PLANT PHYSIOLOGY

of synthetic growth regulator treatmenits on thelevel of free endogenous growth substances inplants. Ann. Botany N.S. 20: 439-59.

6. AUDUS, L. J. AND R. THRESH. 1956. The effectsof synthetic growth substances in the level ofendogenous auxins in planits. In: The Chemistryand Mode of Action of Plant Growth Substances.Wain and Wightman, ed. Butterworths, London.p 248-52.

7. KLEIN, A. 0. AND C. Wi. HAGEN, JR. 1961. Antlio-cyanin production in detached petals of I1iipatienisbalsauinina. L. Plant Physiol. 36: 1-9.

8. NOEL, R. 1951. Contribution a l'etud de la rhizo-genesis. Neoformation des racines dans les frag-ments de hypocotyles d'Im patiens balsamiiina L.cultives in vitro. Arch. Inst. Bot., IUniv. Liege,Belgique 21: 1-164.

9. STRAUSS, J. 1960. Anthocyanin synthesis in cornendosperm tissue cultures. II. Effect of certain

inhibitory and stimulatory agenits. Plant Phvsiol.35: 645-50.

10. THIMANN, K. AND Y. EDMUNDSON. 1951. Thebiogenesis of the anthocyanins. III. The roleof sugars in anthocyanin formation. Arch. Bio-chem. Biophys. 34: 305-23.

11. THIMANN, K. AND Y. EDMUNDSON. 1949. Thebiogenesis of the anthocyanins. I. General nutri-tional conditions leading to anthocyanin formation.Arch. Biochem. 22: 33-53.

12. THIMANN, K. AND B. RADNER. 1958. The bio-genesis of anthocyanin. VI. The role oi ribo-flavin. Arch Biochem. Biophys. 74: 209-23.

13. THIMANN, K. AND B. RADNER. 1955. The bio-genesis of anthocyanin. V. Evidenice of themediation of pyrimidines in anthocyanin svnthesis.Arch. Biochem. Biophys. 59: 511-25.

14. WHITE, P. R. 1943. A Handbook of Plant TissueCulture. Cattell Press, Lancaster, Pa.

Metabolic Processes in Cytoplasmic Particles of theAvocado Fruit VI. Controlled Oxidations

and Coupled Phosphorylations 1, 2, 3J. T. Wiskich4, Roy E. Young, and Jacob B. Biale

Department of Botany and Plant Biochemistry,University of California, Los Angeles

AIn explanation of the climacteric rise of the res-piration rate of ripening fruits has been sought bystudies of subcellular particles isolated fronm the fruit.It is known that in most plant tissues the rate of 0Ouptake can be increased by treating the tissue withuncoupling agents, suggesting that the rate of 03 up-take is limited by the rate of oxidative phosphoryla-tion. If during the process of ripening the restric-tion on oxidation exerted by phosphorylation wasrelieved, a stimulation of the rate of 03 uptake wouldbe expected. Thus, Millerd et al. (24) establishedthat whereas the respiration of avocado tissue slicesof preclimacteric fruit could be stimulated by applica-tion of DNP5, slices from the climacteric peak fruit

1 Received Aug. 6, 1963.2 A preliminary report of this work has appeared (31).3 This research was supported by Grant RG-8224

from the United States Public Health Service and by agrant from the Cancer Research Coordinating Committeeof the University of California.

4 Present address: Department of Botany, Universityof Adelaide, South Australia.

5 Abbreviations: CCP, carbonyl cyanide m-chloro-phenylhydrazone; DNP, 2,4-dinitrophenol; TPP, thiaminepyrophosphate.

were not stimulated by such treatment. From studiesof the isolated mitochondria and of the other fractionsthese workers concluded that there wvas a natuiral un-coupler present in the fruit. Several criticisnms havebeen offered against such an hypothesis (5), andPearson and Robertson (25) suggested that the cli-macteric rise was due to a higher turnover of thephosphorylation cycle. These explanations of the in-creased 0. uptake, involving changes in the control-ling influence of oxidative phosphorylation on themitochonclrial oxidations, are possible, but it is im-probable that the study of the isolated mitochonidriawill reveal the causes of the changes. Nevertheless,much valuable information has been obtained fromthese studies, and in particular, the properties of avo-cado particles have been thoroughly examine(I anddocumented (1, 5, 6, 24, 26).

Ronmani and Biale (26) observed that mitochon-dria isolated from ripe avocado fruit appeared to havean oxidative phosphorylation mechanism which wasinsensitive to DNP. Mitochondria isolated fronm un-ripe avocado fruit were sensitive to DNP, but theuncoupling effect observedl with these particle, was

312

WISKICH ET AL.-METABOLISM IN CYTOPLA\SMIC PARTICLES

reversed with hexokinase (6). Other studies (1)had substantiated the operation of a typical tricar-boxylic acid cycle in these preparations. Thus theeffects of DNP are of particular interest with respectto the mechanism of oxidative phosphorylation.Therefore it was considered necessary to reevaluatethe previous results obtained with mitochondria fromavocado tissue in the light of recent knowledge aboutthe properties of mitochondrial processes.

Since the properties of isolated mitochondria aremarkedly influenced by the isolation procedure (15,18) and since the pattern of behavior of the particlesisolated from avocado fruit showed variation (prob-ably seasonal), no attempt has been made to extendthese results to the physiology of the intact fruit.

Mitochondria isolated from climacteric-peak fruitwere used to study respiratory control and the effectsof DNP on oxidative phosphorylation. Possible ex-planations of the previously reported results (6, 26)are given and the effects of oxaloacetate on oxidationsby particles isolated from the avocado (2) have beenfurther examined.

Methods and Materials

Preparation of Mitochondria. Fuerte avocadofruit, Persea gratissiina., were chilled at 10 for an hourand grated with a kitchen type grater with 3-mmholes.

Grated tissue (100 g) was added to 300 ml of 0.4 M\sucrose containing 5 mm EDTA, 6 mm MgCl,, and4 mm cysteine. The mixture was ground in a mortarwith sand and the pH maintained between 7.0 and7.5 by dropwise additions of 1 N KOH. The brei wassqueezed through washed heavy muslin andl the fil-trate, diluted with 200 ml of 0.4 Ai sucrose containing6 mAt MgCl2, and was subjected to the following cen-trifugings: A) 1,500 X g for 15 minutes-precipi-tate (liscardled; B) 10,000 X g for 15 minutes-supernatant discarded; C) 2,500 X g for 10 minutes-precipitate discarded; D) 10,000 X g for 10 min-utes-supernatant discarded; E) 2,500 X g for 10minutes-precipitate discarded; F) 10,000 X g for10 minutes-supernatant discarded.

All operations were done at 0 to 10. After cen-trifuging, the upper layer of lipid material was re-moved by suction. The precipitates, during the prepa-ration, were suspended in 200 ml of 0.4 M sucrosecontaining 6 mit MgCl2 with the aid of a loose Pot-ter-Elvehjem-type Teflon homogenizer. The finalprecipitate was suspended in 3 ml of this medium andwas green. Replacing the sucrose with 0.5 M man-nitol in the washing procedure allowed an easier sepa-ration of the layers of the precipitates (29). Whenthis was done a brown and a green fraction could beobtained. Preparations isolated from both mannitoland sucrose were used in this work but no differencesin behavior could be detected.

One of the major problems associated with theisolation of these particles was aggregation. The useof a mortar instead of a blendor, the presence of

MgCl2 and the use of large volunmes oI suspendingmedium minimized the tendency of the particles toaggregate. Furthermore, any aggregatedl materialwas removed by low-speed centrifuiging (steps C andE above).

02 uptake was measured polarographically in asealed lucite vessel of 3.0 nil volume using a Clarkelectrode (Yellow Springs Instrumilenit Co.. Cleveland,Ohio), connected to a potentiometric recorder. Thestandard reaction medium contained 10 mM potas-sium phosphate buffer, pH 7.2, 10 miliI Tris-HCI buf-fer, pH 7.2, 5 mri MgCl2. 0.5 ni:t EDTA. an(d either0.25 M sucrose or 0.3 At mannitol. When a-ketoglu-tarate was used as a substrate, malonate -was includedto inhibit the oxidation of succinate.

Esterification of Pi was determiinied 1b ad(linghexokinase and glucose to the above mediumll, fromwhich aliquots were added to cold perchloric acid(final concentration 3 %), neutralizedl with KOH,the KClI4 centrifuged off, andI the glucose-6-P as-sayed by NADP reduction witlh crystalline glucose-6-P dehydrogenase (19).

The nitrogen content of the suspension was de-termined by distillation and titration after (ligestionin sulfuric acid using mercury as catalyst (23).ADP was determined by phosphorylation with

P-enolpyruvate and pyruvate kinase and determiningthe oxidlation of NADH on reduction of the pyruvatewith lactate dehydrogenase.

Fresh Fuerte avocado fruit were obtained fromCalifornia Avocado Growers. Avocados (1o not ripenon the tree and are in the preclimlacteric state whenpicked. The respiratory activity of individual fruitwas followed continuously with the ai(d of a paramag-netic type O2 analyzer (32). Climacteric peak fruitwere selected when they reached their maximuim rateof respiration.

Phosphoenolpyruvate, pyruvate kinase. lactate de-hydrogenase, glucose-6-P dehydrogenase. and organicacids were obtained from California Corporation forBiochemical Research. NAD, NADH. NADP, andhexokinase (type IV) were obtained from SigmaChemical Company. ADP, ATP and AMP wereobtained from Pabst Laboratories. Sodium am)vtalwas obtained from Eli Lilly and Company. Gifts ofoligomycin (Dr. H. A. Lardy) and carbonyl cyanident-chlorophenylhydrazone (CCP) (Dr. P. G. Heytler,E. I. du Pont de Nemours and Company) are grate-fully acknowledged.

Results

Oxidationl and Coutpled Phlosphorylationt. Figure1 shows a typical 02 electrode trace obtained withmitochondria isolated from avocado fruit which wereat the climacteric peak of respiration. It can be seenthat the oxidation of succinate was stimulated by ADPand that as ADP became limiting the rate of oxida-tion decreased. In accordance with Chance and Wil-liams (9), the rate of oxidation of mitochondria inisotonic medium with phosphate, substrate, and a

313

PLANT PHYSIOLOGY

definite amount of ADP added is referred to as state

3. The subsequent oxidation rate, after all of theadded ADP has been converted to ATP making phos-phate acceptor limiting, is state 4. Similar results

were obtained with a-ketoglutarate and malate as sub-strates, as shown in figure 2. The higher respiratorycontrol ratio, defined as the state 3 rate divided by thestate 4 rate, observed with a-ketoglutarate was attrib-uted to the substrate-level phosphorylation being more

tightly coupled than the electron transport chainphosphorylations. It was also observed, with mostpreparations, that a-ketoglutarate was not oxidizeduntil ADP was added, whereas some oxidation of bothmalate and succinate did occur in the absence ofadded ADP. This is also indicative of the strongcoupling between oxidation and phosphorylation at

4,

4,.

0-

n

0

4,

0.

_1Q.

DNP (pM)

FIG. 1. The oxidation of succinate by avocado mi-tochondria. Assayed in 2.7 ml of the standard mediumcontaining sucrose and 138 ,Ag mitochondrial nitrogen.Numbers on the trace are rates expressed as m,u moles02/minute. Mw indicates the addition of mitochondria.Additions are shown as final concentrations.

FIG. 2. Respiratory control with malate and a-keto-glutarate. Assayed in 2.6 ml of the standard sucrose

medium containing 180 gg mitoohondrial nitrogen. Forca-ketoglutarate 67 ,uM TPP and 8 mm sodium malonatewere included in the reaction mixture. Rates are ex-

pressed as mA moles 02/minute.

4

18mM

FIG. 3. The effect of DNP on the state 4 oxidationof succinate. Assayed in 2.6 ml standard mannitol med-ium containing 17 mm sodium succinate, 170 /AM ADPand 266 utg mitochondrial nitrogen.

FIG. 4. The effect of oligomycin on the oxidations ofsuccinate and malate. Assayed in 2.6 ml standard su-

crose medium containing 296 ,ug mitochondrial nitrogen.

314

WISKICH ET AL.-METABOLISM IN CYTOPLASMIC PARTICLES

the substrate-level site of a-ketoglutarate oxidation.An induction period required for maximal a-ketoglu-tarate oxidation can be seen in figure 2 (8).

When the particles were in state 4, the respirationwas stimulated by the addition of ADP or of an un-coupling agent. Figure 3 indicates that 10 ,M DNPhad stimulated state 4 oxidation of succinate and thatapproximately 30 ,uM DNP was optimal while higherconcentrations of DNP showed less stimulation. Itappears, therefore, that the avocado mitochondria iso-lated from climacteric-peak fruit can be no less sensi-

5

+46-M IDNP

- SUCC INATE

6 12 18Minutes

tive to DNP than other plant mitochondria (cf. 26,see 29).

An alternative explanation of the DNP effect isthe stimulation of an adenosine triphosphatase andthe recycling of ADP. This alternative is disprovedby the results of figure 4. Oligomycin, by inhibitingoxidative phosphorylation (21), lowered the oxida-tion rate to that of the state 4 rate, substantiating thatthe state 3 and state 4 changes are due to the couplingbeween phosphorylation and oxidation. Furthermore,figure 4 shows that DNP released the oxidation in-

6

24 30

87

lexokinase50 gm

FIG. 5. The phosphorylating activity of the avocado mitochondria. Assayed in 6.0 ml standard sucrose mediumcontaining 0.2 mg hexokinase, 17 mm glucose, 17 mM succinate, 0.2 mm ADP, and 256 ,Ag mitochondrial nitrogen.

FIG. 6. A comparison of indirect P/O determinations. Assayed in 2.6 ml standard sucrose medium. The mediumin which ATP was determined also contained 17 mm sodium succinate, 4 mm ADP, 0.1 mg hexokinase, 17 mM glu-cose. Rates are expressed as m, moles 02/minute.

FIG. 7. Oxidation of malate by avocado mitochondria. Assayed under the same conditions as outlined for tableII.

FIG. 8. Hexokinase activity of mitochondrial preparations. Assayed in 2.7 ml standard mannitol medium con-taining 175 Ag mitochondrial nitrogen. For ca-ketoglutarate oxidation 67 ,uM TPP was included in the reaction.Rates are expressed as m,u moles 02/minute.

0O.5r

0.4

0.31-

E0.21-

0.1 F-I X1 Xa I

L

X

315

PLANT PHYSIOLOGY

hibited by oligomycin. Under these conditions DNP-stimulated adenosine triphosphatase would be in-hibited by oligomycin (21) and could not have causedthe stimulation of oxidation. This inhibition of theDNP-stimulated adenosine triphosphatase by oligo-mycin was confirmed here. In one experimenit 67 uMDNP increased the rate of ATP hydrolysis by anavocado preparation from 11.5 to 15.0 ug P releasedper minute. The further addition of oligomycin (2,ug/ml reaction volume) re(luce(d this stimulated rateof ATP hydrolysis to 8.0.

In fact, figure 5 slhows that 46 am DNP com-pletely prevented the formationi of ATP with succi-nate as substrate. These experiments were repeatedwith CCP, which at 0.5 MM concentration was as ef-fective as 50 Mu\1 DNP. The ATP found in the pres-ence of DNP or in the absence of succinate was due tothe direct conversion of the added ADP by a(lenylatekinase activity. That adeinylate kinase wvas presentand that the formlation of ATP from ADP by thisenzyme could be inhibited by the addition of AMP isshown in table I. ATP formation by adenylate kinasewas inhibited 95 % when the AMP/ADP ratio was4/1. Since this was a direct assay of mitoclhondrialactivity, any oxiclation of NADPH by the prepara-tion -ouldl lea(l to errors. Parallel experiments

Table IThe Effect of AMP on the Formiiationt of ATP

by Adexylate KinaseAdenylate kinase was assayed in 2.7 ml of standard

sucrose medium containing 17 mm glucose, 0.2 mghexokinase, 0.3 mg crystalline glucose-6-P dehydrogenase,0.1 mms NADP and 239 Ag mitochondrial nitrogen at 200.

AMP/ADP A OD/mimi .0Inhibition01

4

0.2620.1040.0570.017

0617894

showed that NADPH was not oxidized by the mito-chondria.

It is possible to determine ADP/O ratios (10)from 02 electrode traces as shown in figure 6. Whenthis was done the values obtained were usually closeto the expectedl maximal values. However, whenglucose-6-P wvas estimlated the yield was much lessthan expected. Thus the values obtaiine(d from theelectrode traces were regarded with cauti,on. It waspossible that the ATP formiied in situ was not freelyaccessible to the miie(liumii (3, 4). Undcer these cir-cumstances, oxidation would be affectecl by the inter-nal ATP, ADP ratios and the glucose-6-P formcationby the accessibility of ATP to hexokinase.

Effects of Oxraloacetate. Avron and Biale (2)have demonstrated that oxaloacetate can inhibit thesucciniate oxidationi of avocado mlitochondria. Fur-thernmore, \Viskiclh andcl Bonnier (29 in studyingmitochondria isolaited fronii potato andI sweet l)otatofoun(d that freshly isolated lmlitoclhoid(lri-ia may requireATP before any significant rate ot succinate oxi(la-tion is realized. They conclu(le(d that this was (lue toan inhlibitioni of succinate dehvdlro-enase by oxalo-acetate. The freshly isolate(d avocado mitllchondriashowed a similar effect but not a. markedl. How-ever, it was found that witlh avoca(lo iitochoindIriathis inlibition was progressively stiolngei- as the pHof the isolationi mle(liumii was increas-edl fromn 7 to 8.

It was also suggestedI (29 that the progressivedecrease of the rate of nialate oxidl1ation (fig 2, 7) wasdue to the accumiiulation of oxaloacetate. Some evi-dence supporting this claimi wasQoltainedl froml theavocadlo particles. Each experimiielnt in table II repre-sents the (lata obtained frolmi onie O. electrode trace.The adlditionis. their order aln(d the suhse(lueint ratesare showni. Experiment 1 of table II confirmiis theinhibitory effect of arsenite oni a-ketoglutarate oxidla-tion (1, 22 ) an(l shows that tind(ler suich conditionsaddlition of glutamate ha(l little effect onl oxi(lation,indicative of little glutamiiate (lehvldrogen)ase activity.The latter point was colnfirmiiecl in experimiient 2 (tableII) -here there was lno oxidationi of glutalmate in the

Table II02 Uptake in Rclationt to

Initeractionts amitontg Glhtamzate, Ala tc, and O.raloacctateThe reaction mixture consisted of 2.6 ml of the standard manniitol mlediumii containinig 7/ mm malonate, 20 mM

glucose, 0.1 mg hexokinase, 0.38 mm ADP, and 200 ,ug mitochondrial nitrogen. Final concentrations of the added com-pounds, unless otherwise indicated, were: 17 mm a-ketoglutarate; 67 wm TPP; 3 mMi sodium arsenite; 17 m^s gluta-mate; 8 mm oxaloacetate; 17 mM malate. Rates are expressed as m,u moles 02/minute at 200.

Firstaddition

1 a-Ketoglutarate,TPP

2 Glutamate3 Glutamate,

TPP4 Oxaloacetate,

TPPMalate,

Arseniite

Rate Secondaddition

88 Arsenite0 Oxaloacetate

11 Oxaloacetate

0 GlutamateOxaloacetate

86 (1.7 mM)

ThirdRate addition

8 Glutamate12 Arsenite

43 Arseinite

52' Arseniite14 Glutamate 77

Expt. Rate

912

15

17

316

WISKICH ET AL.-METABOLISM IN CYTOPLASMIC PARTICLES

absence of TPP. The addition of oxaloacetate in-duced some oxidation but not enough to observe aninhibition by arsenite. However, if TPP were in-cluded in the medium (experiment 3, table II), glu-tamate was slowly oxidized and this oxidation was

markedly stimulated by oxaloacetate. Under theseconditions arsenite caused a substantial inhibition.Experiment 4 (table II) confirmed this observationand showed that oxaloacetate was not oxidized evenin the presence of TPP.

Therefore, it may be concluded that the avocadomitochondria contained little glutamate dehydro-genase activity but could oxidize glutamate by trans-amination with oxaloacetate to aspartate and a-keto-glutarate and oxidizing the latter product (20).

The marked inhibition of malate oxidation byoxaloacetate is shown in experiment 5 of table II, to-gether with the recovery of this oxidation by theaddition of glutamate. Very little of the recoveredoxidation could have been due to glutamate oxidation(either by dehydrogenase or transaminase activity)as arsenite was present and TPP was not included inthe reaction. The recovery of oxidation was not im-mediate but was progressively increasing, presumablydue to progressive removal of oxaloacetate.

The inhibition of malate oxidation with time andthe beneficial effects of glutamate are shown in figure7. Since the results of figure 7 were recorded withthe same preparation used to obtain the results of tableII, it is apparent again that little of the 02 uptake inthe presence of glutamate could have been due to theoxidation of glutamate itself. Malonate was includedin the above experiments to inhibit the oxidation ofsuccinate formed from the arsenite insensitive frac-tion of a-ketaglutarate oxidation.

Effects of Hexokinase. Biale and Young (6)showed that their preparations from avocado con-

tained hexokinase but not sufficient to transfer all ofthe Pi esterified into ATP. Since it seems unlikelythat hexokinase is a true component of mitochondria,an indirect test for its presence was made with thepreparation described here. Figure 8 indicates thathexokinase was present since the addition of glucosecaused an increase in the state 4 oxidation. However,this activity was insufficient to maintain the level ofADP necessary for state 3 respiration. Both a-keto-glutarate and succinate were used as substrates.

Thus respiratory control may be seen in prepara-

tions containing hexokinase (7) provided that therebe no phosphate acceptor present.

An Exanmination of the Effects of DNP on theOxidation of a-ketoglutarate. The pattern of a-keto-glutarate oxidation by the avocado particles has beendescribed. It was shown that this oxidation was de-pendent on ADP and it was concluded that duringstate 4 oxidation, the substrate-level phosphorylationwas rate limiting. Figure 9 shows that oligomycindid not lower the ADP-stimulated oxidation to thatof the state 4 rate. Thus the electron transport chainphosphorylations cannot be responsible for the very

low level of oxidation during state 4. The oxidation

inhibited by oligomycin (fig 9) could be recoveredby DNP or CCP.

The results of figure 10 show the effect of titratingthe state 4 oxidation of a-ketoglutarate with DNP.Two examples of experiments from which the datawere derived are shown in figure 11. An addition ofADP was necessary to elicit the maximum rate ofa-ketoglutarate oxidation (fig 2). In figure 10, theDNP stimulated rates are plotted as a function of theDNP concentration (solid line with closed circles).The solid line with open circles is a plot of the state4 rates just prior to the addition of DNP to showthat the state 4 rate did not change during the experi-ment. After the DNP rate was followed for approxi-mately 4 minutes, ADP was added again. In figure10 the dash line is the per cent stimulation of the DNPrate caused by the second addition of ADP. Approxi-mately 50 am DNP gave maximal oxidation ratesand that ADP further stimulated oxidation at alllevels of DNP tested. Thus the substrate-level phos-phorylation was the rate-limiting step before andafter the addition of DNP, and because of this no ap-parent inhibition of oxidation was observed with highconcentrations of DNP (cf. succinate in fig 3). Ithas been shown that a discrepancy exists in the appar-ent ADP used as calculated from the attainment ofstate 4 (02 electrode) and amount of ADP phos-phorylated (glucose-6-P assay). Since DNP doesnot uncouple the substrate level phosphorylation whichis rate limiting, DNP must be stimulating oxidationby making ADP available to the substrate level site.Thus, the stimulation of the state 4 oxidation of a-ketoglutarate by DNP may have been due either to astimulation of adenosine triphosphatase activity or toa more favorable ATP/ADP ratio at the active site(3, 4). The experimental pattern for the resultsshown in figure 10 can be seen in figure 11. Even inthe presence of DNP, state 3 to state 4 transitionscan be seen with ADP. This shows that the DNPeffect is not sufficient to provide an ADP supply tothe substrate level phosphorylation capable of main-taining a maximal oxidation rate. Furthermore, fig-ure 11 shows that ADP/O ratio decreases with in-creasing DNP concentration. This could be a reflec-tion of increased adenosine triphosphatase activitywith higher concentrations of DNP.

To decide some of these issues a careful studywas made of the effects of DNP in the presence ofoligomycin. Figure 12 shows that when oligomycinwas added during state 3 oxidation of a-ketoglutaratethere was an immediate small inhibition of oxidation(due to an inhibition of electron transport chainphosphorylations), which was maintained until ADPbecame rate limiting and the coupling of the substratelevel phosphorylation induced the state 4 rate. Oli-gomycin did not inhibit the substrate level phosphory-lation (11). Furthermore DNP did not stimulatethis state 4 respiration in the presence of oligomycin(fig 12). Thus the DNP stimulation of state 4oxidation with a-ketoglutarate was due to thestimulation of adenosine triphosphatase activity

317

PLANT PHYSIOLOGY

50

z

N0

Cl)

enw-J0

E

40 F

301

0 20 40 60 80

DNP (yM)

FIG. 9. The effect of oligomycin on the oxidation of a-ketoglutarate. Assayed in 2.7 ml of standard mannitol med-ium containing 64 AM TPP, 7 mM sodium malonate and 372 ,ug mitochondrial nitrogen. Rates are expressed as mAmoles 0,/minute.

FIG. io. The effects of DNP and ADP on the state 4 oxidation with ca-ketoglutarate as substrate. Assayed in2.8 ml standard sucrose medium containing 64 ,AM TPP, 7 mm sodium malonate, 17 mm sodium a-ketoglutarate and248 Atg mitochondrial nitrogen. The medium was made 0.32 mM in ADP and the system allowed to reach the state 4rate, then DNP was added followed by ADP again. 0 state 4 rate. 0 DNP-stimulated rate. * Final ADP rateas a percentage of the DNP rate.

FIG. 11. Polarographic tracings showing the effects of DNP and ADP on a-ketoglutarate oxidation. Assayed in2.7 ml standard sucrose medium containing 64 ,uM TPP, 7 mm sodium malonate and 248 ,ug mitochondrial nitrogen.Rates are expressed as m,u moles 02/minute.

FIG. 12. The effect of oligomycin on the DNP-stimulated oxidation of a-ketoglutarate. Assayed in 2.7 ml stan-dard mannitol medium containing 64 AM TPP, 10 mm sodium malonate and 200 Ag mitochondrial nitrogen. Ratesare expressed as mA moles 02/minute.

which would be inhibited by oligomycin. This was

confirmed (fig 12) by the immediate inhibition byoligomycin of the DNP-stimulated state 4 oxidationof a-ketoglutarate. The results of figure 9 showingthe expected relationship between oligomycin andDNP were obtained in the presence of excess ADP,

conditions under which the substrate-level phosphory-lation would not become rate limiting.

The ability of DNP to uncouple oxidative phos-phorylation even when a-ketoglutarate was used as

substrate is shown in figures 9 (by recovery of oligo-mycin inhibited oxidation) and 13 (by decrease in

318

10

I DN1~~~A ~~~~DN P

/ \\0 4 %

'PERCENT

0

STATE 410 0-0-O-Oar I I a

30000

200 4

a-za:

wi oo -

100

a-

0

4

X

100on- -

WISKICH ET AL.-METABOLISM IN CYTOPLASMIC PARTICLES

O - indicate a loss of coupling of the substrate level phos-phorylation or an increased adenosine triphosphatase

8e,.LM activity. The loss of oxidative phosphorylation withtime was observed with oligomycin, which inhibited

36ELM the state 3 respiration of a-ketoglutarate (fig 9) offreshly isolated particles. This inhibition of oxida-tion of a-ketoglutarate by oligomycin was lost fairly

iO -0OA rapidly without any apparent loss in the rate of oxida-tion. Thus the level of coupled oxidative phosphory-

144pMt lation was lowered to such an extent that it could notlimit the rate of a-ketoglutarate oxidation. The sta-

e* O bility of preparations made very late in the Fuerteavocado season (June) was low. Respiratory con-trol and coupling were lost very rapidly at 00. The

_______.______.______.______.______._____ reasons for this pattern of behavior are unknown.0 5 10 15 20 25

MINUTES

FIG. 13. The rate of ATP formation at different con-centrations of DNP. Assayed in 3.3 ml sucrose mediumcontaining 64 ,uM TPP, 10 mnM sodium malonate, 20 mmsodium a-ketoglutarate, 0.4 mm ADP, 20 mm glucose, 1mg hexokinase, DNP in concentration noted on the trace,and 108 Ag mitochondrial nitrogen.

glucose-6-P formation). A detailed study of thetime effect is shown in figure 13, where an apparentrecovery of phosphorylation, particularly at low con-centrations of DNP, is apparent. However, the re-sults- of figure 13 are shown as a percentage of thecontrols and the apparent recovery was due to a de-crease in the control phosphorylation rate, as seen forsuccinate in figure 5. Thus in manometric experi-ments with 10 ,uM DNP as used by Romani and Biale(26) any uncoupling of phosphorylation would notbe detected. It has been shown that the oxidativephosphorylation system of these avocado particles wasrelatively unstable even at 00. Thus, we expect thatas oxidative phosphorylation decreased the sensitiv-ity of the system to DNP also decreased.

Stability. During the course of most of this in-vestigation the avocado mitochondria showed mod-erate stability when maintained at 00. Table IIIshows the increase of the state 4 respiration that oc-curred with a-ketoglutarate and the consequent de-crease in the respiratory control ratio. These results

Table IIIThe Loss of Respiratory Control of Mitochondria

after Storage at 00The reaction mixture contained 2.6 ml of the standard

sucrose medium containing 7 mm malonate, 0.38 mm ADP,67 /M TPP, 17 mm a-ketoglutarate, and 180 Ag mitochon-drial nitrogen. Rates are expressed as m, moles 02/minute at 200.

Minutes at 00

0 20 40 60 180

State 4 rate 15 16 17 22 34Respiratory control ratio 5.1 4.8 4.7 3.8 2.5

Discussion

Plant mitochondria showing the property of res-

piratory control have been isolated from sweet potatoand white potato (29) and from cauliflower bud (7).Wiskich and Bonner (29) described some of the con-

ditions which were critical in obtaining preparationsshowing respiratory control. Apart from the factorswhich mask respiratory control (e.g., ATP-hydrolyz-ing enzymes) and which may be precipitated withthe mitochondria (7), little is known about the con-

ditions necessary for isolating particles showingrespiratory control. It is certain that the structuralorganization, at all levels, of the mitochondrion playsa major role (12). Thus plant tissues with theirvariable composition and need of severe treatmentfor disruption of the cells have yielded mitochondrialpreparations with inconsistent properties and a lackof respiratory control. The differences in propertiesmay be a reflection of small changes in structure (30)and are probably correlated with the conditions used(13).

Whereas it was possible to prepare ADP con-trolled mitochondria from avocado fruit at theclimacteric peak when tissue slices show no response

to DNP (24), the same procedure failed to yieldADP controlled mitochondria from preclimactericfruit [tissue slices from which respond to DNP (24) ].This failure may have been because only those cellsof the preclimacteric fruit subjected to the highershearing forces were disrupted. A direct conse-

quence of this, and perhaps the cause of the lack ofevidence of respiratory control, may be the observa-tion of Avron and Biale (6) that mitochondria iso-lated from preclimacteric avocado fruit possess a more

active adenosine triphosphatase than mitochondriaisolated from climacteric peak fruit. Since the res-

piratory rate of the parent plant tissue is, in mostcases, limited by oxidative phosphorylation and there-fore stimulated by DNP, the property of respiratorycontrol in the mitochondria prepared from such tissueis essential for critical investigations. However, a

mere stimulation of mitochondrial oxidations by ADP(14, 16, 28) is not necessarily indicative of respiratorycontrol (8). This effect may be due to a low level ofendogenous adenylate (and may be indicative of

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PLANT PHYSIOLOGY

coupling) or to other reactions not related to respira-tory control (29).

The Climnacteric Rise of Respiration. Since theclimacteric peak fruit yielded ADP controlled par-ticles, it could be claimed that DNP failed to stimulatethe oxidation rate of clinmacteric peak avocado slices,Inot because of endlogenous uncoupling (24) but be-cause of a higher turnover rate of endogenous phos-phate or phosphate acceptor (25). However, such ap)hysiological interpretation of these results is notwarranted because it cannot be applied to results ob-taiinecl with preclimacteric peak avocado slices andwith nmitochondria isolated therefrom. The interpre-tation of results obtained with isolated plant mito-chondria requires a precise definition of the conditions,particularly if the overall activity of the preparationhas not been investigated.

From the results reporte(l here it appears that themiiitochondria of intact, climacteric peak fruit have notlost their ability for oxidative phosphorylation andthat oxidation can be controlled by phosphate or phos-phate-acceptor level. If endogenous uncouplers occurin the supernatant fraction of a mlitochon(drial prep-aration from avocado fruit (24), it remains to beestablished that theY are effective in situ. It is un-likely that oxidative phosphorylation is the onlymechanism which controls the turnover of the Krebscycle.

It is generally considered that cellular oxidationcan be stimulated by increased turnover of ATP.However, it is interesting to speculate whether cell-ular oxidation can be stimulated by a reversible un-coupling, a reversible activation of a specific ATP-hydrolyzing enzyme or by activation of nonphos-phorylating oxidation pathways. The studies re-ported here are quite inaclequate for a resolution ofthese possibilities. This is particularly important inplant tissues where it has been shown that oxidationsinsensitive to respiratory chain inhibitors can occur,and that in some instances the supernatant fraction isalso capable of presumably nonmitochon(drial oxida-tions (15).

Uncoupling by DNP. In contrast with earlierreports (26) it has been established that DNP canuncouple the oxidative phosphorylation of mito-chondria prepared from climacteric peak avocadofruit. The failure by Romani and Biale (26) todemonstrate this effect is explained by a combinationof the use of a relatively low concentration of DNP,and the unstable oxidative phosphorylation of avocadomitochondria.

Figure 13 shows that 18 uAM DNP has relativelylittle effect on the total amount of phosphate esterifiedafter 25 minutes incubation, as is usual with mano-metric experiments. This apparent recovery ofcoupling in the presence of DNP is due to the progres-sive decrease of rate of phosphorylation in the control.A similar effect has been observed with rat liverniitochondria (27). Higher concentrations of DNPproduced a more marked effect. The apparent con-tinual decrease in phosphate esterification with in-creasing concentrationls of DNP can be taken to in-

dicate a high (legree of electron tranisport chainphosphorylation with little substrate level phosphory-lation. Hovever, a more probable explanation is theincreased adenosine triphosphatase activity with in-creasing DNP competing more withl hexokiinase forthe ATP. The effects of DNP on the state 4 oxida-tion of a-ketoglutarate (fig 10, 11) have been showvnto be due to the stimulation of adenosine triphospha-tase activity). Furthermore, it has been show,vn thatthe DNP stimlulated adenosine triphosphatase is sensi-tive to oligomycini (21), allowing a clifferentiationbetween effects on the electron transport chain phos-phorylation andcl on substrate level plhosplhorylationis,the latter being insensitive to both DNP and( oligomy-cin (11). Preliminiary experiments with a potentuncoupling agent (CCP) described by Heytler et al.(17) duplicated the effects of 50 ,uti\m DNP at a coni-centration of 0.5 Aum.

P/O Ratios. High P/O ratios have not beenobtained with avocado mitochondria. The presenceof high concentrations of EDTA or of bovine serumalbumin (5) did not appear to offer any protection.In fact, ain absolute requirement for EDTA in theisolation medium was not establishedl. The P'/0ratios observed here by indirect calculation fromii the02 electrode data of ADP-controlled preparations ap-proached the expected maximum. Hovever, it isnot necessary to have a high (legree of coupling forwell controlled imiitoclhondria if compartmiientation ofATP at the active site occurs (3, 4). The controlshown by this type of information may be due to theproperties of only one of the phosphorylation sites.Thus the very low rate of state 4 oxidationi of a-keto-glutarate was clue to the controlling influence of thesubstrate level site only. None of the electron trans-port chain plhosphorylations was capable of exertingsuch a strong restriction on oxidation as evidlenced bythe experiments with oligomycin (fig 9). Oligo-mycin has been shown to inhibit the oxidation ofmalate to a rate similar to the state 4 rate (fig 4),thus establishing the effectiveness of oligomycin.

The discrepancy between the indirect calculationof P/O ancl the anmount of glucose-6-P formed stillrequires explanation. It was shown that 50 yM DNPdid not stimulate the state 4 oxidation of a-ketoglu-tarate to the state 3 ratio (fig 11). Furthermore, 100,M DNP was no more effective in stimulating state4 oxidation of a-ketoglutarate than was 50 j}A DNP(fig 10). On the other hand, hexokinase (50 tigl2.7ml reaction volume) did stimulate the state 4 oxida-tion of a-ketoglutarate to the state 3 rate (fig 8).This amount of hexokinase was capable of a higherADP turnover than was the maximum DNP-stim-ulated adenosine triphosphatase activity. Moreover,the hexokinase stimulated oxidation was maintained.Therefore, the reason for the discrepancy is probablynot due to compartmentation of ATP (3, 4) nor to apenetration barrier between ATP and hexokinase.Although some results suggesting penetration bar-riers have been obtained, they have not been subjectedto a detailed investigation. The presence of phos-phatases or some other enzymes as contaminants of

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WISKICH ET AL.-METABOLISM IN CYTOPLASMIC PARTICLES

the preparation would also reduce the yield of glucose-6-P. Such contaminating enzymes are pertinent tothe reversal of the DNP effect by hexokinase (6).Further studies are necessary before these problemscan be resolved.

An alternative possibility is that the endogenousadenosine triphosphatase of these particles is recy-cling ADP at a rate slower than the rate of ADPphosphorylation. Thus once the bulk of the addedADP is phosphorylated the oxidation rate wvould de-crease to the level where rates of hydrolysis and offormation of ATP were equal. Respiratory controlwould be evident and yet a significant amount of ATPmay not be available to hexokinase for glucose-6-Pformation. However, under these conditions it wouldbe expected that the oligomycin inhibited oxidationrate wvould be much less than the state 4 rate. Thisexpectation was not realized for the oxidation ofeither succinate or malate in the presence of oligo-mycin (fig 4). Thus, convenient but indirect meas-urements of P O as offered by the O.2 electrode tech-nique must be verified witlh direct assays (see also10).

The Kinetics of llalate Oxidation. W\iskich andBonner (29) observed the progressive dlecrease in therate of malate oxidation and suggested that this wasdue to product accumulation. Attempts to removethe oxaloacetate were unsuccessful. A similar pro-gressive decrease in the rate of malate oxidation wasobserved here.

It was established that very little oxidation wasdue to glutamate (elehydrogenase alone, and that oxalo-acetate stimulated glutamate oxidation through anarsenite-sensitive pathway (table II). Thus it isclaimed that a transamination is involved yieldinga-ketoglutarate and aspartate (20) and that the oxy-gen uptake is due to the oxidation of a-ketoglutarate.In the presence of glutamate and arsenite the oxida-tion of malate was maintained, presumably due to theremoval of oxaloacetate. Very little of this oxida-tion was due to substrates other than malate.

Summary

Particles showinig respiratory control as evidencedby stimulations of oxidation on successive addi-tions of adenosine diphosphate have been isolated fromripe fruit of the avocado. Dinitrophenol has beenshown to uncouple the electron transport chain phos-phorylations of these particles. Failure to observethis in previous studies has been due to a combina-tion of factors associated with the particles and withthe assay technique.

It has been established that dinitrophenol stim-ulates an adenosine triphosphatase activity which issensitive to oligomycin, and that neither dinitrophenolnor oligomycin affect the substrate-level phosphory-lations. The effects of 50 gM dinitrophenol wereduplicated by 0.5 iM carbonyl cyanide mn-chloro-phenylhydrazone.

It has been shown that oxidative phosphorylationis unstable in these preparations and that substrate

level phosphorylations are more tightly coupled andmay account for most of the adenosine triphosphateformed. This condition explains the observation ofgood respiratory control with low P/0 ratios.

Product inhibition of malate oxidation has beenconfirmed. This inhibition was overcome by theaddition of glutamate and arsenite. Transaminationoccurred between glutamate and oxaloacetate and ar-senite inhibited the oxidation of a-ketoglutarate. Itwas established that in the avocado particles littleglutamate oxidation operated via a direct glutamatedehydrogenase pathway.

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