Plant Physiol. (1981) 68, 610-6150032-0889/81/68/0610/06/$00.50/0

The Coupling of Electron Flow to ATP Synthesis in Pea andMaize Mesophyll Chloroplasts" 2I. INTERACTION OF ADENINE NUCLEOTIDES AND ENERGY TRANSFER INHIBITORS WITH THECOUPLING FACTOR COMPLEX

Received for publication November 28, 1980 and in revised form March 26, 1981

RICHARD M. COLE3, WENDY A. MACPEEK, AND WILLIAM S. COHENT. H. Morgan School of Biological Sciences, University of Kentucky, Lexington, Kentucky 40506

ABSTRACT

The rate of nouphosphorylating electron transport (in the absence ofADP and inorganic phosphate) in weD-coupled (ATP/2e-= 0.9-1.1) maizemesophyD choroplasts Is not modulated by external pH (6.5-8.5), lowlevels of ADP or ATP, or energy transfer inhibitors, e.g. triphenyltin andHeg ions. In contrast nonphosphorylating electron flow in pea chloroplastsis sensitive to alterations In medium pH, and to the presence of adeninenucleotides and energy transfer inhdbitors in the assay medium AlthoughATP is without effect on the rate of basal electron transport in maizechloroplasts, steady-state proton uptake is stimulated 3- to 5-fold by lowlevels of ATP. These results suggest that differences may exist in themanner in which the coupling factor complex controls proton efflux fromthe intrathylakoid space in Cs and C4 mesophyll chloroplasts.

A number of recent studies have examined the relationshipbetween proton flux through the coupling factor complex (CFo-CF1) and electron flow in chloroplasts (2, 7-11, 24, 25, 27-29). InC3 mesophyll chloroplasts which have developed a large trans-membrane proton concentration gradient, there appears to be apathway for proton efflux through CFo-CF, that is not coupled toATP synthesis (8, 11, 17, 19, 24, 27, 28). This pathway is mostprominent at alkaline pH values and is reflected by high rates ofnonphosphorylating electron transport (2, 17, 28). The binding ofsmall amounts of ATP or ADP (5 /M) to CF1 appears to preventa major part of this leak by altering the conformational state ofthe coupling factor (8, 17, 24, 27, 28). Studies of the effects ofenergy transfer inhibitors, e.g. phlorizin, alkyl tins, Hg2+ ions, etc.on basal electron flow also suggest a role for CFo-CF, in thecontrol of proton leakage from the intrathylakoid space via thispathway (8, 11, 27, 28). Our aim was to compare the effects ofadenine nucleotides and energy transfer inhibitors on the rate ofnonphosphorylating electron flow and on proton fluxes in meso-phyll chloroplasts isolated from both a C3 plant (pea) and a C4plant (maize).

1 This research was supported by National Science Foundation GrantPCM76-17214.2A preliminary report of this work was presented at the annual meeting

of the American Society of Plant Physiologists, Columbus, Ohio, July1979.

3 Submitted to the Graduate School of the University of Kentucky inpartial fulfillment of the requirements for the Master of Science degree.

MATERIALS AND METHODS

Pea chloroplasts were isolated from 2- or 3-wk-old pea seedlings(Pisum sativum L. var. Laxton' Progress) grown in a controlledgrowth facility (5). Maize seedlings (Zea mays L. var. Pioneer3535) were grown in a greenhouse in the summer under naturalconditions, and in the winter months in the greenhouse withsupplemental illumination provided by metal halide lamps. Seed-lings were harvested at the 10- to 14-day stage, and only theterminal 4 to 5 cm of second and third leaves were used forchloroplast isolations (3). The leaf tissue was removed, chilled for30 min in ice water, and then cut into 2-mm sections whilesubmerged under the grinding medium. The medium for homog-enization contained 400 mm sorbitol, 50 mM Mops4 (pH 7.4), 10mM NaCi, 2.5 mm MgCl2, 1 mm MnCl2, 2 mM EDTA, 10 mMDTE, and 0.25% BSA. The chopped leaf sections plus the medium(150 ml) and 2 g of Polyclar AT were then placed in a Plexiglaschamber (5 x 5 x 12 cm). The leaf tissue was homogenized usinga Polytron tissue distintegrator (PT-20) for 5 to 6 s at a setting of5.5. The homogenate was filtered through a combination of 4layers of cheesecloth and one layer of Miracloth, and the filtratewas centrifuged for 60 s at 500g. The pellet was discarded and thechloroplasts were sedimented by centrifugation (l,500g for 8 min).The chloroplasts were washed once with 40 ml of medium con-taining 400 mm sorbitol, 50 mm Mops (pH 7.4), 10 mm NaCl, 2.5mM MgCl2, 1 mm MnCl2, 2 mm EDTA, and 0.5% BSA, andsedimented (l,500g for 8 min). Final suspension was in a smallvolume ofwashing medium; Chl was estimated in acetone extractsas described by Amnon (1).

Electron transport was monitored either by following 02 con-centration changes polarographically (4) or by measuring ferro-cyanide appearance as described by Portis and McCarty (25). Thereaction mixture for assaying electron transport contained 100 mMsorbitol (peas) or 50 mm sorbitol (maize M), 50 mm buffer (Mes atpH 6.5, Mops at pH 7.0 to 7.5 and Tricine at pH 8.0 to 8.5), 20mM KC1, 5 mM MgCl2, 5 mm K2HPO4, and chloroplasts equivalentto 13 to 22 pg/ml Chl. The acceptor systems employed were: H20

FeCy (1 mM K3Fe(CN)6; H20 -* MV (0.1 mM MV plus 0.5 mMNaN3), DADH2 -* MV (1 mm DAD, 2.5 mm neutralized ascor-bate, 4 ,lM DCMU, 0.1 mm MV, 0.5 mm NaN3), and DQH2 -MMV (0.5 mm durohydroquinone, 4 ,U DCMU, 0.1 mM MV, 0.5mM NaN3)).

Photophosphorylation, NEM inhibition of phosphorylation,

4Abbreviations: Mops, [3-(N-morpholino)propane sulfonic acid]; DTE,dithioerythritol; MV, methyl viologen; DAD, diaminodurene, 2,3,5,6-tetramethyl-p-phenylenediamine; NEM, n-ethylmaleimide; DCCD, dicyclo-hexyl carbodiimide; DQ, duroquinone, 2,3,5,6-tetramethyl-p-benzoqui-none.

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COUPLING IN PEA AND MAIZE CHLOROPLASTS

proton uptake (measured with a glass electrode) and adeninenucleotide exchange were assayed as described elsewhere (5, 6).

Triphenyltin chloride and deoxyphlorizin were gifts from Dr. J.M. Gould and Dr. G. D. Winget, respectively. All other reagentswere of the highest purity commercially available.

RESULTS

Maize mesophyll (Al) chloroplasts, isolated with the aid of aPolytron tissue distintegrator, exhibit rates of noncycic and cyclicphotophosphorylation that are comparable with those observedwith pea mesophyll chloroplasts (Fig. 1). In both pea and maizeM chloroplasts maximal coupling (ATP/2e-) is observed at pH8.0 to 8.5.The effects of the pH of the medium on rates of nonphospho-

rylating (-ADP-Pi) and phosphorylating (+ADP+Pi) electronflow (water to MV) in pea and maizeM chloroplasts are shown inFigure 2. The rates of both types of electron transport in peachloroplasts are strongly influenced by changes in medium pH,whereas with maizeM chloroplasts only phosphorylating electronflow is sensitive to external pH. Effects of the pH of the mediumon the rate of overall electron transport appear to be closelyassociated with an effect on the PS I portion of the electrontransfer chain (10). In both pea and maize M chloroplasts, anincrease in the pH of the assay medium from 7.5 to 8.1 resulted inhigher rates of phosphorylating and nonphosphorylating electronflow with either reduced diaminodurene (DADH2) or durohydro-quinone (DQH2) as the PS I electron donor (data not shown).

Nonphosphorylating electron flow in maize M chloroplasts isnot sensitive to the presence of 200 pM ATP in the assay medium,whereas electron flow under the same conditions in pea chloro-plasts is markedly inhibited (Table I). Basal electron flow in maizeM chloroplasts was also not affected by ADP concentrations upto 1 mm or by including arsenate or phosphate with ATP (datanot shown).

The rate of nonphosphorylating electron flow in C3 mesophyllchloroplasts can also be decreased by certain energy transferinhibitors (2, 8). For example, Figure 3 shows the effect oftriphenyltin chloride on basal electron flow in pea chloroplasts.The inhibitory effect is most pronounced at pH 7.5 to 8.5. At pH8.1 phosphorylating electron flow in maize M chloroplasts isseverely inhibited by 2 gM triphenyltin, 50 /AM DCCD, or 100 pMdeoxyphlorizin, whereas nonphosphorylating electron flow is un-affected (data not shown). The combination of an energy transferinhibitor triphenyltin and ADP (8) was also without effect on therate of nonphosphorylating electron flow in maizeM chloroplasts,whereas the combination inhibited basal electron flow in peachloroplasts to a greater extent than triphenyltin alone (Table II).

Nonphosphorylating electron flow in C3-mesophyll chloroplastscan also be modulated by the energy transfer inhibitor HgCl2 (10,13, 27, 28). Table III shows that HgCl2, at the 5 ,UM level, stimulatesbasal electron flow in pea chloroplasts 50 to 70%o. The increase inelectron flow induced by Hg2+ ions was not observed either in thepresence of an uncoupling agent (10 mm methylamine) or in thepresence of triphenyltin (2 gM). HgCl2, at concentrations of up to10 gm, was without effect on the rate of nonphosphorylatingelectron transport in maize M chloroplasts. In other experiments,we observed that phosphorylating electron flow in both pea andmaize M chloroplasts was substantially inhibited by 5 tLM HgC12(data not shown).Inasmuch as we failed to observe an effect of adenine nucleo-

tides on the rate of nonphosphorylating electron flow in maize Mchloroplasts, it was of interest to study the effects of ATP on theextent of steady-state proton uptake. A number of workers havecorrelated inhibition of basal electron flow by ATP or ADP withan increase in the extent of proton uptake in the steady state (5,8, 17). Although ATP did not alter the rate of basal electron flowin maizeM chloroplasts, the extent ofproton uptake was increased3- to 5-fold (Table IV). The ATP-induced increase was alsoobserved in the presence of 0.2 mm deoxphlorizin, which was

pH pHFIG. 1. Effect of pH on rates of phosphorylation and on coupling efficiences (ATP/2e-) in pea and maize chloroplasts. The reaction mixture for

assaying photophosphorylation was similar to the one described under "Materials and Methods" for measuring electron transport. In addition, itcontained I mM ADP, 5 mM [32PJ K2HPO4 (1-2 x 106 cpm), 1.5 mm K3Fe [CN]6 or 0.025 mm pyocyanine and pea or maize chloroplasts equivalent to24 ,ug/ml and 15 ,ug/ml Chl, respectively.

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COLE, MACPEEK, AND COHEN

140

120k

I.

-cy)

CREN

0cnI)0E

10o

80o

60F

40

2C

65 7U 75 8U 85pH

FIG. 2. Effect of pH on rates of nonphosphorylating and phospho-rylating electron flow in pea and maize chloroplasts. The reaction mixturescontained: pea or maize chloroplasts equivalent to 21 and 19 ,ug/ml Chl,respectively. Phosphorylating electron flow was assayed in the presence ofI mM ADP and 5 mM K2HP04. The buffer systems described under"Materials and Methods" were employed.

Table I. Effect ofATP on Nonphosphorylating Electron Transport in Peaand Maize Chloroplasts

The Chl concentrations were 21 iLg/ml Chl and 19 itg/ml Chl for peaand maize chloroplasts, respectively. When added, ATP was present at 0.2mM.

Electron Transport Rate

pH Peas Maize

-ATP +ATP -ATP +ATP

jumol 02 consumed/mg Chl h6.5 30 29 63 577.5 96 46 65 678.5 159 78 67 72

added to trevent any possibility of inward proton translocationdue to Mg +-ATPase activity.

Nucleotide interaction with CF1 can also be monitored byexaiining the effect of the nucleotide on the development of theirreversible NEM inhibition of photophosphorylation (14-16, 18,20, 29). Table V shows that with maizeM chloroplasts, 20 si ATPdoes not provide the same degree of protection from the NEMinhibition as it does in either pea or spinach chloroplasts. Increas-ing the ATP concentration to 100 AM or replacing ATP with anequivalent amount ofADP gave similar results (data not shown).Although phosphate and arsenate alone have little effect on thedevelopment of the NEM inhibition, both anions enhance theprotective effect of ATP in maize M chloroplasts, in pea chloro-plasts, and in spinach chloroplasts.

Energy-dependent adenine nucleotide exchange was also ex-

80sb-,__

0E 60E

CY0'A 40

E

20_

__-0'~~

R

/~~~~*TRIPHENYLTIN

6.5 70 75pH

80 85

FIG. 3. Effect of pH on the triphenyl tin inhibition of nonphospho-rylating electron flow in pea chloroplasts. When added, triphenyltinchloride was present at a final concentration of 2 jsm.

Table II. Effect of Triphenyltin andADP on Electron Transport in Peaand Maize Chloroplasts

The reaction mixtures contained pea or maize chloroplasts equivalentto 18 and 13 ug/ml Chl, respectively. Maize M chloroplasts were isolatedwith ascorbate in the grinding medium to eliminate any effects of thiolson the inhibitory action of triphenyltin (9). When added, triphenyltinchloride was present at a concentration of 2 pm.

Electron TransportRate

Source of .d.Chloroplasts -Tri- +Tri-

phenyl- phenyl-tin tin

MUmol 02/mg Chlb hPeas H20 to MV 185 107

H20 toMV + I mM ADP 117 93

Maize H20 to MV 62 62H20 toMV + I mm ADP 62 62

amined in both pea and maize chloroplasts (12, 21, 26, 32).Comparative studies indicated that the amount of labeled ADPbound per mg Chl in pea thylakoids was approximately twice thatobserved with maize thylakoids (Table VI). Both pea and maizethylakoids were capable of releasing ADP from prelabeled mem-branes in a light-mediated process (26) that was sensitive touncoupling agents and insensitive to energy transfer inhibitors(data not shown).

DISCUSSION

A strong correlation exists between the rate of proton effluxfrom the intrathylakoid space of C3 mesophyll chloroplasts andthe rate of overall electron flow. There appear to be three differentpathways for the efflux of protons (Fig. 4): (a) passive diffusionthrough the membrane; (b) efflux through the CFo- CF1 complexin a reaction that is coupled to the synthesis of ATP from ADPand Pi; and (c) efflux through the CFo-CF1 complex via apathway that is not coupled to ATP synthesis. By examining theeffects of low levels of ATP on the internal proton concentrationin spinach chloroplasts at both pH 7 and at pH 8, Portis et al. (24)came to the conclusion that leakage via pathway No. 3 increasesas the medium pH rises. This increase in proton leakage via

I

PEA (COUPLED) -

I MAIZE (COUPLED)

PEA (BASAL) -

_ MAIZE (BASAL)

I --

IbU.| X uU I Il i

U.

U' -.- .- -.1- --- n-.ni

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COUPLING IN PEA AND MAIZE CHLOROPLASTS

Table III. Effects of HgCl2 on Electron Transport in Pea and MaizeChloroplasts

Pea and maize chloroplasts were isolated as described under "Materialsand Methods." For this study, the washing and resuspension mediumcontained 400 mM sorbitol, 50 mM Hepes-NaOH (pH 7.5) and 10 mmNaCl.

Source2hlo Ti LM HgC12 +Hg2+Expt. of Chlo- phenltin -Hg_2+roplasts p 0 5

electron transport ratepmol 02/mg Chl.h

I Peas - 114 180 1.58+ 78 78 1.00

II Peas - 71 (272)8 115 (254) 1.62 (0.93)+ 42 46 1.10

III Maize - 98 91 0.93+ 96 NDb

IV Maize - 28 32 1.14

'Assayed in presence of 10 mM methylamine.b Not determined.

Table IV. Effect ofA TP on the Extent ofProton Uptake in Pea andMaize Chloroplasts

The assay medium for proton uptake (in 2 ml) contained: 50 mMsorbitol, 30 mM KCI, 5 mM MgCl2, 0.1 mM MV, I mm Tricine-NaOH andchloroplasts equivalent to approximately 35 jig/ml Chl. The starting pHwas adjusted to 7.5 ± 0.1.

Extent of Proton

Expt. Source of Chloroplasts Uptake

-ATP +ATP`

neq H+/mg ChlI Peas 150 521

Maize 146 735

II Maize 237 907Maize + 0.2 mM Deoxyphlorizin 286 849

a The ATP concentration was 100 pM in experiment I and 25 pm inexperiment II.

Table V. Effect ofA TP, Arsenate and Phosphate on the Development ofNEM Inhibition of Photophosphorylation in Pea and Maize ChloroplastsPreillumination of the chloroplasts in the presence of ATP, arsenate, or

phosphate had no effect on the rate of phosphorylation. The control ratesof phosphorylation (preilluminated minus NEM) were 254 ymol/mg Chlh and 848 iLmol/mg Chl-h for maize and pea chloroplasts, respectively.

Addition to Preillumination Source of ChloroplastsStage Peas Maize Spinacha

% inhibition ofNEM64 53 60

20 M ATP 33 43 231 mm arsenate 67 57 581 mM phosphate 67 56 57ATP/arsenate 12 2 15ATP/phosphate 3 36 9

aUnpublshed experiments of A. Grebanier.

Table VI. Energy-Dependent Nucleotide Binding in Pea and MaizeChloroplasts

Binding of labeled ADP to thylakoid membranes was carried out in areaction mixture, which contained (in 3 ml): 50 mM KCI, 25 mM Tricine-NaOH (pH 8.1), 5 mm MgCl2, 0.033 mm pyocyanine, 0.015 mm [3H]ADP(1.3 x 105 cpm/nmol) and chloroplasts (either pea or maize) equivalent to300 ug Chl. Samples were illuminated with heat-filtered white light for45 s and nucleotide binding was determined as described elsewhere (6).

nmol [3H]ADP bound/mg ChlSource of Chlo-

roplasts Light Dark Light mi-nus Dark

1 Maize 0.589 0.257 0.332II Maize 0.572 0.246 0.326II Peas 0.963 0.308 0.655IV Peas 0.820 0.211 0.608

STROMA

FIG. 4. Simplified scheme showing the relationship between electronflow, proton movements and phosphorylation in chloroplasts. CFo, prote-olipid proton channel, CF,, chloroplast coupling factor, eT, chloroplastelectron transfer chain. Pathway No. 1 represents passive leakage ofprotons through the thylakoidmembrane, pathway No. 2 represents protonmovements through the active site of CF1 which promote phospho-rylation and pathway No. 3 represents leakage of protons from the intra-thylakoid space via CFo in a manner which does not contribute tophosphorylation.

pathway No. 3 was presumably associated with a change in theconformation of CF1 that could be partly reversed in the presenceof ATP. The ability of triphenyltin to reverse a Hg2+-inducedincrease in the rate of proton efflux (reflected by an increase inthe rate of nonphosphorylating electron flow) led Underwood andGould (1 1, 27, 28) to conclude that interaction of triphenyltin withCFo was associated with a decrease in proton leakage via pathwayNo. 3. The DCCD-induced reversal of the increase in the rate ofproton leakage in 0-phenylenedimaleimide-treated spinach chlo-roplasts is also consistent with an increase in proton flux viapathway No. 3 (19).

Using pea (C3) mesophyll chloroplasts we observed that non-phosphorylating electron flow is increased as the pH of themedium is raised from 6.5 to 8.5, and that this increase is sensitiveto both small amounts of ATP (or ADP) and alkyl tins. Inaddition, we have observed that triphenyltin will reverse the Hg2+-

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COLE, MACPEEK, AND COHEN

induced stimulation in basal electron flow in pea chloroplasts. Inmaize M chloroplasts nonphosphorylating electron flow, in con-trast, is not modulated by medium pH, low levels of adeninenucleotides, alkyl tins, or Hg2' ions. These observations suggestthat pathway No. 3 is operative in pea chloroplasts, but may notbe operative in maizeM chloroplasts. Control of proton efflux viapathway No. 3 appears to be regulated by the gamma subunit ofCF1 (14, 19). Perhaps in maize M chloroplasts interaction of CF1with CFo via the gamma subunit results in a tighter junction witha smaller degree of nonspecific proton leakage.

Although the effect of the pH of the medium on the rate ofnonphosphorylating electron flow cannot be measured directlyunder phosphorylating conditions, McCarty and Portis (19) havedeveloped a theoretical treatment which permits one to estimatethe contribution of nonphosphorylating electron flow to the rateof overall electron flow. Using their method of analysis, we havedetermined that in pea chloroplasts as the medium pH is raisedfrom 6.5 to 8.5 the rate of both nonphosphorylating and phos-phorylating electron flow increase. The rate of phosphorylatingelectron flow increases to a greater extent, and thus the couplingefficiency increases (Table VII). With maizeM chloroplasts as thepH of the medium is raised from 6.5 to 8.5, the rate of phospho-rylating electron flow increases, but the rate of nonphosphorylat-ing electron flow decreases. The increase in coupling efficiency inmaize M chloroplasts results from a combination of a smallerincrease in the rate of phosphorylating electron flow (comparedwith pea chloroplasts) concomitant with a decrease in the rate ofnonphosphorylating electron flow. This analysis points up anotherdifference in the coupling of electron and proton movements inmaize M chloroplasts compared with pea chloroplasts.McCarty and Co-workers (19, 24) have correlated the inhibitory

effect of ATP on nonphosphorylating electron flow in spinachchloroplasts with an increase in the steady-state extent of protonuptake. Because this effect is most prominent at pH values above7.5, it has been inferred that the ATP-induced alterations in theconformation of CF1 primarily affect proton leakage via pathwayNo. 3. Although we failed to observe a decrease in the rate ofbasal electron transport in maize M chloroplasts in the presenceof ATP, we did observe a large increase in the extent of protonuptake (Table VII), and a decrease in the rate of passive protonefflux (Cole and Cohen, unpublished). If the ATP-induced in-crease in proton uptake in maize M chloroplasts is leading to a"true" increase in the internal proton concentration, this increaseis apparently not correlated with a change in the rate of nonphos-phorylating electron flow.During the initial stages of this investigation, we assumed that

Table VII. Effect ofMediumpH on the Rate of NonphosphorylatingElectron Flow Under Phosphorylating Conditions in Pea and Maize

ChloroplastsThe rate of nonphosphorylating electron flow was estimated using the

following equation Rb = Re - 3/2 Rep, where Rb is the rate of nonphos-phorylating electron flow under coupled conditions, Re is the overall rateof electron flow under coupled conditions, and Rep is the rate of phospho-rylation. The data from the experiments presented in Figure I were usedin the analysis; data from other experiments gave comparable results. Rb,Re, and Rep are expressed in ,umol/mg Chl- h.

Source of pH Re Rep Rb ATP/2e-Peas 6.5 149 22 116 0.30

7.5 359 156 125 0.878.5 613 314 142 1.03

Maize 6.5 184 18 154 0.207.5 358 152 130 0.85

the absence of ATP (or ADP) effects on basal electron flow inmaize M chloroplasts might be correlated with a failure of thenucleotides to interact with coupling factor. Subsequent studiesindicated that ATP could alter the extent of proton uptake inmaize M chloroplasts and that maize M chloroplasts were capableof exchanging labeled ADP in an energy-dependent manner. Anotable exception with regard to nucleotide-CF1 interactions inmaize M chloroplasts was the failure of adenine nucleotides toprevent partially the irreversible NEM inhibition of photophos-phorylation. In both spinach and pea chloroplasts significantprotection against the NEM inhibition is observed when 1 to 5/LM ATP or ADP is included in the preillumination stage withNEM (5, 6, 20). Some additional protection is afforded wheneither arsenate or phosphate is included with the adenine nucleo-tide. In maize M chloroplasts, addition of nucleotides in theabsence of phosphate or arsenate did not prevent the NEMinhibition. Inclusion of either phosphate or arsenate with the ATPdid offer significant protection in maize M chloroplasts. Thedecrease in the level ofNEM inhibition in the presence of adeninenucleotides has been correlated with a change in the conformationof the coupling factor protein when the nucleotide binds to anoncatalytic site on the a-subunit of the enzyme (15, 16, 22). Themodified conformation in the presence of the nucleotide appar-ently alters accessibility of the sulfhydryl reagent to a criticalresidue on the y-subunit of the protein. The presence of anarsenate or phosphate at the fl-subunit may further decrease theaccessibility ofNEM to the y-subunit. With maizeM chloroplasts,a change in conformation of CF1 may require the presence of anadenine nucleotide on the a-subunit as well as a phosphate orarsenate at the fl-subunit. Although it is tempting to assume thateffects observed with low concentrations of adenine nucleotides inelectron transport studies, in proton uptake studies, and in protec-tion experiments have in common an interaction of the nucleotidewith a non-catalytic subunit of the coupling factor, our data withmaize M chloroplasts and some of the data of McCarty and co-workers (14-16) appear to be consistent with a more complex setof interactions.

Acknowledgments-We wish to thank Drs. J. M. Gould and A. Grebanier forsupplying us with unpublished data. We also wish to thank Dr. R. E. McCarty forhelpful discussions.

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COUPLING IN PEA AND MAIZE CHLOROPLASTS

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Plant Physiol. Vol. 68, 1981

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