Plant Physiol. (1979) 64, 757-7620032-0889/79/64/0757/06/$00.50/0

Oxidative Phosphorylation in Pea Cotyledon SubmitochondrialParticles'

Received for publication March 7, 1979 and in revised form June 14, 1979

CHARLES GRUBMEYER2, DARA MELANSON, IAN DUNCAN, AND MARY SPENCER3Department of Plant Science, University ofAlberta, Edmonton, Alberta T6G 2N2 Canada

ABSTRACT

Mitochondria and submitochondrial particles (SMP) from pea cotyle-doDs were shown to catalyze oxidative phosphorylation as measured by32p; uptake into pbosphate esters. ATP synthesis was sensitive to theelectron transport inhibitor KCN, the uncoupler carbonyl cyanide m-chlo-rophenylhydrazone, and the coupling factor inhibitor oligomychr. Experi-ments with the adenine nucleotide translocator inhibitor atractylosideindicated the SMP were inside-out. Mersalyl completely inhibited ATPsynthesis by SMP, and a separate experiment indicated that mersalyl hasa direct effect on the ATPase complex. The kinetics of ATP synthesisindicated a high affinity for phosphate (K. = 0.18 milliar). ADPkinetics gave a biphasic curve with K. values of about 4.8 and 160micromolar. 02 uptake and ATP synthesis had a pH maximum of 7.6 whilethe ratio of m cromoles phosphate esterifed to microatoms 02 taken upwas highest at pH 7.2. Sodium chloride inhibited both ATP synthesis and02 uptake but stimulated the ATPase reaction. The SMP also catalyzeda slow ATP-phosphate exchange reaction.

Studies from several laboratories have shown that oxidativephosphorylation in plant mitochondria is similar in many respectsto that in animal mitochondria (12, 13, 15). While intact plantand animal mitochondria have been well characterized, they arenot ideal systems for studying the mechanism or the control ofoxidative phosphorylation. Access of all charged substrates to theinner matrix is governed by specific carriers that may, under someconditions, control apparent substrate affinities and maximal ratesof oxidative phosphorylation (29).

In contrast, the use of submitochondrial particles is particularlyadvantageous in that substrates in solution have direct access tothe catalytic site on the ATPase-ATP synthetase enzyme complex.Schuster et al. (23) have used beef heart and rat liver SMP4 todetermine the kinetic parameters and a kinetic mechanism foroxidative phosphorylation. They found, as have other workers (3,22), that the Km values with the beef heart and rat liver SMP weresubstantially higher than those normally obtained with the intactmitochondria. SMP capable of substrate oxidation and responseto ATP have been prepared from mung bean mitochondria (1, 27,

'We gratefully acknowledge grant A-1451 from the National ResearchCouncil of Canada in aid of this research, a Postgraduate Scholarship toCG and Postdoctoral Fellowship to DM.

2Present address: Department of Biochemistry, Public Health ResearchInstitute of the City of New York, Inc., 455 First Avenue, New York, N.Y.

3To whom reprint requests should be addressed.4Abbreviations: SMP: submitochondrial particles; RCR: respiratory

control ratio; TPP: thiamine pyrophosphate; CCCP: carbonyl cyanide m-chlorophenylhydrazone; p-CMBS: para-chloromercuribenzoate, sulfonicacid salt; P/O: ratio of pmol phosphate esterified to jtatoms 02 taken up.

28), and Passam and Palmer (20) have demonstrated both sub-strate oxidation and oxidative phosphorylation in SMP fromJerusalem artichoke. In order to supplement studies on the ATPasesystem of pea cotyledon mitochondria (8, 16, 17), a characteriza-tion was recently begun of the ATPase activity of SMP (9;Grubmeyer and Spencer, unpublished data).

In this report, pea cotyledon SMP are shown to catalyze highrates of succinate and NADH oxidation, phosphorylate ADP, andperform an ATP-Pi exchange reaction. The kinetic parameters forATP synthesis were determined from analysis of these data. Inaddition, the properties, including sensitivities to inhibitors, of theSMP are reported.

MATERIALS AND METHODS

Pea seeds (Pisum sativum L. cv. Homesteader) were germinatedat 27 C in the dark for 4 days in Vermiculite. Cotyledons (1,200ml) were harvested and mitochondria prepared as previouslyreported (25). The mitochondria were resuspended in 60 ml of0.25 M sucrose and centrifuged at 20,000g for 7 min. The darkbrown pellet was cleaned of a light brown layer by suction andthe mitochondria (approximately 250 mg of protein) were resus-pended in 10 ml of 0.25 M sucrose.

For the preparation of SMP, the mitochondria could either beused immediately or stored overnight at -20 C without change inthe yield or activity of the resulting SMP preparation.The above mitochondrial suspension was diluted to 60 ml in a

beaker with ice cold SMP buffer (0.25 M sucrose, 50 ims Tes,brought to pH 7.0 at 20 C with Tris). The beaker was placed in anice bath and sonicated for two 1-min bursts, separated by a 1-mincooling period, at 90%o of full power on an Artek Sonic Dismem-brator (model 300) equipped with a full size tip. The sonicate wascentrifuged for 7 min at 20,000g at 0 C in a Beckman model J-21Bcentrifuge to remove the small amount ofunbroken mitochondria.The translucent supernatant layer was centrifuged for 1 h at100,000g at 4 C in a Beckman L2-65B ultracentrifuge and thepellet (SMP) was resuspended in SMP buffer to a concentrationof 3 to 6 mg protein/ml. The SMP were stored in 0.5-ml aliquotsin stoppered glass tubes at -20 C for no longer than 2 weeks.ATP synthesis was measured by esterification of [P12jPi. The

reaction was in a total volume of 1 ml. The assay medium was: 0.3M sucrose, 4 mM MgCl2, 20mM glucose, 4 mM K2HPO4 (containingapproximately 200,000 cpm [IP]Pi, 50 mm Tes, 5 units of hexo-kinase, 2 mm ADP, and either 0.88 mM NADH or 8 mM succinate,brought to pH 7.2 at 25 C with Tris. Changes in this assay mediumwere as noted in the figures and tables. The reaction was startedby the addition of 70 to 150 jg protein (SMP or mitochondria),and was allowed to proceed for 6 to 15 min at 25 C. To stop thereaction, 1 ml of quench solution (0.3 M HCI, 0.12 M glycine, and1.8 M NaClO4) was added and the tubes were placed on ice for 5min. After a 6-min centrifugation to remove precipitated protein,the supernatant layer was transferred to a clean test tube and 2.0ml of molybdate reagent (2.1 N H2SO4, 0.6 M NaClO4, and 12.5

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Plant Physiol. Vol. 64, 1979

mM ammonium molybdate) was added to react with the Pi (19).The tubes were left 2 to 6 min at room temperature to ensure

formation of the phosphomolybdate complex. This complex wasthen removed by three extractions with 4-ml portions of isobutan-olyl alcohol-benzene (1:1). The esterified [32P]Pi was measured bythe addition of a 1--ml portion of the aqueous residue to 10 ml ofAquasol, and counted by liquid scintillation (Searle 6872 liquidscintillation system). Except where noted, blank tubes containedall assay components plus 1 jig of oligomcyin.Measurement of 02 uptake during oxidative phosphorylation

was made in a final volume of 3 ml of the ATP synthesis assay

medium, except that no [32PJPi or hexokinase was present. A YSlmodel 53 oxygen monitor equipped with a Clark-type electrodewas used to follow 02 consumption.ATPase was measured using the phosphate release procedure

previously described (8, 16). The assay medium was 0.3 M sucrose,4 mm MgCl2, 50 mm Tes, 20 mm glucose, and 3 mm ATP, broughtto pH 7.2 at 20 C with Tris. The reaction was run for 10 min at 25C.

Protein was measured by a simple dye-binding procedure (24),which gave good agreement with the method of Lowry et al. (14).The experiments described were all run two or more times, and

every assay contained two or three replicates of each treatment.The data presented are from representative runs, and variationbetween runs with the same batch of mitochondria or SMP wasminimal. Different batches varied in their activities.

All biochemicals were obtained from Sigma Chemical Co.Bakers' yeast hexokinase was Sigma product H-4502 (200-300units/mg protein). Aquasol and [32PJPi in 20 mm HCI were

obtained from New England Nuclear. Inorganic chemicals wereFisher certified grade.

RESULTS

02 Uptake by Mitochondria and SMP. The measurements of02 uptake for our preparations of mitochondria are shown inTable I. Mitochondria showed RCR values of2 to 3 with succinateand 3 to 4 with the NADH-linked substrate malate plus TPP (notshown). Oligomycin, a specific inhibitor of the ATPase-ATPsynthetase complex, did not inhibit state IV respiration but didprevent the increase in respiration normally observed upon theaddition of ADP. The rate of 02 uptake, in the presence of anoptimal concentration of uncoupler (16 jiM CCCP), was onlyslightly greater than the rate in state III. Atractyloside, which

competitively inhibits ADP transport into the mitochondrion (29),produced a one-third inhibition of the state III rate when used ata concentration of 20 jiM in the presence of 1 mm ADP. We alsofound that 10 jiM mersalyl, a sulfhydryl blocking reagent thatinhibits phosphate transport into mitochondria (11, 29), preventedany increase in mitochondrial respiration upon the addition ofADP.With SMP-oxidizing NADH, ADP produced only a slight to

moderate stimulation of 02 uptake (Table I). Between prepara-

tions of SMP this ADP response varied from 0 to 40%o. (Nostimulation was noted with succinate.) No state III to state IVtransition was observed (not shown). In the presence ofuncoupler,however, respiration of SMP was increased substantially beyondstate III rates. Oligomycin inhibited the initial state IV slightly,and prevented any increase in respiration on addition of ADP. Itdid not affect the rate of uncoupled respiration. With NADH assubstrate, the rate of02 uptake by SMP was stimulated by additionof 10 jig of oxidized Cyt c. Mersalyl (10 jIM) completely blockedthe increase in respiration observed on addition ofADP plus Pi toSMP (Table I) and had no effect on uncoupled rates of respiration.At a concentration of 20 ,lM, atractyloside had no effect onrespiration of the SMP.

Oxidative Phosphorylation by Mitochondria and SMP. Bothmitochondria and SMP were capable of good rates of oxidativephosphorylation, as measured by I32P]Pi esterification (Table II).The assay was linear with respect to time and concentration ofmitochondria or SMP up to the point of 02 depletion (results notshown). The test system was set up to use 40 to 60%Yo of theavailable 02- With SMP, the assay required hexokinase for max-imal activity, since these particles catalyze an active ATPasereaction even in the presence of oxidizable substrate (26; Grub-meyer and Spencer, unpublished results).

Oxidative phosphorylation by mitochondria or SMP was se-

verely reduced by either uncoupler or oligomycin (Table II). Inall further experiments blanks contained all assay componentsplus 1 jug oligomycin, which ensured that no other Pi-esterifyingsystem was interfering. Addition of excess hexokinase kept inter-ference by the ATP hydrolysis reaction to a minimum.A series of experiments with atractyloside showed that our pea

SMP were indeed inside-out, and thus not kinetically governed bytransporter systems. To ensure that the pea ADP transporter wasnot insensitive to atractyloside the experiments were also per-formed with whole mitochondria (Tables I and II). Atractyloside(20 liM) inhibited mitochondrial 02 uptake and oxidative phos-

Table I. Oxygen Uptake by Mitochondria and SMPfrom Pea CotyledonsAssay was at 25 C with 02 electrode in 3 ml of the following medium: 0.3 M sucrose, 4 mm MgCl2, 20 mm glucose, 4 mm K2HP04, and 50 mm Tes

(pH 7.2) at 25 C (with Tris). Where noted the assay also contained 0.88 mM NADH, 8 mM succinate, or 2 mm ADP. The same batch of mitochondriaor SMP was used for both treated and untreated samples within any one treatment. Where noted, the same batch was used for different treatments.

02 Uptake

Preparation Substrate Treatment Treated Untreated

No ADP +ADP ANDop +ADP

nmol/min * mg protein

Mitochondria Malate None' 20 68Succinate I iLg Oligomycina 61 61 69 154

16 ,UM CCCP 192 75 14820,LM Atractyloside 260 289 242 52010 ,UM Mersalyl 88 88 110 246

SMP Succinate 16 psm CCCPb 83 45 45NADH 16 jiM CCCPb 332 153 189

lILgOligomycin 112 112 131 16110 ,ig Cyt c 159 200C 88 117IO,UM Mersalyl 66 56 61 88

a Same batch of mitochondria.b From same batch of SMP.c Rate with 16 ,iM CCCP was 313.2 nmol/min-mg protein.

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OXIDATIVE PHOSPHORYLATION BY PEA SMP

Table II. Oxidative Phosphorylation by Mitochondria and SMPAssay was carried out for 6 to 15 min at 25 C in a total volume of I ml.

Medium: 0.3 M sucrose, 4 mM MgCl2, 20 mm glucose, 4 mM K2HPO4(initially with about 200,000 cpm 132P] Pi), 50 mm Tes, 5 units of hexoki-nase, 2 mM ADP, and either 0.88 mM NADH or 8 mM succinate, at pH7.2. The reaction was started by the addition of 70 to 150 jg of either SMPor mitochondria. The same preparation of SMP was used for both treatedand untreated samples within any one treatment.

ATP SynthesisPreparation Substrate Treatment

Treated Untreateduwis

Mitochon- Malate None 0.194dna Succinate 16 iLM CCCP 0.001 0.364

I jig Oligomycin 0.015 0.32720 tiM Atractyl- 0.083 0.128oside

10,M Mersalyl 0.034 0.303SMP Succinate I jLg Oligomycin 0.022 0.078

NADH 16t,M CCCP 0.005 0.3581 yg Oligomycin 0.012 0.31620 piM Atractyl- 0.161 0.172oside

20 pM Atractyl- 0.024 0.022osidea

20 pM Atractyl- 0.050 0.059osideb

20 1,UM Atractyl- 0.114 0.119osidec

10 iLM Mersalyl 0.010 0.23310tgCytc 0.281 0.1960.2 mM KCN 0.002 0.174

aADP at 200M.b ADP at 20 gLm.":ADP at 200 jim.

phorylation by 30%o, but had no effect on the phosphorylation bySMP. Thus, the adenine nucleotide transporter is not involved inthe synthesis of ATP by SMP.When mersalyl was present at a concentration of 10 ,UM, phos-

phorylation by either mitochondria or SMP was almost completelystopped, at both low (0.1 mM) and high (4.0 mM) phosphateconcentrations. The ATPase reaction of SMP was also inhibited.(Mersalyl binds to exposed sulfliydryl groups, such as one wouldexpect to be on the surface of inside-out particles. It is a transportinhibitor for phosphate as a result of capacity for blocking the Pi-OH- antiporter [11].) It has previously been shown that the solubleATPase of peas is inhibited by the sulfhydryl blocking compoundp-CMBS (8).

Addition of oxidized Cyt c to the assay medium resulted in a30%o increase in oxidative phosphorylation when SMP were oxi-dizing NADH. A 0.2 mm concentration of KCN completelyinhibited ATP synthesis.When NADH was used as substrate for the SMP, P/O ratios of

0.5 to 1.4 were obtained, while succinate gave values ranging from0.2 to 0.5. In the majority of experiments NADH was used assubstrate. Although the addition of Cyt c increased O2 consump-tion by 50%o, the smaller increase in phosphorylation resulted in alowered P/O ratio.

Kinetics of Oxidative Phosphorylation by SMP. The Km for Piwas found to be 0.18 mm in pea SMP (Fig. 1). The positivecooperativity toward Pi, noted by Schuster et al. with beef heartand rat liver SMP (23), was not detected in our experiments. Sincethe experiments of Schuster et al. were conducted at concentrationsof ADP below 200 pM, our Pi kinetics experiments were repeatedat 50 AIM ADP, and again linear kinetics were found (not shown).The ADP kinetics of oxidative phosphorylation were not as

simple as the Pi kinetics. In repeated experiments a biphasic plot

was consistently obtained that suggested that the curve for ATPsynthesis was the result of the combined operation of two separateprocesses. Derivation of the apparent Michaelis-Menten kineticconstants by extrapolating the two straight line segments of thecurve to intersect at the x and y axis is shown in Figure 2. Valuesfor Kmi, Vmax 1, Km2, and Vmax 2 were 9 tiM, 0.26 units, 39 t,M, and0.33 units, respectively. When dealing with such curvilinear plotsthis procedure is inaccurate since the values obtained for process1 would contribute to process 2 and vice versa (5). The separateparameters were analyzed by computer to obtain a theoreticalcurve that was used as a basis for adjusting the assumed values forKmi, Vmax 1, Km2, V.a 2. This method has been used successfullyin the kinetic study of multicomponent transport processes (5, 21).The new assumed values of Km1 = 4.8 tiM and V.i1 = 0.21 unitsand Km2 = 160 ,llM and Vmx2 = 0.12 units have separated theoriginal curve into two separate components, each unaffected bythe other. It is now apparent from V,,., and V,,,2 that theoriginal curve was the result oftwo-third contribution from processI and a one-third contribution from process 2. It is possible thatsuch a curve could reflect two populations of particles, one inside-out and one right-side-in. The kinetics of right-side-in particlescould be govemed by an adenine nucleotide transporter with ahigh affinity for ADP, while the inside-out particles could give asecond, higher Km. To test this, an attempt was made to inhibitoxidative phosphorylation at low levels of ADP by adding 20 t,Matractyloside. Table II shows that even at very low ADP levels,atractyloside had very little effect.ATPase and Oxidative Phosphorylation. Inasmuch as it has

been found that the ATPase reaction and substrate oxidation canoccur at the same time (26, Grubmeyer and Spencer, unpublishedresults), experiments were undertaken to determine what, if any,relationship exists between rates of ATP hydrolysis, ATP synthe-sis, and 02 uptake.

Table III shows that each of the three reactions studied had adifferent pH curve. As expected from earlier work on the solublepea mitochondrial ATPase (16), ATPase activity ofSMP increasedsteadily over the pH range 6.8 to 8.0. The rate of phosphorylation,however, showed a peak at pH 7.6. In addition, 02 uptake in thepresence of ADP and Pi, or with uncoupler, was maximal at pH7.6. P/O ratios were highest at pH 7.2.The stimulations of ATPase by Cl- and HCO3 as noted in

several systems for the soluble F1 enzyme provided another testsystem for the relationship between ATP hydrolysis and ATPsynthesis in pea SMP. Table IV shows that both NaCl andNaHCO3 stimulated ATP hydrolysis at pH 7.2. Phosphorylation,however, was slightly reduced. To show that this reduction wasnot caused by the enhanced ATPase activity competing withhexokinase for the ATP, the level of hexokinase was increased 2-

30 -i.---

25-

20-

.2.1

10

5

0 -5 0 5 10 15 20 25 20

1/[Pij (mM)-'FIG. 1. Phosphate kinetics of ATP formation by pea cotyledon SMP.

Assay as in Table II. Substrate was 0.88 mM NADH.

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Plant Physiol. Vol. 64, 1979

0 10 20 30 40 50

1/[ADPI (mM)-l

FIG. 2. ADP kinetics of ATP formation by pea cotyledon SMP. Assayas in Table II. Substrate was 2 mM NADH. Apparent ADP kinetics: 1:V. i = 0.26 units, Km. = 9 ,AM; 2: V,a. 2 = 0.33 units, Km2 = 39 ,LM.Calculation of ADP kinetics: 1: Vm. i = 0.21 units, Km1 = 4.9 a,lM; 2: Vm,x 2

= 0.12 units, Km2 = 160 units.

Table III. Effects ofpH on A TP Synthesis and A TP Hydrolysis by PeaCotyledon SMP

Assay as in Tables I and II. Substrate was 0.88 mM NADH.02 Uptakce

pH ATP Hy- ATP02+AkepH drolysis Synthesis NoPO

ADP +ADP +CCCP

units units nmol/mini mg protein ratio

6.8 0.086 0.176 86.4 86.4 116.9 1.027.2 0.141 0.244 78.2 86.4 120.2 1.417.6 0.190 0.273 108.6 136.6 206.6 1.008.0 0.276 0.224 99.6 114.4 179.4 0.98

Table IV. Effects of Bicarbonate and Chloride Ions on OxidativePhosphorylation and A TP Hydrolysis by SMPfrom Pea Cotyledons02 uptake was measured in the presence of 0.88 mm NADH and 2 mM

ADP. Assays as in Tables I and II.Treatment ATP Hy- ATP 02 UpP/-drolysis Synthesis take

units units nmol/min- ratiomg protein

Control 0.196 0.130 83.0 0.7820 mm NaHCO0a 0.238 0.124 68.2 0.91100 mM NaCl 0.261 0.087 64.1 0.6820 mM NaHCO0a 0.085 43.2 0.99+ l00 mmNaCl

a Twenty mM added NaHCO3 gives a solution with HC03- = 17.4 mmat pH 7.2 and 25 C.

Table V. Investigations ofA TP-[rP]Pi Exchange by Pea CotyledonSMP

Assay medium used was as described in Table II, except hexokinasewas absent and ADP, ATP, and NADH were added only as indicatedbelow. The reaction was started by the addition of 150,tg SMP to makea final volume of 1 ml and incubation was for 10 min at 25 C.

In the presence of 2 mm ADP, 0.88 mm NADH, and 5 units ofhexokinase, net ATP synthesis was 0.117 unit/mg protein. In the presenceof 2 mM ADP, 0.88 mM NADH but no hexokinase, net ATP synthesis was0.077 unit/mg protein.

Pi incorporated

Conditions No nucleo- 2 mm 2 mM ADP +tide addi- ATP 2 mM ATP

tions

iLmol/min * mg protein

No substrate 0.003 0.019 0.0240.88 mmNADH 0.008 0.040 0.0560.88 mM NADH + 0.016

1 ug Oligomycin

fold, but this had no effect. Results with the 02 electrode indicatedthat both NaCl and NaHCO3 were inhibitors of 02 uptake bySMP (Table IV).Both intact mitochondria and an isolated ATPase enzyme from

rat liver mitochondria catalyze an exchange reaction between theterminal phosphate of ATP and Pi in the apparent absence ofsubstrate oxidation (7). With pea SMP, whenNADH was excludedfrom the reaction medium and ATP was present instead of ADP,there was a low level of incorporation into ATP (Table V). Thisproceeded at a rate well below that of oxidative phosphorylationeven when ATP was not removed through the hexokinase reaction.Since the amount of Pi formed by ATP hydrolysis was insignifi-cant in comparison to the added Pi no correction for Pi pooldilution was made. Cooper and Lehninger (7) also reported thatADP affects the rate of ATP-32Pi exchange and that this effectdepends on the relative concentrations of Pi, ADP, and ATP.With pea SMP under the conditions of our experiments, thecombined addition of ATP and ADP (Table V) gave similarresults to the addition of ATP alone.

DISCUSSION

The rates of oxidation of NADH and succinate by peak SMP(Table I) are similar to those reported by Wilson and Bonner (27,28) for mung bean SMP prepared by osmotic shock. With the peaSMP addition ofADP stimulated oxidation of NADH. There wasa lack of respiratory control. Succinate oxidation by pea SMP wasvery much reduced compared with that by intact mitochondria,and was not enhanced by ADP. Passam and Palmer (20), whoprepared SMP from Jerusalem artichoke by sonication found thatthe rate of oxidation of succinate was only half the rate catalyzedby the mitochondria. They suggested that a segment of the succi-nate oxidase pathway may have been damaged during preparationof the SMP. Since oxidation of NADH in the pea system wasincreased by the addition ofCyt c, an observation made previouslyby Beyer et al. (1) for mung bean, this indicates that some loss ofmembrane components may 4,ave occurred during the isolationprocedure. It is to be expected that soluble (matrix) mitochondrialenzymes would be lost during the preparation of SMP, as a resultof sonication and subsequent centrifugations.Our preparation of pea SMP were capable of oxidative phos-

phorylation as shown in Table II. Values ranged from 0.172 to0.358 unit5 of SMP protein with NADH and in the presence of

5One unit of ATP synthetase activity is defined as the amount (mgprotein) to esterify I ,umol of Pi/min under the above assay conditions.One unit of ATPase hydrolysis activity is defined as the amount mgprotein required to hydrolyze 1 ttmol ATP/min under the above assayconditions.

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OXIDATIVE PHOSPHORYLATION BY PEA SMP

excess (2 mM) ADP. This variation reflected different batches andstorage times of SMP. (Comparison studies were done with SMPof the same batch and age.) Passam and Palmer (20) found thatSMP from sonicated Jerusalem artichoke mitochondria phospho-rylated ADP at a rate of 0.103 units protein. Although Wilsonand Bonner (27, 28) showed that their mung bean SMP catalyzedenergy-linked reactions (NADH - NADP+ transhydrogenase andreverse electron transport), oxidative phosphorylation was notreported.

Results with inhibitors showed that the reactions under inves-tigation were those of oxidative phosphorylation. An electrontransport inhibitor (KCN), an uncoupler (CCCP), and an ATPase-ATP synthesis inhibitor (oligomycin), all severely inhibited theuptake of [mP]Pi into organic phosphate esters.The pea SMP appear to have a much stronger affinity for Pi

than do any of the reported animal preparations. For example,Bygrave and Lehninger (3), who investigated the Pi kinetics ofintact and broken rat liver mitochondria, found that the concen-tration of Pi required for half-maximal velocity increased from0.25 mm for whole mitochondria, to 3.0 mm and 6.0 mm withdigitonin-treated and sonicated mitochondria, respectively. Simi-larly, Schatz and Racker (22), using beef heart SMP, have foundKm values for Pi of 2 to 10 mM, with the Km proportional to therate of respiration. Schuster et al. (23), although not reporting aKm value, show data indicating a Km of over 10 mM Pi for beefheart SMP. Our own experiments indicated a Km of over 2 mMwith rat liver SMP. In contrast to the animal preparations wefound that SMP from peas have a Km of 0.18 mM, which suggeststhat pea SMP have an intrinsically high affinity for Pi. Whetherthis is a basic property of the pea enzyme system, or indicates thatthe phosphorylating system of mammalian SMP is more easilydamaged by sonication, is not known.The ADP kinetics of ATP synthesis in pea SMP resulted in a

biphasic curve with Km values of approximately 4.8 pM and 160pM. Since this did not appear to result from a mixed population ofinside-out and right-side-in particles we concluded that it is abasic property of our pea SMP preparations. There could be twopossible explanations for this. Pea SMP could, for instance, shownegative cooperativity toward ADP, or secondly, there may betwo sites for ADP binding, one high affinity binding site and onelow affinity site. The low affinity site could be responsible fornonspecific binding of ADP. Under normal physiological condi-tions of low ADP levels in the mitochondria it would be assumedthat the high affinity site is mainly involved in ATP synthesis.A wide range of Km values for ADP has been reported for

mammalian SMP. With sonicated rat liver mitochondria, Bygraveand Lehninger (3) found a Km for ADP of 300 pM, althoughLineweaver-Burk plots were not shown and the data of Schusteret al. (23) suggest a Km of below 30 pM (at 10 mm Pi) for beef heartSMP, with linear kinetics. Catterall and Pedersen (4) found a Kmfor ADP of 3.8 ,UM for phosphorylation by a purified preparationof inner membrane vesicles of rat liver. Since in the latter twocases, both research groups used low concentrations of ADP(below 200 tIM) the possible influence of a second component inthe synthesis of ATP could have remained undetected.pH effects did not demonstrate a simple relationship between

ATP synthesis and hydrolysis (Table III). While it is possible thatthe reduction of ATP synthesis at pH 8.0 is caused by low 02consumption, since P/O ratios were the same at pH 7.6 and 8.0,further knowledge is needed about the rate-limiting steps in theSMP system before interpretation of the pH effects is clear.

Since both ATP synthesis and hydrolysis are catalyzed by thesame enzyme, and previous work (10) had demonstrated thatNaCl and NaHCO3 stimulated ATP hydrolysis by the pea enzyme,experiments were done to determine whether these salts had asimilar effect on the synthesis of ATP. (It should be noted that theexperiments could be conclusive only if ATP synthesis and notsubstrate oxidation were rate-limiting.) The stimulatory effect ofuncoupler on NADH oxidation in the presence of ADP (Table I)

indicated that state III respiration was, in fact, limited by ATPformation. The actual effect of the salts on ATP formation,however, was inhibitory and not stimulatory (Table IV). Thisresult is in agreement with the observations of Christiansen et al.(6) for beef heart SMP; but, in contrast to their system, we foundthat 02 uptake was also inhibited (Table IV). Since both 02 uptakeand ATP formation were inhibited by NaCl in the pea system, itis inconclusive as to whether the ATP synthetase system wasdirectly affected by use of the salt. High ionic strength (I > 0.1)has previously been shown to inhibit pea cotyledon Cyt oxidase(2), and similar effects of NaCl and NaHCO3 on oxidative phos-phorylation have been noted in cauliflower mitochondria (18).The results presented in this paper demonstrate that the pea

SMP preparations catalyze high rates of succinate and NADHoxidation and also show that the kinetics of ATP formation arecomparable to those from animal SMP. Pea SMP appear, there-fore, to be a good source of enzyme with which to study theATPase-ATP synthetase complex.

Acknowledgment-We wish to thank Dr. J. Weiner for his help in the analysis of some of thekinetic data.

LITERATURE CITED

1. BEys RE, GA PEmas, H IKulA 1968 Oxido-reduction states and natural homologue ofubiquinone (coenzyme Q) in submitochondrial particles from etiolated mung bean (Phaseohasaures) seedlings. Plant Physiol 43: 1395-1400

2. BomHoFF GH, M SPENcEs 1977 Optimum pH and ionic strength for the assay of cytochromec oxidase from pea cotyledon mitochondria. Can J Biochem 55: 1114-1117

3. Bygrave FL. AL Lehninger 1967 The affinity of mitochondrial oxidative phosphorylationmechanisms for phosphate and adenosine diphosphate. Proc Nat Acad Sci USA 57: 1409-1415

4. CATTERALL WA, PL PEDEasEN 1972 Adenosine triphosphatase from rat liver mitochondria. II.Interaction with adenosine diphosphate. J Biol Chem 247: 7969-7976

5. CstuSTENSEN HN 1975 In Don Mills, ed, Biological Transport, Ed 2. WA Benjamin, NewYork, pp 139-141

6. CIUsTsNsEN RO, A Loynm, E RACKE 1969 Effect of anions on oxidative phosphorylationin submitochondrial particles. Biochim Biophys Acts 180 207-210

7. CooPER C, AL LEHNINGER 1957 Oxidative phosphorylation by an enzyme complex fromextracts of mitochondria. V. The adenosinse triphosphate-phosphate exchange reaction. IBiol Chem 224: 561-578

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