Vlinked photooxidation of




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TABLE VI

Effects of addition of CRS and MV on NADP-photoreductionby acetone-treated lamellae'

Reaction system NADP-reduction{p equi/mg chl/hr)

Expt. 1Non-treated lamellaeAcetone-treated lamellaeAcetone-treated lamellae+CRSAcetone-treated lamellae+extract

Expt. 2Non-treated lamellaeAcetone-treated lamellaeNon-treated lamellae+MV (10-5M)Acetone-treated lamellae+MV (10-6M)

705

34

22

° In experiment 1, CRS purified by a Sephadex column or extracts of coldincubation of the treated lamellae were added in amounts saturating the CRS-photophosphorylation (10~6 M ca). In experiment 2, H2-palladium black couplewas used for anaerobiosis. The reaction mixture in H2 were preincubated inthe dark for 15 min so as to remove contaminating O2. The lamellae used weretreated with 60% acetone.

VA

LI

£0.5—

RE

LA

/

p

M

- 200

- IOOJ;

50 100 0ACETONE CONCENTRATION (%)

100

Fig. 2. Effects of treatment with various concentrations of acetone on C-2-linkedphotooxidation of DPIP. Curve A, chlorophyll content of treated lamellae; CurveB, activity for the reaction without dye; Curve C, activity for the reaction withdyes (10-<M MV in chart a, 10~4M DQ in chart b); Curve D, stimulation by addeddyes (%). Experimental procedures, see-Table IV, Fig. 1 and text.




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278 Y. FBJITA AND P. MURANO Vol. 8 (1967)

TABLE VII

Stimulating effects of MV and DQ on 02-linked photooxidationof DPIP by the acetone-treated lamellae"

Additions

Expt. 1NoneMV, 5X1O~7M

3 X 1 0 - 6 M

1X1O"5M2xlO~5M8xlO-4M

Expt. 2NoneDQ, 3X1O-6M

1X10-5M3X1O-"M8X1O-4M

02-uptake(ft equi/mg chl/hr)

112

123

146

213

291

303

107

127268

321

319

Relativerate

1.0

1.1

1.3

1.92.62.7

1.01.22.53.03.0

" Lamellae treated with 60% acetone were used. Experimental procedures,see text.

TABLE VIII

Effect of addition of CRS on the 02-linked photooxidationof DPIP by acetone-treated lamellae*

Addition* 02-uptake StimulationAdditions {ft e q u i / m g c h l / h r ) [o/o)

None 90 —MV, 5X1O-6M 213 136CRS, 0.2 114 27CRS, 1.0 129 47

° Experimental procedures, see text. The lamellae treated with 40% acetonewere used. On treatment, the lamellae lost 90% of the activity for NADP-photoreduetion. CRS preparation purified by a Sephadex column was used.CRS-concentration, which saturated the photophosphorylation (10~6M ca), wasexpressed as 1.0 in the table.

reduced DPIP was not affected by the treatment with polar solvents whenthe reaction was measured in the presence of added dye of negative redoxpotential. However, when the reaction was run without the dye, the photo-oxidation was also significantly suppressed by the solvent-treatment (Fig. 2aand b, Curves B). The addition of sufficient amounts of the dye (5 X10"5 M)restored the suppressed activity at least partially (Curves C). Stimulation by




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TABLE IX

Effects of added dyes and ferredoxin on Oz-linked photooxidationof DPIP in acetone-treated lamellae

NoneFerredoxin, 4xlO-5MDQ, 4xlO-5MMV, 4xlO~5M

O2-uptake(f equi/mg chl/hr)

9883

274269

Relativerate

1.00.92.82.8

" Experimental procedures, see text. The lamellae treated with 60% acetonewere used. Ferredoxin concentration was estimated from the absorption dif-ference (oxidized minus reduced) at 463 mft (11).

the added dye was more marked in the reaction which had been more stronglysuppressed by the treatment (Curves B vs D).

The reaction in the non-treated lamellae was stimulated insignificantly, ifever, by the addition of the dye. The reaction was stimulated much moremarkedly in the lamellae prepared by longer sonication.

Stimulating effects at various dye-concentrations are shown in Table VII.The stimulation attained a maximum at a dye-concentration of about 5xlO~5Meither with MV or DQ.

Addition of purified CRS preparation also significantly stimulated the reactionin the acetone-treated lamellae (Table VIII), although the stimulation was notso marked as that with the dye. However, ferredoxin could not support thereaction (Table IX). Ferredoxin has been known to stimulate the electronflow from photosynthetic system to O2 (12). Thus ferredoxin could be expectedto stimulate the O2-linked photooxidation of reduced DPIP, if it would be reducedphotochemically.

DISCUSSION

Our findings described above can be explained at least in two ways. Thefirst is that an electron carrying factor functions between the photochemicalreaction (photooxidation of P700) and the reduction of ferredoxin or Os. Thetreatment of lamellae with dilute solution of polar solvents modifies the activityof the factor; it loses the activity for ferredoxin-reduction but still significantlyretains the activity for Cvreduction. Thus the treated lamellae lost the activityfor NADP-photoreduction (Tables III and IV), whereas they were still activein the O2-linked photooxidation of reduced DPIP in the absence of dyes (Fig.2a and b, Curves B). When the factor has been inactivated, its function maybe partially replaced with those of the dyes such as FMN and viologens adsorbedon the treated lamellae, because the added dyes restored slightly the NADP-photoreduction (Table VI), and significantly the O2-linked photooxidation ofreduced DPIP (Fig. 2a and b). The observed correlation between the loss of




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photochemical activities and the solubilization of CRS caused by the treatmentwith various polar solvents (Tables IV and V and Fig. 1) suggests that thefactor is identical with CRS in the lamellae. The isolated CRS, however, losesits activity for NADP-reduction during treatment with the organic solvents.The back-addition of the isolated CRS cannot restore the NADP-photoreductionin the treated lamellae (Table VI) whereas the Cvlinked photooxidation canbe significantly restored by added CRS (Table VII). The finding that actualremoval of CRS from the lamellae by subsequent water-extraction is notrequired for the suppression of the photochemical reactions (see p. 276) can beexplained in a similar way. The " inactivation" of the factor may be at-tributed not only to the denaturation of the factor itself but also to themodification of the lamellar structure. The structure of the lamellae may bemodified so that the added CRS cannot reach the proper site for its originalfunction.

The second probable explanation is that a rapid back flow of electronsagainst the photochemical reaction takes place after treatment of the lamellaewith polar solvents. The back flow of electrons, from the reduced product ofthe photochemical reaction to DPIP or to the oxidant (P700) suppresses thereaction rate measured either as NADP-reduction or as Cvuptake. It hasalready been reported that CRS can form such back flow when mammaliancytochrome c is used as the electron donor in the reaction of photochemicalsystem I in Anabaena cylindrica (2). We also obtained evidence indicatingthat similar back flow occurs in the spinach reaction with plastocyanin as theelectron donor (Table II). However, reduced CRS seems not to be oxidizedby DPIP in the non-treated lamellae (see p. 272). If CRS should form suchback flow with DPIP in the present case, the modified CRS remaining in thelamellae after the solvent treatment must have induced an additional reactivityof the DPIP-reduction. In Anabaena system, however, the reactivity of CRSto mammalian cytochrome c is not changed by the treatment for CRS-solubilization (unpublished data). It is unlikely that the reactivity of CRS ischanged by the same treatment only with respect to its action towardsDPIP.

Another possible back flow of electrons may occur between the reductantof photochemical reaction and the oxidant. Although we cannot preciselyevaluate this possibility at the present time, the simultaneous occurrence ofthe back flow and CRS-solubilization on the treatment indicates that CRS isclosely related to the photochemical system.

In either of explanation, the involvement of an electron carrying factorbetween P 700-photooxidation and ferredoxin-reduction has to be considered.Two modes of reaction have been proposed concerning the action of photo-chemical system I. The one involves the photooxidation of P 700 linked tothe reduction of a " primary reductant", which is assumed to be more tightlybound to the lamellar structure than f erredoxin and have a sufficiently negativeredox potential that it can reduce various negative-potential electron acceptorsincluding ferredoxin (13). The other is the assumption that ferredoxin itself




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is the primary reductant (14). The results of the present experiment are infavor of the former theory. If the first interpretation made on the presentresults is true, the factor in question or CRS in vivo may be identical withthe "primary reductant". The second interpretation, on the other hand,does not necessarily allow such assumption. The final decision on the actionof CRS in the reaction of photochemical system I awaits further investigation.

The authors wish to express their thanks to Prof. JACK MYERS of the Universityof Texas to his advices to this work.

REFERENCES

( 1 ) Y. FUJITA and J. MYERS. 1966. Cytochrome c redox reactions induced byphotochemical system 1 in sonicated preparations of Anabaena cylindrica.Arch. Biochem. Biophys., 113, 730-737.

( 2 ) Y. FUJITA and J. MYERS. 1967. Kinetic analysis of light-induced cytochromec redox reactions in Anabaena lamellar fragments, ibid., 119, 8-15.

( 3 ) Y. FUJITA and J. MYERS. 1966. Comparative studies of cytochrome c redoxreactions by photochemical lamellar preparations obtained from blue-green,red, and green algae, and spinach chloroplasts. ibid., 113, 738-741.

( -4 ) M. LOSADA and D. I. ARNON. 1964. Enzyme systems in photosynthesis. InModern Methods of Plant Analysis. 7. Edited by H. F. LlNSKENS, B. D.SANWAL and M. V. TRACEY. p. 569-615. Springer, Heidelberg.

( 5 ) S. KATOH, I. SHIRATORI and A. TAKAMIYA. 1962. Purification and some pro-perties of spinach plastocyanin. J. Biochem. (Tokyo), 51, 32-40.

{ 6 ) Y. FUJITA and J. MYERS. 1966. Some properties of the cytochrome c reducingsubstance, a factor for light-induced redox reaction of cytochrome c in photo-synthetic lamellae. Plant & Cell Physiol., 7, 599-606.

( 7 ) M. AVRON. 1960. Photophosphorylation by swiss-chard chloroplasts. Biochim.Biophys. Acta, 40, 257-272.

{ 8 ) S. KATOH and A. SAN PIETRO. 1966. Activities of chloroplasts fragments. IHill reaction and ascorbate-indophenol photoreductions. J. Biol. Chem., 241,3575-3581.

{ 9 ) J. S. C. WESSELS. 1965. Studies with small fragments prepared by digitonintreatment of spinach chloroplasts. In Currents in Photosynthesis. Proceedingsof the Second Western-Europe Conference on Photosynthesis. Edited by J. B.THOMAS and J. C. GOEDHEER. p. 129-136. Ad. Donker, Rotterdam.

(10) B. KOK, H. J. RURAINSKI and A. E. HARMON. 1964. Photooxidation of cyto-chromes c , /and plastocyanin by detergent treated chloroplasts. Plant Phosiol.,39, 513-520.

{11) F. R. WHATLEY, K. TAGAWA and D. I. ARNON. 1963. Separation of the lightand dark reactions in electron transfer during photosynthesis. Proc. Natl.Acad. Sci. U. S., 38, 680-685.

{12) C. A. FEWSON, C. C. BLACK and M. GIBBS. 1963. Further studies on thephotochemical production of reduced triphosphopyridine nucleotide and adenosinetriphosphate by fragmented spinach chloroplasts. Plant Physiol, 38, 680-685.

{13) B. KOK. 1965. Concentration and normal potential of primary photooxidantsand reductants in photosynthesis. In Current in Photosynthesis. Proceedingsof the Second Western-Europe Conference on Photosynthesis. Edited by J. B.THOMAS and J. C. GOEDHEER. p. 383-392. Ad. Donker, Rotterdam.D. I. ARNON. 1965. Ferredoxin and photosynthesis. Science, 149, 1460-1470.




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