The Necessity for -Carotene in the 518 Nanometer ...Plant Physiol. Vol. 48, 1971 LD E 6C 50 40 3C 2C IC Totol /3-Carotene:3-Carotene Extracted t 1'| B-Carotene Remaining 1 - 0 10 20

Plant Physiol. (1971) 48, 553-558

The Necessity for -Carotene in the 518 NanometerAbsorbance Change1

Received for publication March 9, 1971

GARY R. STRICHARTZ2Johnson Research Foundation, The University of Pennsylvania, Philadelphia, Pennsylvania 19104

ABSTRACT

The identity of the pigment responsible for the light-induced518 nanometer absorbance change was investigated by extrac-tion and reconstitution of spinach chloroplasts. Heptane ex-traction of carotene and quinones from lyophilized chloro.plasts removes absorbance changes at 518 and 475 nanometersactivated by both laser flash and continuous illumination.Some electron transport activity is always present, even in caro-tene- and quinone-depleted chloroplasts, but the light-inducedpH increase disappears following the first extraction step. Re-addition of pure 8-carotene partially restores the 518 and 475nanometer absorbance changes.

Duysens (10) first reported reversible absorbance changes at518 nm arising from the illumination of Chlorella cells by redlight. The absorbance increase at 518 nm (hereafter calledA,,,), accompanied by a corresponding absorbance decrease at475 nm, has since been observed in a variety of plants (4, 14)and algae (7, 12, 23). The pigment producing A,8 has not beenidentified, although either /3-carotene (6, 26) or chl a or b (13,18) has been proposed.The present investigation shows that the removal of /3-caro-

tene from chloroplasts completely inhibits the 518 nm absorb-ance change while only partially inhibiting electron transferand, by implication, chlorophyll photooxidation. Furthermore,reconstitution of the chloroplasts using pure /3-carotene re-stores an absorbance change which is spectrally identical withA518-

MATERIALS AND METHODS

Chloroplast Isolation. Chloroplasts were isolated from 60 gof deribbed spinach leaves (Spinacia oleracea). It was impera-tive that the tissue came from fresh leaves which were picked,washed, and stored in the cold as rapidly as possible. Marketspinach was unsuitable for these experiments. The leaves werehomogenized in a blender in 200 ml of 0.4 M sorbitol, 10 mmNaCl, 20 mm MOPS.3 pH 7.8. The homogenate was filtered

This research was supported by United States Public HealthService Training Grant GM2G27709. Submitted in partial fulfill-ment of requirements for the degree of Doctor of Philosophy to theGraduate School of Arts and Sciences of the University of Pennsyl-vania.

I Present address: Department of Physiology and Biophysics,University of Washington School of Medicine, Seattle, Wash. 98105.

3 Abbreviations: MOPS: morpholinopropane sulfonic acid; TMA-OH: tetramethyl ammonium hydroxide; BCP: bromcresol purple.

through four layers of cheesecloth and the filtrate was centri-fuged at 500g for 30 sec. The supernatant was then centrifugedat i5OOg for 10 min, and the resulting pellet was resuspendedin a minimal volume of isolating medium to give a final con-centration of 2 to 4 mg chl/ml.

Extraction of Chloroplasts. Isolated chloroplasts were ly-ophilized to dryness. Part of the original pellet was not lyophi-lized, but was stored in the dark at 0 C to be used as a controlsample. An unlyophilized control sample was necessary be-cause lyophilized chloroplasts invariably had lower activitiesthan samples which were lyophilized and then rapidly swirledin heptane before drying and resuspension (cf. 2, 3).

Lyophilized chloroplasts, containing about 10 mg of chl,were ground to a powder and shaken in 10 ml of heptane for2 min on a reciprocal shaker. The suspension was centrifuged,the extract was decanted, a chloroplast sample was withdrawn,and fresh heptane was added. After the final extract wasdecanted, residual heptane was evaporated under vacuum. Thedried, extracted chloroplasts were resuspended in a mediumidentical to the isolating medium but at pH 7.0. The entire ex-traction was conducted at 0 to 4 C.

Reconstitution. Reconstitution was by a method similar tothat of Emster et al. (11). Extracted chloroplasts were incu-bated with heptane solutions containing various reconstitutingcompounds. In a typical experiment chloroplasts containingapproximately 2 mg of chl were reconstituted by incubation in2.0 ml of a heptane solution containing 0.4 mg of /3-carotene.This was equivalent to a /3-carotene/chl ratio five times that ofthe unextracted material. During the incubation period of 30min, at 0 C, the chloroplasts were agitated frequently to mini-mize settling. They were centrifuged, dried, and resuspendedin the same manner as the extracted chloroplasts. Further in-cubation of resuspended, reconstituted chloroplasts for 10 minat 25 C frequently improved the restoration of A,,, and ofelectron transport activity.

Chemical Assays. Plastoquinone was assayed by the methodof Redfearn and Friend (25). Chloroplast /8-carotene was as-sayed by the same procedure by measuring the absorbance at450 nm of the ethanol solution of the extract and using amolar absorptivity of E = 1.35 X 105 M1 cm' (31). Chloro-phyll was measured by the method of Nishimura (21).

Optical Measurements. Kinetics of absorbance changes andlight - dark difference spectra were measured on a dual wave-length spectrophotometer. Absolute spectra and continuousdifference spectra were measured on a Perkin-Elmer model 356spectrophotometer. Hill reaction activity was measured by ab-sorbance changes due to ferricyanide reduction at 420 and 440nm in a dual wavelength spectrophotometer.

Rapid, light-induced pH changes were measured on a singlebeam instrument with BCP (5). Flash experiments on the dualwavelength instrument used the light from a Braun HobbyF270 xenon flash lamp (Braun AG Company, Frankfurt/M,

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Plant Physiol. Vol. 48, 1971

LD

E

6C

50

40

3C

2C

IC

Totol /3-Carotene

:3- Carotene Extracted

t

| B-Carotene Remaining1'1 -0 10 20 30

Extraction Time

FIG. 1. Removal of ,-carotene from lyophilized chloroplasts byheptane extraction. Total chlorophyll was 7.67 mg. Refer to "Re-sults" for details of the assay procedure.

West Germany). This flash was filtered through a Corning 2030glass filter to give a 200-/csec pulse delivering 0.76 millijoulesof red light. Laser flash experiments were conducted using theinstrument described by DeVault (9).

Actinic illumination was provided by a tungsten lamp withappropriate interference filters from Baird Atomic Corp.,Cambridge, Mass., or with a Corning glass filter, number 2030,which passed all wavelengths above 650 nm. All actinic con-tinuous illumination was filtered through 3 cm of water to min-imize heating.

Chemicals. Chemicals were of reagent grade. MOPS was ob-tained from Calbiochem, Los Angeles, Calif. TMA-OH waspurchased from Eastman Chemicals, Rochester, N.Y. Theheptane used for extraction was either Fisher SpectranalyzedCertified reagent or Eastman Organic Chemicals Highest Pu-rity. Potassium ferricyanide was obtained from the J. T. BakerChemical Co., Phillipsburgh, N.J., and BCP from A. H.Thomas, Philadelphia, Pa. Type IV crystalline /3-carotene waspurchased from the Sigma Chemical Co., St. Louis, Mo.

RESULTS

Extraction. Figure 1 shows the removal of /-carotene fromlyophilized chloroplasts by heptane extraction. The /3-carotenein the extracts was assayed spectrophotometrically, whereas the/3-carotene remaining was calculated by measuring /3-carotene/chl in the chloroplasts and multiplying this ratio by the amountof chlorophyll still remaining in the extraction vessel when theparticular sample was taken. The sum of the /3-carotene ex-tracted and /3-carotene remaining is plotted as a broken linein Figure 1.The estimation of chl/,B-carotene content in unlyophilized

chloroplasts was determined by pigment extraction accordingto the method of Redfearn and Friend (25), a procedure con-siderably more vigorous than the heptane extraction. The useof methanol in the former process removes protein-bound pig-ments, plastoquinone, and carotene, which are not entirely ex-tractable by the heptane. In subsequent figures appearing inthis paper, the values given for "pigments remaining" in hep-tane-extracted samples refer to these more tightly bound moie-ties.The difference spectrum of unextracted chloroplasts minus

extracted chloroplasts is shown in Figure 2. The two chloro-plast suspensions were made equal in chlorophyll concentrationby adding chloroplasts to give equal absorbances at 678 nm;this wavelength is at the maximum of the long wavelengthchlorophyll absorbance band, a band which is not affected bythe extraction process. The integrity of this band confirms theevidence, suggested by the spectrophotometric scanning of theheptane extracts, that no chlorophyll is removed during theextraction.

The chloroplast difference spectrum is compared to two ab-

solute spectra in Figure 2. The spectrum of one heptane ex-tract closely resembles the spectrum of pure /3-carotene inethanol. Small variations observed in these two spectra resultfrom differences in solvent polarity (unpublished observation).The difference spectrum shows a distinct red shift of the twopeaks at longer wavelengths relative to both the absolute spec-trum of /3-carotene and that of the extract. A spectrum similarto the difference spectrum may be produced by dissolving /3-carotene in rather polar organic solvents, such as dimethyl-sulfoxide.

Extraction reduces A508 drastically (Fig. 3A). Less than 10%of A,,, activity remains after only 2 min of extraction. The

.061 unextracted - extracted

*Q5-

04 /

03-4a I/ heptane \.02- extract

.01 3-carotene Xin EtOH .

0 - - - i400 450 500

X nm

FIG. 2. The difference spectrum of unextracted minus extractedchloroplasts ( ) (13 ,ug chl/ml); the absolute spectra of f-caro-tene in ethanol (- -); and of one heptane extract from the ex-traction of lyophilized chloroplasts (-). The latter have beenscaled to give the same absorbance as the difference spectrum atthe center peak.

100-

A

'a..t

0

'e

0 2030 DrivenO 640nm Driven,'A 720nm Driven

B

Hill Reactivity (0)Light-Induced pH Increase (a)

C

C020Extraction Time (Minutes)

30

FIG. 3. The effect of extraction on light-activated chloroplastfunctions. A: Absorbance changes at 518 nm; B: absorbancechanges at 475 rn; C: Hill reaction rates (o) and light-induced pHchanges (-). For A and B the reaction cuvette contains 0.4 Msorbitol; 10 mM NaCl; 20 mM MOPS, pH 7.2; and chloroplastfragments (50-100 ,ug chl/ml). For C, the Hill activity assay con-tains the above ingredients plus 21 AM KaFe (CN)6. The cuvette forthe assay of the pH change contained 50 mM KCI, 50 mm BCP, 25,ug/ml pyocyanine, and chloroplast fragments (43 ,ug chl/ml). Inpart C the 100% control activities correspond to 9 ,umoles of ferri-cyanide reduced per mg of chlorophyll per hr and 0.2 HI removedper chl per sec for the Hill activity and pH increase, respectively.

554 STRICHARTZ

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Plant Physiol. Vol. 48, 1971 3-CAROTENE AND THE 518 NM ABSORBANCE CHANGE

ae 1ffi Extent A

ls_____ iRate108'

\ 640nm-Driven Cyt f Reductiono j Rate

50

Extent

2b5r 4

-3

o

EL 0 10 20 30Extraction Time (Minutes)

FIG. 4. Effect of extraction on light-driven cytochrome f oxida-tion and reduction and on the plastoquinone content of chloroplasts.For A and B the reaction cuvette contains 0.4 M sorbitol; 0.1 MNaCl; 20 mm MOPS, pH 7.0; and chloroplasts (50 ,ug chl/ml). The"rates" compared are the inverses of calculated half-times for re-actions occurring during continuous illumination. Plastoquinone wasassayed as noted in "Materials and Methods."

extraction profile of A,8 and that of the absorbance change at475 nm (Fig. 3B) are the same, regardless of whether the ac-tinic illumination was by 640 nm, 740 nm, or broad band redlight (filter 2030).The extraction profile of light-induced electron transfer is

markedly different from that of A.8. Although the Hill reactionis reduced by almost 50% after the first extraction step, itmaintains this level of activity throughout the remaining ex-tractions (Fig. 3C). The behavior of the Hill reaction is paral-leled by that of cytochrome f reduction driven by 640 nmlight, as shown in Figure 4B.The changes in plastoquinone concentration during extrac-

tion are shown in Figure 4C. The initial plastoquinone contentof the chloroplasts is reduced by half during the extractionprocess, yet there appears to be an inhomogeneity of the plasto-quinone distribution since the remaining 50% is not removedby further extraction. Unexpectedly, the oxidation of cyto-chrome f by system I is also decreased by extraction (Fig. 4A).

Light-induced pH changes are strongly inhibited by the ex-traction process (Fig. 3C). The initial control value of 0.2 H+/chl * sec matches the reported values for proton uptake bychloroplasts (19).

Reconstitution. Figure 5 shows that the addition of ,8-caro-tene restores some of the 518 nm absorbance change. The ab-sorbance change is only 15 to 20% of the unlyophilized sam-ple, which is taken as the control. The absorbance decrease at475 nm is also restored to the same extent by addition of ,B-carotene to the extracted material.The light - dark difference spectrum from 450 to 540 nm is

shown in Figure 6 for control and reconstituted chloroplasts.The spectra are identical within experimental error.

Flash-induced absorbance changes at 518 nm in the controland reconstituted chloroplasts are shown in Figure 7. The half-time of the decay in the control (unlyophilized) sample is0.56 + 0.05 sec; for the reconstituted sample the half-decaytime is 0.12 + 0.04 sec. Extrapolation of semilog plots of thecurves in Figure 7 to the time of the flash shows that the re-constituted material has an initial change which is 61% of theinitial absorbance change of the control when normalized forchlorophyll concentration. Evidently the lower activity of re-constituted chloroplasts under continuous illumination is a

manifestation of both a smaller number of "active units" and afaster rate of decay of the signal produced in these units.

Experiments using laser flash excitation detected absorbancechanges through an instrument with a 3-,tsec response time.These experiments confirm that extraction does reduce the ex-tent of the initial absorbance change below the level of de-tection (0.0004 A).The trace of the flash-induced signal in the extracted sample

shows no fast jump following light absorption. However, thereis a slow rise of smaller magnitude. This type of signal is also

Off

' Off

A A

On On

Control

OffOff, Off , Off r

v ' v

On On A 1.6%

Iminm On

Extracted +,8-Carotene

FIG. 5. Light-driven absorbance changes at 518 nm in unlyophi-lized, extracted, and reconstituted chloroplasts. The chlorophyllconcentrations are 78, 98, and 113 gg/ml, respectively. Illuminationwas by broad band red light.

Light-Dark Difference Spectra

--Control Chloroplasts*-Extracted Chloroplasts

+ B-Carotene10- 02

< ~~~~~~~~~~0l

_-01 <

-10- T--02

450 470 490 510 530(nm)

FIG. 6. Light minus dark difference spectrum of unlyophilizedand reconstituted chloroplasts. Chlorophyll concentrations are 78and 113 ,ug/ml, respectively, in 0.3 M sorbitol, 10 mM NaCl, and 20mM MOPS, pH 7.0. Illumination was by broad band red light.

Control T1!i 0.005 0 D

:.hX-Ill*,-l-.i.l.:.:....s-!-.l-e-iI...:..:... 8

Osec

Extracted T000500D

t O2seciFlosh Carotene 0002500

Flash

FIG. 7. Absorbance changes in unlyophilized, extracted, and re-constituted chloroplasts activated by a xenon flash. The chlorophyllconcentrations are as in Figure 6. The top and the middle trace arethe averages of four experiments, the bottom trace the average ofsix.

555




seen in the reconstituted sample. Whether it reflects a processwhich occurs in the same units that show the fast absorbancechange, or whether the trace is the sum of reconstituted andunreconstituted species in the same sample cannot be deter-mined.

Figure 8A and 8B illustrate the relative extent of restoredactivity in systems which were reconstituted with different com-pounds. Only the samples which contained the /-carotenewere reconstituted with respect to 518 nm absorbance changes.These figures should be compared with Figure 9A, whichshows the extent of cytochrome f oxidation by far red light, adirect assay of photosystem I activity. In the presence of thequinone the cytochrome f oxidation is greatly enhanced, butthe extent of the 518 nm absorbance change is the same.The 83-carotene content of the different samples is shown

in Figure 9B. The control sample has a /3-carotene content of0.040 + 0.003 /3-carotene/chl. This yields between 4 and 8/-carotene molecules per photosynthetic unit (200-400 chlmolecules). Exhaustive extraction of the lyophilized materialusing heptane and an analysis of the difference spectrum ofunextracted - extracted chloroplasts gives a similar value,0.039 + 0.005 83-carotene/chl. Extraction reduces the /3-caro-tene content to 0.0006 + 0.002 /3-carotene/chl. This yieldsapproximately 1 /3-carotene molecule for every 2 photosyn-thetic units.

Activities reported here are referred to the unlyophilizedchloroplasts as the control sample. Lyophilization of chloro-

0

4-00

A

AA518-540nm

5o()() 81)7E) c c4- 4- ) 0

0 03 4-o c0 o- 0 or'< 0 2 -0 0cr- 0 I0

+ + CQ0

>, 125-C) 100-- 75-0 50-

zO25-

A720nm

Onoff

v1vI0'AL:100 :3

IRIw >1

HAA54-540

5) (L) a)

c QL0

0 0 Cr 0cX 0) 0 %.

C) =

c+

o U /3-Carotene, 40- L4cx -; 0 Content,100-0

< 75-

50-0

25-

8-0

B

-I{

AA500-475

-LL 111F

5)(1

CX0 C C0

U. =3 L)0

+ C

FIG. 8. Relative absorbance changes at 518 nm (A) and 475 nm(B) in unlyophilized, extracted, and reconstituted chloroplasts. Thethree sections of each bar represent, from right to left, the activitiesfrom illumination by 720 nm light, 640 nm light, and broad bandred light. The slants at the top of each bar signify the indeterminacyof each measurement; the midpoint of the slant is the mean valueof three changes. Titles below the abscissa refer to the compoundspresent in the reconstituting solutions.

DISCUSSION

Absorbance Changes at 518 nm. The similarity between theabsorption spectra of a /3-carotene solution and that of a hep-tane extract of chloroplasts limits the possible contaminationfrom other carotenoids in the extract to less than 2%. Chro-matographic assays show that in extractions performed at 21 Cthe xanthophylls and other oxygen-containing carotenoids areonly partially removed. Under these conditions, however, /-carotene is extracted as a homogeneous pool. At 0 C less than1 % of this pool becomes more tightly bound (8).The difference spectrum of unextracted - extracted chloro-

plasts is red-shifted compared to that of /3-carotene, and thelong wavelength peak is particularly accentuated. Since thelarge majority of the extracted material is /3-carotene, the dif-ference spectrum implies that the environment of the /3-caro-tene in the membrane is different from that of liquid heptane.Perhaps the /3-carotene is normally in a condensed pigmentphase among layers of carotenoid and chlorophyll or near aregion of more polar protein molecules. Although protein-carotenoid complexes are found in extracts of spinach chloro-plasts (16, 22), there is no direct evidence that they exist invivo. Ji et al. (17) have shown difference spectra of unex-

556 STRICHARTZ



Plant Physiol. Vol. 48, 1971 /3-CAROTENE AND THE 518 NM ABSORBANCE CHANGE

tracted - heptane extracted chloroplasts, both samples havingbeen previously extracted with 70% acetone to remove chloro-phyll. The discernible peaks of these difference spectra areshifted less than 5 nm to longer wavelengths compared tohexane solutions of ,8-carotene. Thus, in the absence of chloro-phyll the in vivo g-carotene absorbance spectrum is not nearlyso red-shifted, although acetone treatment could produce ef-fects other than chlorophyll removal.The reconstituted absorbance change appears to be identical

to A. in its spectrum, its response to both photosystems, andthe time range of its decay. The extent of the restored absorb-ance change activated by continuous illumination is only 20%of the control signal, but reduced activity apparently resultsfrom a faster rate of decay of the signal in the reconstitutedsample as well as a small initial signal.The acceleration of this decay rate may indicate an increased

ion permeability following heptane extraction, since Junge andWitt (18) have shown that agents which selectively increase theion permeability of the membrane increased the decay rate offlash-induced A.,n. Likewise, extraction abolishes the light-in-duced pH change, perhaps by rendering the membrane so per-meable to protons that any gradients are dissipated beforereaching a detectable size. Certain uncouplers of both photo-phosphorylation and light-induced pH changes are supposed tofunction similarly (27, 28). Still, some aspect of membranestructure must be preserved since both Hill activity and photo-phosphorylation are restored by readdition of plastoquinone(20).The /3-carotene content of the reconstituted sample is ap-

proximately 35% of the control sample. However, the activityof the flash-induced 518 nm absorbance change in the recon-stituted sample is 60% of the control sample. If /3-carotene isdirectly responsible for the absorbance change, not all of thepigment in the control sample is active in producing the ab-sorbance change. Further evidence for this is reported byWiekard et al. (30), who showed that more than 100% of thecontrol activity of A,. could be restored by addition of plasto-quinone to chloroplasts which were only partially depleted of/3-carotene. This may result from the fact that not all /3-caro-tene molecules in the chloroplasts are active in A.. as well asthe known ability of plastoquinone to restore more than 100%of the Hill activity in extracted samples (24).

Electron Transfer. Quinones are extracted from lyophilizedchloroplasts by heptane. The major quinone component, andthe one which appears functionally most important, is plasto-quinone. Preliminary experiments with only partially extractedchloroplasts showed that An,, could be restored by addition ofduroquinone or benzoquinone as well as the quinones Q10 andQ, which have isoprenoid side chains of 50 and 30 carbons,respectively (G. Strichartz, unpublished observation). How-ever, none of these four quinones is active in restoring photo-phosphorylation or Hill activity in extracted chloroplasts (20).Therefore, any restoration of electron transfer in fully ex-tracted chloroplasts, as well as the enhancement of As,, inpartially extracted chloroplasts, most probably derives fromquinone activity at a site different from that of native plasto-quinone. The results of heptane extraction on the system II-activated reduction of cytochrome f and on the Hill reactionagree with current schemes for a site of plastoquinone betweensystem II and cytochrome f (3, 24).

Extraction also affects system I-activated cytochrome f oxi-dation. There are several possible explanations. Trebst (29) hassuggested that there are two sites of plastoquinone activity. Thefirst is between system II and cytochrome f; plastoquinone atthis site is more difficult to extract than that located at the

second site of action, after the primary acceptor of system I.This proposal for two sites of action is supported by the rela-tive effects of extraction on the rate of system I-activated cyto-chrome f oxidation (Fig. 4A) and on system II-activated cyto-chrome f reduction (Fig. 4B).

Paralleling this hypothesis is the proposal by Arnon (1) thatquinones might be the functional cofactors of cyclic photo-phosphorylation in vivo. The removal of cyclic cofactor qui-nones by extraction could limit the rate and extent of systemI-mediated cytochrome f oxidation, particularly in the absenceof noncyclic electron transfer to NADP.

Acknouledgments-The author gratefully acknowledges the generosity of Pro-fessor W. D. Bonner, Jr., for providing laboratory facilities and the support andencouragement of Professor Britton Chance. Special appreciation is appropriate toMr. D. Specca, Burlington, New Jersey, for free access to his spinach field.

LITERATURE CITED

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2. BISHOP, N. I. 1959. The reactivity of a naturally occurring quinone (Q-255) inphotochemical reactions of isolated chloroplasts. Proc. Nat. Acad. Sci.U.S.A. 45: 1696-1702.

3. BISHOP, N. I. 1961. The possible role of plastoquinone (Q-254) inthe electron transport system of photosynthesis. In: G. E. W. Wolestenholmeand C. M. O'Connor, eds., Ciba Foundation Symposium, "Quinones in Elec-tron Transport." Churchill, London. p. 385.

4. BONNER, W. D. A_ND R. HILL. 1963. Light-induced optical changes in greenleaves. In: Photosynthesis Mechanisms in Green Plants, Publication 1145.National Academy of Sciences, National Research Foundation, Washington,D.C. p. 82-90.

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14. HILDRETH, W. W. 1967. Laser-induced kinetics of cytochrome oxidation andthe 518 mg absorption change in spinach leaves and chloroplasts. Biochim.Biophys. Acta 153: 197-202.

15. HIN-D, G. AND J. 'M. OLSON. 1968. Electron transport pathways in photosynthe-sis. Annu. Rev. Plant. Physiol. 19: 249-282.

16. Ji, T. H., J. L. HESS, AND A. A. BENSON. 1968. The nature of /3-carotene asso-ciation in chloroplast lamellae. In: K. Shibata, A. Takamiya, A. T. Jagen-dorf, and R. C. Fuller, eds., Comparative Biochemistry and Biophysics ofPhotosynthesis. University Park Press, State College, Pa. pp. 36-49.

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18. JUNGE, W. AN-D H. WITT. 1968. On the ion transport system of photosynthesis-Investigations on a molecular level. Z. Naturforsch. 32b: 244-254.

19. KARLISH, S. J. D. AND M. AVRON. 1968. Analysis of light-induced proton up-take in isolated chloroplasts. Biochim. Biophys. Acta 153: 878-888.

20. KROGMAN, D. W. AND E. OLIVERO. 1962. The specificity of plastoquinone as acofactor for photophosphorylation. J. Biol. Chem. 237: 3292-3295.

21. NISHIMURA, M. 1958. A nomogram for chlorophyll determinations of acetoneextracts. Kagaku No Tyoiko (Special Issue 34-"Colorimetric Methods inBiochemistry") 2: 135.

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STRICHARTZ

22. NISHIMaRA, M. AND K. TAKAMATSU,. 1957. A carotene-protein complex iso-lated from green leaves. Nature 180: 699-700.

23. PRATT, L. AND N. I. BiSHOP. 1968. The 520 nm light-induced absorbancechange in photosynthetic mutants of Scenedesmus. Biochim. Biophys. Acta162: 369-379.

24. REDFEARN, E. R. 1965. Plastoquinone. In: R. A. Morton, ed., Biochemistryof Quinones. Academic Press, London and New York. pp. 149-181.

25. REDFEARN, E. R. AND J. FRIEND. 1962. Studies on plastoquinone. I. Determina-tion of the concentration and oxidation-reduction state of plastoquinone inisolated chloroplasts. Phytochemistry 1: 147-151.

26. SAGER, R. AND M. ZALOKAR. 1958. Pigments and photsynthesis in a carotenoid-deficient mutant of Chlamydomonas. Nature 182: 98-100.

27. SHAVIT, N., R. DILLEY, ANOD A. SAN PIETRO. 1968. Ion translocation in iso-


lated chloroplasts. Uncoupling of photophosphorylation and translocationof K+ and H+ ions induced by nigericin. Biochemistry 7: 2356-2363.

28. SHAVIT, N. AND A. SAN PIETRO. 1967. K+-dependent uncoupling of photo-phosphorylation by nigericin. Biochem. Biophys. Res. Commun. 28: 277-283.

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