Plant Physiol. (1970) 46, 25-30

Preparation and Some Properties of Submitochondrial Particles

from Tightly Coupled Mung Bean MitochondriatReceived for publication May 23, 1969

S. B. WILSON AND W. D. BONNER, JR.Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania 19104

ABSTRACT

Osmotic shock was found to be better than freezing andthawing, a French press, or sonic oscillation for the prepa-ration of submitochondrial particles from mung bean(Phaseolus aureus) hypocotyl mitochondria. Particles pre-pared by osmotic shock rapidly oxidize reduced nicotina-mide adenine dinucleotide and succinate, but they oxidizemalate slowly. NADH oxidation was slightly stimulated bycytochrome c, ATP, and ADP; succinate oxidation wasmarkedly increased by ATP, slightly by ADP and cyto-chrome c; and malate oxidation required the addition ofNAD+ NADH oxidation is inhibited weakly by amytal, com-pletely by antimycin A and KCN, but not by rotenone.Chlorsuccinate, malonate, antimycin A, and KCN inhibitsuccinate oxidation. The action of antimycin A and KCN isincomplete, while chlorsuccinate and malonate were com-petitive inhibitors. Antimycin A combined stoichiomet-rically with particle protein in the ratio of 0.23 millimicro-mole per milligram of protein.Oligomycin and bis(hexafluoroacetonitryl) acetone, a po-

tent uncoupler of oxidative phosphorylation, were withouteffect on oxygen uptake but did influence the ATP-stimu-lated onset of respiration when succinate was substrate.Fresh particles were markedly inhibited by oxtylguanidine,indicating energy conservation, but this inhibition de-creased on storage of the particles.Spectra show the presence of cytochrome components

the same as those of the intact mung bean mitochondrion,but present at higher concentrations. The molar concen-trations of the particle cytochromes were two to three timesthose of the intact mitochondrion and the molar ratios werecalculated as 0.9:1.0:1.0:2.8 for cytochromes a:b:c: flavopro-tein, respectively.

Submitochondrial particles prepared from animal mitochon-dria have been found to show many of the properties and func-tions of the intact mitochondria and have been extensively studiedin elucidating processes of electron transport and phosphoryla-tion (cf. 14, 16).Ikuma and Bonner (8) have described a procedure for the

production of tightly coupled mitochondria with reproduciblecharacteristics from dark-grown mung bean hypocotyls (Phaseolusaureus var. Jumbo). These mitochondria have been characterizedwith respect to malate, NADH, and succinate as substrates and

1 This work was supported by a grant from the National ScienceFoundation.

to a range of uncouplers and inhibitors of electron transport andphosphorylation (8-10, 17). Studies of the spectral properties ofthe electron transfer components have been reported (11).

This paper compares four methods of producing submito-chondrial particles and describes a simple method which con-verts mung bean mitochondria to submitochondrial particleswith reasonable yield. The particles produced have a relativelyhigh activity for the oxidation of substrates and can be stored at-15 C and still retain much of their activity. The apparentMichaelis constant (Km) for malate, succinate, and NADHhave been measured together with the inhibition by examples ofeach of the four classes of inhibitors previously characterizedwith intact mung bean mitochondria. Malonate and chlorsuc-cinate were used as inhibitors of succinoxidase, amytal, androtenone to inhibit NADH-cytochrome C reductase. AntimycinA was used as an inhibitor of electron transport between cyto-chromes b and c, and cyanide as an inhibitor of cytochromeoxidase.

Octylguanidine was used as an inhibitor of energy transferacting close to the respiratory chain (15, 8), 1799,2 as an un-coupler of energy transfer from electron transport, and oligo-mycin as an inhibitor of phosphorylation.Accompanying spectra show that the submitochondrial

particles have electron transfer components similar to those ofthe intact mung bean mitochondria.

METHODS

Initially mitochondria were isolated from dark-grown mungbean hypocotyls 5 to 6 days old as described earlier (8). Later,increased yields of mitochondria were obtained by disruptingthe tissue for 5 sec with a Polytron mixer instead of hand grind-ing.For a comparison of preparative methods, submitochondrial

particles were made in a disruption medium containing 0.25 Msucrose, 5 mM ATP, 1 mm EDTA. All operations were carriedout at 4 C with freshly isolated, tightly coupled mitochondria.

Five cycles of freezing and thawing were carried out by freezingthe mitochondria suspended in the sucrose medium with liquidnitrogen, then thawing in cold water.Two treatments with a French Press at 8000 p.s.i. were per-

formed with an Aminco French pressure cell.Ultrasonic treatment for 1.5 min at 4 amp was given at 30-

sec bursts with a 1-min break between each burst with a BransonSonifier model LS-75.The osmotic shock treatment consisted of slowly adding cold

glycerol to a suspension of the mitochondria until the finalglycerol concentration was about 80%. After the mitochondriawere allowed to soak in this medium for 30 min, the suspensionwas injected with a hypodermic syringe fitted with a No. 18

2 Abbreviations: 1799: bis(hexafluoroacetonitryl) acetone; BSA: bo-vine serum albumin.

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WILSON AND BONNER

Table I. Comparison of Submitochonidrial Particles Produced bySeveral Methods

Oxygen ConsumptionAMethod Yield

Succinatel NADH Malate

Freezing and thawingFrench pressSonifier

Osmotic shock

Improved osmotic shock

Very lowVery goodGood freshFrozenModerate freshFrozenGood freshFrozen

Mung bean mitochondrial FreshFrozen

mrAmoles/min-e*g protein

ND'Nil46236852413111511

'ND NDND ND98 Nil67 Nil149 Nil86 1Nil555 16.0255 4.6280 8737 2.3

I Not determined.

needle into 20 volumes of a rapidly stirred disruption me-dium.

In the improved osmotic shock method, subsequently adoptedas routine for preparing submitochondrial particles, the sucrosedisruption medium was replaced by 0.1 % BSA and 1 mM EDTAadjusted to pH 7.2 with 0.1 M KOH. The particles producedappeared similar to those prepared in the sucrose medium.

After disruption of the mitochondria by one of the abovemethods, any remaining intact mitochondria were removed bycentrifugation at 10,000g for 15 min, and the submitochondrialparticles were isolated at 100,000g for 60 min. The isolatedparticles were washed by resuspension in wash medium (0.3 Mmannitol, 0.1% BSA, 1 mm EDTA, pH 7.2) and reisolated at100,000g. Further particles could be recovered by resuspendingthe mitochondrial residue in wash medium and centrifuging at10,000g and 100,000g. The isolated particles were either used forexperiments or stored at -15 C until required.

Table 1 summarizes the results obtained from the differentmethods used for mitochondrial rupture, and Figure 1 showsthe flow sheet for the improved osmotic shock method. Proteincontents were determined by the Lowry procedure (13) with BSAas the standard.Oxygen uptake was measured by the polarographic method

with a Clark-type oxygen electrode (Yellow Springs InstrumentCo.) in Lucite chambers of 1- or 3-ml capacity stirred by a mag-netic stirrer. Oxygen tension was recorded with a linear recorder,and respiratory rates were calculated from the traces on thebasis of 240 ,lM oxygen in the aerated medium.The Michaelis constant, Km, for succinate, malate, and NADH

was determined from the rate of oxygen consumption after suc-cessive small additions of the substrates to the reaction mixturegiven below. NADH solutions were standardized spectrophoto-metrically at 340 nm with 6.22 as mm extinction coefficient (3).Because of the low malate dehydrogenase activity, only succinateand NADH were used as primary substrates to study the effectsof inhibitors. Inhibition by malonate, chlorsuccinate, amytal,rotenone, antimycin A, KCN, and octylguanidine was measuredby following the oxygen uptake for at least 1 min after successivesmall additions of the inhibitor (dissolved in water or ethanol andadjusted to approximately pH 7.2). The inhibitor constant, Ki,was calculated according to the method of Dixon (6). Molecularweights of 153, 250, 394, and 500 were assumed for chlorsuc-cinate, amytal, rotenone, and antimycin A, respectively.The basic reaction medium contained 0.3 M mannitol; 10 mM

KCl; 10 mm potassium phosphate buffer, pH 7.2; 5 mM MgCl2.After the addition of submitochondrial particles, 10 mm suc-

Fresh Mitochondrial Suspension

Soak in 80% Glycerol 1/2 hour

Osmotic shock by injection into 20-30 volumes disruption medium

Centrifuge 10,000g 15 mins

L

Pellet Mitochondriol residueResuspended in wash mediumCentrifuge 1O,OOg 15mins

IL

Pellet discard9% original protein

SupernatantCentrifuge 100,000g 60mins

J-

Pellet SupernotanResuspend in wash medium discord

Supernatant 1

Centrifuge 100,000g 60mins

Supernatont Pelletdiscard Submitochondrial particles

Resuspend in wash mediumor Store at -15°C23% original protein

FIG. 1. Improved osmotic shock technique of the production of sub-mitochondrial particles; carried out at 0 to 4 C. The final distributionof protein is shown. Disruption medium 0.1% BSA, 1 mm EDTA,pH 7.2. Wash medium 0.3 M mannitol, 0.1% BSA, 1 mm EDTA,pH 7.2.

cinate or 1 mm NADH, 0.2 mm ATP, and 2 mm cytochrome cwere added to initiate respiration. When malate was used assubstrate, the basic mixture and submitochondrial particles weresupplemented with 20 mm malate, and 2 mm NAD+.

Difference spectra were recorded at liquid nitrogen tempera-tures with a split beam spectrophotometer (3). When necessary,spectra were replotted on a flat base line with isosbestic pointsat 615, 530, and 450 nm (5). Concentrations of respiratory com-ponents were calculated by the method of Chance and Williams(4). Wave length pairs were modified to take account of thefact that the absorption peaks of plant respiratory carriers do notcoincide with those of animal carriers (11).

Chlorsuccinate was kindly donated by Dr. E. Racker of Cor-nell University, octylguanidine sulphate by Dr. B. Pressman ofthe Johnson Research Foundation, and 1799 by Dr. P. Heytlerof DuPont de Nemours, Wilmington, Delaware.

RESULTS

Critique of Production of Submitochondrial Particles. Themethods are compared for their ability to produce a reasonableyield of particles with high rates of oxidation of succinate andNADH which could be retained after overnight storage at -15 C.The osmotic shock technique was superior in the specific ac-tivity of succinate and NADH oxidase and further developedto improve yield. Unlike the intact mitochondria, the particlescould be stored at -15 C and still retain much of their activity.It was also possible to store the glycerol suspension of mito-chondria for up to a month at -15 C and subsequently producevery active particles. The energy-linked functions which havebeen described elsewhere (18) were always rapidly lost onstorage. Table I shows the ability of fresh and frozen submito-chondrial particles, produced by the improved osmotic shockmethod, to oxidize NADH, malate, and succinate comparedwith the activities of fresh and frozen mung bean mitochondria.

Substrate Requirements for the Oxidation ofNADH, Succinate,and Malate. Particles produced by the improved osmotic shockmethod could oxidize NADH and were further stimulated byadded cytochrome c and ATP (Fig. 2A). There was no oxidation

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MUNG BEAN SUBMITOCHONDRIAL PARTICLES

ImM NADH

SM

I

0.2mM ADPTt2T9Cyt.c

24 M 02 \02mMA

-IF0OmM Succinote lOin

; 0.2mM ADP SMPSMP

60sec0.2mM ATP

24OMM 02

2~Lg Cyt.c

(B

20mM Malote0.2mM ATP

SMP;

0.2mM NADI

0.2mM ADP

M Succinote0.2mM ATP

2 g9 Cyt.c

0.2mM ADP

FIG. 2. Substrate requirements of respiration of fresh and aged(24 hr at -15 C) submitochondrial particles (SMP). Oxygen electrodetraces showing: A: NADH oxidation; B: malate oxidation; C: suc-cinate oxidation, fresh particles; D: succinate oxidation, particles aged24 hr at -15 C.

LA -mg9lOmM Succinote

I 0.2mM ADP

SMP

02mM ATP

I24P1A

IOmM Succinate T

02mM ATPSMP

20M1M 1799

c

(B +5mM Mg++lOmM Succinote

\l-

M 02 2,ig Oligomycin

lOmM Succinote

l 0.2mM ATP

2Esg Oligomycin

U)

+5mM fMiKA,+OmM,V, 60 -

FIG. 3. Oxygen electrode traces showing the effect of magnesium1799, and oligomycin added to submitochondrial particles oxidizingsuccinate: A: without MgCl2; B: with 5 mm MgCl2, 2 ,g of oligomycinadded after 0.2 mm ATP and 0.2 mM ADP; C: with 5 mM MgC12,20 ,g of 1799 added prior to adding 0.2 mm ATP; D: with 5 mMMgCl2, 2,ug of oligomycin added prior to adding 0.2 mM ATP.

of malate without the addition of NAD+ (Fig. 2B). Succinateoxidase was greatly stimulated by added ATP after a slight lagand slightly stimulated by cytochrome c (Fig. 2C). ADP slightlystimulated both NADH and succinate oxidation, but respiratorycontrol was not observed. Omission of Mg2+ from the reactionmedium prevented the ATP stimulation of succinate oxidation(Fig. 3).Values of the Km apparent for succinate, malate, and NADH

oxidation by the submitochondrial particles are shown in TableII, together with the values for apparent Ki for malonate andchlorsuccinate compared with values obtained with intact mito-chondria. Both malonate and chlorsuccinate behaved as com-

petitive inhibitors.Effect of Inhibitors on Uncouplers of Energy Transfer. Sub-

mitochondrial particles oxidizing succinate or NADH in thepresence of ATP were unaffected by 1799, a potent uncoupler ofoxidative phosphorylation. The addition of 1799 prior to the

addition of ATP produced a slow increase in respiration (Fig.3), but not as large as that observed on the addition of ATP. Ithas been observed (9) that uncouplers exert their maximal effecton intact mitochondria only when the mitochondria have beenexposed to ATP.

In agreement with the observations of Lardy et al. (12) withmammalian submitochondrial particles, oligomycin up to 4 yg/mlwas without effect on oxygen consumption by mung bean sub-mitochondrial particles when either NADH or succinate was

substrate in the presence of ADP. Oligomycin prevented the risein respiration occurring when ATP was added in the presence ofsuccinate (Fig. 3).The effects of octylguanidine on NADH and succinate oxida-

tion of fresh particles and on succinate oxidation by particlesstored 24 hr are shown in Figure 4. The half-maximal inhibition offresh particles occurred at 0.64 mm for NADH oxidation and 1.12mM for succinate oxidation compared with values of 0.36 and0.68 for NADH and succinate oxidation, respectively, by intactmitochondria (17). Stored particles lost much of their octyl-guanidine sensitivity in a manner analogous to that observed forintact mitochondria.

Effects of Inhibitors of Electron Transfer. Both malonate andchlorsuccinate inhibited succinate oxidation by submitochondrialparticles, the values of the inhibitor constant Ki for malonatebeing in agreement with that observed for intact mitochondria(10) (Table H). Chlorsuccinate, however, had a value of K.higher for the particles than for the mitochondria. Unlike mam-malian submitochondrial particles, mung bean submitochondrialparticles did not oxidize chlorsuccinate.Rotenone up to 90 ,M was without effect on the submitochon-

drial particles oxidizing NADH. Amytal was also relatively inef-fective (Fig. 5); the half-maximal inhibition occurring at 7 mmcompared with 2 to 2.5 mm for intact mitochondria with malate as

substrate. The inhibition by amytal, however, varied betweenpreparations. Because of the poor response to these inhibitorswhen NADH was being oxidized, no attempt was made to studytheir effect on succinate oxidation.

c Succ. Oxid. Aged Particles

0 Succ. Oxid. Fresh Particles

* NADH Oxid. Fresh Particles

,20 DL- 80-

100-,v0 2.0 4.0 60

Octyl Guanidine Concentration (mM)

FIG. 4. The effect of octylguanidine on the respiration of submito-chondrial particles supplied with succinate or NADH as substrate.Particles fresh and aged (24 hr at -15 C).

Table II. Km and Ki Values for Mung Bean SubmitochondrialParticles anid Mitochondria

Km K1

Succinate Malate NADH suCchirat Malonate

X 1OMX X 10'M

Submitochondrial 0.44 3.0 0.07 0.73 0.14particles

Mitochondria 0.4 2.0 0.07 0.50 0.13

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WILSON AND BONNER

cytochromes a and a3 (11), a large multiple band at 561 to 547nm due to cytochromes b and c (11), and a large band at 515nm with a Soret peak around 420 nm. Room temperature spectrashow a shift of a few nanometers toward the red end of the spec-trum compared with those produced at -190 C. Comparison oflow temperature NADH-reduced-oxidized and dithionite-re-duced-oxidized spectra (Fig. 9) shows that a high percentage ofthe various cytochrome components can be enzymically reducedcompared with intact mitochondria, which contain a componentwhich can only be reduced by dithionite (11). The proportion ofthe respiratory pigments which could be enzymically reducedvaried depending on the age and condition of the particles. Fromroom temperature spectra the concentrations of the respiratorypigments were calculated to be 0.3, 0.37, 0.32, and 0.97 m,umoles/mg protein corresponding to molar ratios of 0.9:1.0:1.0:2.8 forcytochromes a + a3, b, c, and flavoproteins, respectively. Theconcentrations are approximately two to three times those re-ported earlier for intact mung bean mitochondria (11), a feature

Amytol Concentration (mM)

FIG. 5. Inhibition by amytal of respiration of submitochondrialparticles oxidizing NADH.

A ~~~~~[B (NADH Oxidation

0- 2mgo 20- \ mg

05mg

69 0- 0,25mg

2-80-

100O G5 lb

Succinote Oxidotior

-

0 05

Antimycin "A Concentration 81M

0

Substrate

C: - Succ< 0

4- NADH

-F

,- O2- Slope/ 023mLmole

-C;) /mg Protein

D i 0 20

Protein mg

FIG. 6. The effect of antimycin A on the respiration of submito-chondrial particles supplied with succinate or NADH as substrate.A: NADH oxidation at various protein concentrations; B: succinateoxidation at various protein concentrations; C: plot of antimycin con-

centration causing half-maximal inhibition of respiration and proteinconcentration.

Figure 6 shows that the effect of antimycin A on the submito-chondrial particles varied somewhat with batches, but was similarto that found for intact mitochondria, the inhibition being de-pendent on the concentration of submitochondrial particles. Theslope of a plot of half-maximal inhibition concentration againstprotein concentration was found to be 0.23 m,umole/mg ofprotein, similar to that observed for intact mitochondria. Valuesfor the half-maximal inhibition were identical for both succinateand NADH, indicating that a similar antimycin A site is utilizedfor the oxidation of both inhibitors. Succinate oxidation, how-ever, is incompletely inhibited by antimycin A.Cyanide inhibited NADH oxidation almost completely, but the

half-maximal concentration varied somewhat between batches.For the example used in Figure 7, this was 11 ,UM NADH, close tothat observed for the intact mitochondria. Succinate oxidationwas also variable in its response to KCN but was always lesssensitive to KCN inhibition (half-maximal 19 ,UM), with 15 to 20%of the respiration being insensitive to high concentrations of theinhibitor.

Spectral Properties of Submitochondrial Particles. Submito-chondrial particles prepared from mung bean mitochondriapossess properties similar to those found in the intact mito-chondria. Low temperature spectra show considerable enhance-ment and improved resolution compared with those at room

temperature (Fig. 8). Reduced-oxidized spectra measured at lowtemperature, -190 C, have a sharp peak at 598 nm attributable to

c

0

-0

C:

0)

a)

av

* Succ. Oxid.* NADH Oxid.

50 IC

KCN Concentration(LM)

FIG. 7. The effect of cyanide on respiration of submitochondrialparticles supplied with NADH or succinate. Above: Succinate; below:

NADH.

,LODD - 05

427

1

\ ~~~~~518

t / /

450 50AOD500

450 500 550 600

B

x(nm)FIG. 8. Difference spectra of submitochondrial particles; dithionite-

reduced-aerobic (oxygen-treated). A: Measured at -190 C, 3 mm lightpath; B: measured at room temperature, 10 mm light path.

0*4

20-

C:0

. 40-C:

0c 60-

a)c0-

80-

1000 20

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MUNG BEAN SUBMITOCHONDRIAL PARTICLES

TAO.D.=-005\.00

517 560

,525I

S2°- Ox

---NADH Anaerobic - Ox.

500 550 600

X(nm)

FIG. 9. Difference spectra of submitochondrial particles measured at

-190 C with a 3 mm light path. Continuous trace: dithionite-reduced-aerobic (oxygen-treated); broken trace: reduced by 1 mm NADH-aerobic (oxygen-treated).

400 450 500 560 600

X(nm)

FIG. 10. Difference spectra of submitochondrial particles measuredat -190 C with a 3 mm light path. A: Reduced by 1 mm NADH +0.2 mm KCN and oxygen-treated; 1 mrm NADH-aerobic (oxygen-treated); B: reduced by 1 mm NADH + 1.5 ,ug of antimycin A andoxygen-treated; 1 mm NADH-aerobic (oxygen-treated).

in agreement with the correspondingly high NADH oxidase ac-

tivity of the particles. Similar observations have been reported forsubmitochondrial particles prepared from beef heart mitochon-dria (7). The molar ratios, however, indicate relatively less ofcytochrome c and flavoprotein, both of which are easily removedfrom mitochondria. Unlike the intact organelle, pyridine nucleo-tides do not appear to be present in submitochondrial particlessince externally added NAD+ is required to initiate malate oxida-tion.

Further resolution of the cytochrome components was achievedby the use of antimycin A or cyanide as inhibitor (Fig. 10). Inthe presence of antimycin A, NADH-reduced-oxidized spectrashow only the cytochrome b components. These can be observedas a double peak at 553 and 557 nm with a shoulder at 561 nm,

a weak 13-band at 532 and 525 nm, and a single Soret band at 427nm. Comparison of the spectrum with a similar treatment of intactmitochondria shows a smaller amount of the 561 nm b componentin the particles. When antimycin A is replaced by KCN cyto-chrome oxidase and cytochrome c remain reduced. Cytochrome

oxidase can be observed as a band at 597 nm and peaks at 443and 436 nm in the Soret region. The sharp peak at 547 nm is thecombined bands of cytochromes c with ,3-bands at 507 and 510 nmand a Soret band at 461 nm. The cytochromes b were not reducedin the presence of KCN. In the intact mitochondria the reductionof the b cytochromes is markedly energy-dependent.

DISCUSSION

The results reported in this paper clearly demonstrate that ac-tive, stable submitochondrial particles can be prepared frommung bean mitochondria. The particles produced have beencharacterized with respect to substrates and inhibitors suffi-ciently to allow their use in further studies. Although the particleswere still active after prolonged storage, the variable sensitivitiesto inhibitors probably resulted from a slow deterioration, par-ticularly of the energy-linked functions. Particles prepared byosmotic shock of mung bean mitochondria show many of theproperties of the original mitochondria but are less sensitive thanthe original organelle to some inhibitors and uncouplers, a fea-ture most marked in the NADH-cytochrome b segment of theelectron transport chain, where sensitivity to amytal is greatlyreduced. It has been reported earlier (10) that mung bean mito-chondria are only poorly inhibited by rotenone and are insensitivein the absence of ADP (state 4), and the submitochondrial parti-cles are unaffected by rotenone.The respiration of the particles is unaffected by oligomycin and

uncouplers, suggesting a loss of energy coupling resulting fromthe disruption of the structure of the mitochondrion. However,the particles are still inhibited by octylguanidine and are affectedby ATP, ADP uncouplers, and oligomycin in the early stages ofrespiration, particularly when succinate is used as substrate.These observations indicate that some energy coupling is still re-tained in fairly fresh particles. In another paper of this series (18)the energy-linked functions of the submitochondrial particles willbe examined in greater detail.

Disruption of the mitochondrial structure results in greateraccessibility of substrate to the inner membrane fragments andalso to a loss of easily solubilized components. Thus, there is aloss of mitochondrial protein which results in a high specificactivity of NADH oxidase and an increased concentration, on aprotein basis, of respiratory pigments. In the fragmentationprocess pyridine nucleotide, flavoprotein, and cytochrome c arealso lost. Spectral evidence shows that large amounts of enzymi-cally reducible cytochrome c remain in the mung bean submito-chondrial particles, but a large portion of this absorption bandmay be contributed by the second, firmly membrane-bound c-typecytochrome (11).Osmotic shock has proved to be a relatively gentle technique

for the disruption of cells and organelles (1). When particles areprepared from mammalian mitochondria by sonic oscillation,fusion of the broken membranes gives rise to spherical submito-chondrial particles which are inside-out compared with the intactmitochondrion. No evidence is at present available to indicate thesituation when plant mitochondria are disrupted by osmoticshock. Refusion of the broken membranes must occur, butwhether the vesicles are inside-out or retain the same configura-tion as in the intact mitochondrion remains unknown.When succinate was used as substrate, antimycin A and KCN

did not completely inhibit oxygen consumption by the particles.The proportion of respiration insensitive to the two inhibitorsvaried between preparations, but was much greater than that forNADH as substrate. Differential effects of antimycin and KCNhave been observed elsewhere (10) for mung bean mitochondriautilizing malate or succinate. Submitochondrial particles were95 to 100% sensitive to antimycin A and KCN when NADH was

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30 WILSON AND BONNER

supplied as substrate, but 15 to 30% insensitive to the inhibitorswhen succinate was substrate.

LITERATURE CITED

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2. CHANCE, B. 1957. Techniques for the assay of the respiratory enzymes. In: S. P.Colowick and N. 0. Kaplan, eds., Methods in Enzymology, Vol. IV. AcademicPress, New York. pp. 273-329.

3. CHANCE, B. AND B. SCHOENER. 1966. High and low energy states of cytochromes.I. In mitochondria. J. Biol. Chem. 241: 4567-4573.

4. CHANCE, B. AND G. R. WILLIAMS. 1955. Respiratory enzymes in oxidative phos-phorylation. I. Kinetics of oxygen utilization. J. Biol. Chem. 217: 383-393.

5. CHANCE, B. AND G. R. WILLIAMS. 1955. Respiratory enzymes in oxidative phos-phorylation. II. Difference spectra. J. Biol. Chem. 217: 395-407.

6. DixoN, M. 1953. The determination of enzyme inhibition constants. Biochem. J.55: 170-171.

7. ELLIOT, W. B. AND D. W. HAAS. 1967. Preparation and properties of digitoninparticles from beEf heart. In: R. W. Estabrook and M. E. Pullman, eds., Meth-ods in Enzymology, Vol. X. Academic Press, New York. pp. 179-181.

8. IKuMA, H. AND W. D. BONNER. 1967. Properties of higher plant mitochondria.1. Isolation and some characteristics of tightly coupled mung bean mitochon-dria. Plant Physiol. 42: 67-75.

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9. IKUMA, H. AND W. D. BONNER. 1967. Properties of higher plant mitochondria.IL. Effects of DNP, m-CI-CCP, and oligomycin on respiration of mung beanmitochondria. Plant Physiol. 42: 1400-1406.

10. IKUMA, H. AND W. D. BONNER. 1967. Properties of higher plant mitochondria.{II. Effects of respiratory inhibitors. Plant Physiol. 42: 1535-1544.

11. LANCE, C. AND W. D. BONNER. 1968. The respiratory chain components of higherplant mitochondria. Plant Physiol. 43: 756-766.

12. LARDY, H. A., D. JOHNSON, AND W. C. MCMURRAY. 1958. Antibiotics as toolsfor metabolism studies. I. A survey of toxic antibiotics in respiratory phos-phorylative and glycolytic systems. Arch. Biochem. Biophys. 78: 587-597.

13. LAYNE, E. 1957. Spectrophotometric and turbidimetric methods for measuringproteins. In: C. P. Colowick and N. 0. Kaplan, eds., Methods in Enzymology,Vol. III. Academic Press, New York. pp. 447-454.

14. LEE, C. P. AND L. ERNSTER. 1968. Studies of the energy transfer systems of sub-mitochondrial particles. 11. Effects of oligomycin and aurovertin. Eur. J. Bio-chem. 3: 391-400.

15. PRESSMAN, B. C. 1965. The effect of guanidine and alkyl guanidine on the energytransfer reactions of mitochondria. J. Biol. Chem. 238: 401-409.

16. STOREY, B. T. 1967. Kinetics of ubiquinone reduction by succinate in electrontransport particles. Arch. Biochem. Biophys. 121: 261-270.

17. WILSON, S. B. AND W. D. BONNER. 1970. Effects of guanidine inhibitors on mungbean mitochondria. Plant Physiol. 46: 21-24.

18. WILSON, S. B. AND W. D. BONNER. Energy-linked functions of submitochondriatparticles prepared from mung bean mitochondria. Plant Physiol. 46: 31-35.

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