swelling and contraction corn mitochondria · miitochiondria hield coldi fromi the beginning in 0.4...

Plant Physiol. (1966) 41, 255-266

Swelling and Contraction of Corn Mitochondria"C. D. Stoner2 and J. B. Hanson

Department of Agronomy, University of Illinois, Urbana, Illinois

Received June 14, 1965.

Sunhinary. A survey has been ii)ade of the p)rol)erties of cori wlitoc(hon(rlia in .swell-ing and contraction. The mitochoni(lria swell spontalneouisly in KCl but not in sucrose.Aged mitochondria will swell rapidly in sucrose if treate(l Nvith citrate or ED'FA. Swell-ing does not impair oxidative phosphorylation if bovine ser-niiii albumin is p)resellt.

Contraction can be maintained or initiated with ATP X- Mg or anl oxi(lizable sub-strate, contraction being imore rapid with the subst,rate. Alagnesiuni is n1ot re(uire(l forsubstrate powered contraction. Contraction powered by ATP is accompanied by tllerelease of phosphate. Oligonmycin inhibits both ATP-powered contractioni and the re-lease of phosphate. However, it does not affect substrate-powere(l contraction. SuEb-strate powered contraction is inhibited by electron-tranlsport inhibitors. T'hle unlcoupler.carbonyl cyanide m-chlorophenyl hydrazone, accelerates swelling and(I inhibits both AT1Pand substrate-powNiered contraction. However, the concentratioi's require(d are well inexcess of those required to produce uncoupling and to accelerate adeniosine triphosphatase;the concenitrationis required inhibit respiration in a phosphorylatig,- meditui.

Phosphate is a very effective inhibitor of succinate-powered contraction. Neitheroligomycini nor Mfg affects the lphosphate inhibition. Phosphate is less inhibitory \ithtlle ATP-powered contraction.

'rhe results are (liscussed in termiis of a hypothesis that conitractioni is associate( \\ ith a

nonphosphorylate(l higlh energy intermediate of oxi(dati\e pllosph) or1rlatioll.

Sxwelling anld conitractioni of animnal niitochonidriahave been extensively investigated, and are the sub-ject of 2 excellent reviews (2. 15). It is establishie(dthat within the linmits imposed by their extensibilityand compressibility mitochondria behave as osl11o-meters, and that they are relatively permeable to saltssuch as KCl, but much less so to sucrose and otherpolyhydroxy compounds. Osmotic adjustment is ob-tained within a few seconds on changing osmolarity,but there is a slow swelling (spontaneous swelling)which can be accelerated by a wide variety of swell-ing agents, and which is largely reversible by ATPor phosphorylating respirationi. The swelling is somle-how linked to electron transfer and inhibitors of res-piration prevent swelling. The contractile mecha-nism is thought to reside in the membrane, and islinked in some fashion to the intermediate system ofoxidative phosphorylation. Swelling seems to resultfrom relaxation of the contractile mechanism, possiblya mechanoprotein (15), which is reestablished by alnATP-consuming process with a concomitant extru-sion of water.

This research was supported in p)art by theAtomic Energy Commission (AT/l1-1 /7(0) an( thc National Science Foundation (G-16082).

2 Present address: Heart Bioclhcmi!stry Laboratory.University Hospital, Ohio State University, Columbus,Ohio.

255

Very littlc of t his brief sunmiary can be apililedto plant mitochondria. Hon(la and Muenster ( 10)studie(l the relationshil) between swelling and( su ccin-ate oxidationi of lupine mitoclhonidria uisinig bothl)acked volunm.e anid light-scatterinig measuremlienits.\Vhile packed volume measuremlienits indicated thatsucciniate somiietimes prevente(l osmlotically inducedswelling and induced the mitochondria to contract,parallel optical studies indicated that succinate in-(luced the mlitochondria to sewell. Osmliotic swellingbrought abouit either anl activation or inhibition ofsucciniate oxidation depending onl the concentrationof succinlate used. These studies l)ointed out theneed for a more complete characterization of swellingand contraction phenomenia in p)lanit miitochondria.

Lyons and Pratt (18) have studied the effect ofethylene on swelling and contraction of rat liver an(dcauliflower mitochon(dria. Both types swelled spon-taneously in KCl and contracted upon ad(litioln ofATP and Mg. Swelling did n1ot occur in sucrose.In related work, LyoIns, Wheaton and Pratt (19) in-vestigated the extent of mitoclhonidrial swelling as re-lated to chilling resistance of various p)lant tissues.

Chloroplasts undergo reversible liight-scatteringclhanges upoIn illuminatioln, and Packer et al. (24 )isuggest that intermilediates of p)hosp)horylation in bothimitochondria aind chloroplast mienmbranes are callableof bringing about structural changes inl the imem-branes. Dilley (4) has recently related chloroplast

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256 P~~~~~~~ILANT PHYSIOLOGY

shrinka-e to a -ligt (dependent potassiuim efflux.As pairt o)f a stud) oni memb)rane lprolperties of

corin mitochondria xx e thiought it would be desirableto h1ave iniformiationi on reversible swelling, and theexiperinments are relported here. Corn iimitochonidriaappear to differ fi-omi miost animial preparations inthat they 4how rapidI spontaneous swelling, and( willreadIiIv conitract oni additioni of suhstrate alone. I i owx-ever, we suispect that these distinictionis may result fromat greater lermeal)ility of coriinmitochondria, ratherthani fromi differenices in the basic miechanism conicerlie(l.

Materials and Methods

Isoltotioo of l i/oc/i oodria. C( rmi eedl ing- Zeuiiiiovs. \VPF) X M114) xxere groxxmmi ini the (lark at 28to 3)()IoI paper towels ,;atliratedl wxith 0.1nmxm CaCl..A~bont 1 5)) of 3 anid onielhalf (lay shoots wxere grounidinl a co-Ild m1ortar wxith 301) iml o-f 0.4 M, siicrose + 5 m--\21)TA m 0.1 ,,, Tris + 0.05 M~\ mialeate (pH 7.5).TIhe miitoclion(lr-ia were sedimented at 12,000 X g for1 0 minuittes after clearing the homiogeniate at 2000 Xy for l() n mutes. an-d xvere xxashed onice in the grind-ing- niedlitini anid Mn~in(.4 xi snecr mse -- m i Rl )TA.Finial suspenision xWas inl 0.4 Ni sucrose.

O)XIdative Ph/osp/m ory/uttooo. Respiration xx,as mieas-ture(l wx thi the \Warburg resl)irometer at 300. Thecvessels comitainied ini 2.5 iul volum,e: 450 ~uiioles su-crose, 125 JLnmoles glncose. 40 Mmiioles each of pyrtivateanid mialate. S0 1kimoles KI ITON), 2.5) /,woles MgSO412.5 Itioles \A1)1 0.6 pki-u.ole. NAt), 0.4 w1mole thiamine111piyrophosphate, 0. 1 [tnmole CoA, anid 25) KM tmnitshiexokiniase, adjustedI to pH 17.5. Respiration xwasgenlerally folloxxed for 20 miniiutes after 1 0 miniiitesequilib1 ratiOin.

S-av/lUigiiy d Conltractioni. Chianges ini lighit scat-tering( xxerc followed biy clianges ini I) at 520 iii

xxith a Colemi-an MAodel 11 spectrophotoniieter. Thestepxwise iprocedture consisted of: 1) placinig 4.0 ml of0.25 xr KCl +4 0.025 M.\ Tris-HICl buffer, pH 7.5. inmiatchedI tuies; 2 ) bringinig the xolume to 4.95 milxvith xwater or exl)erimental additives; 3 ) ejuilibrat-iing the soluitionis at 28' in a xwater lbath; 4) inijecting0.05; ml (i)f miitochiondrial suispenisioni conitainingi 0.12 to0.15 img N ;5 ) miixing- hx shiaking- and( 6) folloxvingchang-es inl ( )l). Ni expmeriim,ents xxhere pHA xxas varied,T1ris-maleate buffer xvas used. In a fexx early ex-

lperiments. 0.1 ml of miitochond(ria xvas added to 4.9 ml1o)f m-ie(liumi. Betxveen mieasturemienits the tubes werehield ini the xvater bath. Conitractioni xvas iniitiated bymlding- thei ini(licate(l additives in a sm-all volumiie ( 0.02-0.05; nil ), anld correctio)ns xxere madt(e for the smiallchange ini 1) (tlue to dilutioni. Ethanol xvas used asso:lvenit fo-r somie reagenits, but a series of exlperimientsshoxxed the smmdll amiounit uised to he xvithout sig-niificanit effect omi sxxelling or- contractionl- ethaniolbdauks xxere tised ini conitrol treatmenits.

For- gravimetric determiniiations l)arallel treatmienits\kwere uise(l xxith larger aliquots of miitochonidria (ca.0.9 mlg N A..t the en(l of the sxwelliing-conitraction

p)eriod. the soluitions xxvere centrifug-ed at 25,000 Xg for- 0(1 m11intes, the utl e careftillx drained and the

tttim o)f the l1ster id tube containing the lpellet xxascuit a\xvaxv. Fresh, oxen (lrv and tare xxveigh,ts xo)btaine(l. Calculations xxvere based oni the chang-e inixxater conitent o)f the pellet upon sxxelfing and conitraction. and( nno effoitwixas nmale toi estimiate u-cclniledsalts mnid xxater.

h/eti~ii i(isuo)x. 1 -i)phate buiffer 1.)) .m.p11 7.5 ) xx s ue( in these sxx\velling contraction stid-ies because Tris reacts xxvith the osimfixativxe. Athe eml(l of the sxxelling-contraction treatment, the sn,pemsinms \xvcrc chilledI oni ice, and 4 7( s )I In (.2 miIKCl xxas madded xxitli vigorous stirring to a flinal coil-centration o)f 0.5 (7 ( )so,1 An equlivalenit mliqtuot ofmiitochiondria hield coldI fromi the beginning in 0.4 ~msticrose +0.01 xm potassitimn phlosphate (pH 7.5 )wxass~imilarly fixed. The mito-chondri-a xxere collectedI asa thini l)e1let (ca. 0.2 mum thiick ) in flat-bottomied testtubes in a sxvinging bucket rotor at 1500 X g for 15miniutes. TIhe clear superniatant xxas (lecailte(l andfreslh fixative (I % (W)s() in KCI o)r sucrose pdiis bufferas above) added. A fter 3() uinuittes oni ice, the fix-ative xxas replaced xxith half stremigtli buffered lKl'CIor suicrose for 15) minutes. 'Ihle lpellets xxvere (1hywdrated and emibedded in mtethyxacrxlatd xxith the miSCo)f 0.01 % uiranvl niitrate ( 317 and a miitrogemm atmos-phemre (23) to hielp pmrexvemnt iimnex-cmi polymnieriza-tiomm.Sectionis xx ere cuit oni an I,K 11 microtommie. Stained for-1 houir xvith 4 (7 tranyvl acetate, amid viexve(l xxvitli aIIlitachi 118 6 electroli microscope.

l,'d'(sCiiO 1ic TriP/iosPhiitasc' Deterinio'at'ioms. 'Ihlebasic miiediuimmi xxas the samie as that uisedI for sxvellinigciimitraIctmomi sttidie~~. Pinial comncentrations imi 5 miil xxer~e(0.2 _m KCI. 0.02 m Tris IHCI (p11 7.5), -5 niiN ATtP,I1 Mux MgCI2, 10 MINI SLIcrose (added xv-itli the miito-chiomidria ) am(Il 0.15 to 0.2(1 mug miitochomidrial N.Media xvere equilibrated and liel(l at 28'. 1Determuina-tomis of Pi (6) xx ere miiade omi 2 mnil aliqtmots at 1imitiiute amidi at 31 miiimiiutes after add imig the miuitochomi-

dna. The reaction xxvas stopped xvith ice cold S (trichloroacetic acid ( fimial comncemntratiomn) amidI theniitochiomidrial p)recip)itate cleared by centriftigatiomi.

Results

,S'Poiitaieoiis S7vcc//iiny and( /ts Rcvcr-sal tilt/i 11.I/PIPrelimimiary xvork estLablishedI that cormi miitoclimidnlMado0 mint sxxell appreciably in sucrose, but xwill swx (1spmontanieoislv xvhemn tramis ferredI to isoi)smiola r 1K Cl.1,Nomis et al. (19) miadle simmlilar- fimidimigis xxith avariety of plamit mu1itochomidria. \Whemi the same )s-muiolarity is immaimitaimiedI by varyimng prolportiomns o)f suicrose amnd KCI. the dlegree of sl)omntmameous sxwellinig imicreases xvitli the comicemitratiomi o)f KCI (fig ) Thieiniitial 01) also rises xvitli imncrease(l l)ropourtiomn of1KCl, xxhlich is l)robabily dule to the (decreaisilg refrac-tive imidex of the mediumim (33).

Does the (lecrease imn 01) xxliemm mitochioudia(ri areimmjected imito KCI solutions actually represent sxvell-imig?IThe literature omi swxellimmg of amiimiiial miitochmio

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STONER AND HANSON-SWELLING IN CORN ATITOCHONDRIA

osmolarity (mM) .6

sucrose KCL

207 200_

0

.5

407 0

t 407400

.4

4

ATPMgBSA

.2

0Tris c/- 8

.1

PO4

2pellet

AT\ MgA,BSA, initially dry HL0i wt.

_ /_ mg.s--95 55.2

9.5 58.8

ATPMg.B.S.A/

/

.8

.7

0

0

.6

no

0 10 20MINUTES

oddition9.6 69.4

a I I .530 40

5

/

swelling

/ contraction

6p H

a7

3KCL(mM)

II' ATP 200MgBSA /

125I,

aXfi '< 2 " 50

. 0.

1

0 10 20 30MINUTES

6

pHATP

70Mg7.0 *BSA

% 1,~~~~~~~~~~~~~~~~.\1 f

75

0

MINUTES

FIG. 1. The pronmotion of swelling in corn shoot mitochon1dria by KCI as indicated by loss of OD. Basicmedium was 0.02 M Tris, pH 7.5 with KCI and sucrose as indicated.

FIG. 2. Relationship between OD change and water content of mitochondrial pellet. Basic medium was 0.2 M

KCl + 0.02 M Tris, pH 7.5. Contraction initiated or maintained with 3 mm ATP, 2 mm MgCl2 and 1 mg/ml BSA.FIG. 3. Mitochondrial swelling and contraction in varying concentrations of KCI. Media were buffered with

0.02 M Tris-HCI, pH 7.5. Contraction initiated with 5 mm ATP, 3 mm MgQl2 and 2 mg/ml BSA.FIG. 4. Comparison of swelling and contraction in 0.02 M Tris or potassium phosphate buffer, pH 7.5. Con-

traction initiated with 1 mm ATP, 1 mm MgCl2 and lmg/ml BSA.FIG. 5. Swelling and contraction as a function of pH. The curves present changes in OD 5 minutes after initiation

of swelling or contraction in separate experiments. Swelling was carried out in 0.2 -m KCI in 0.02 M Tris-maleatebuffer. For contraction, mitochondria were allowed to swell in 0.2 M KCI + 0.005 M Tris-HCI, pH 7.5. At 20minutes 0.05 ml of 1 M Tris-malate buffer of varying pH was added, and contraction was initiated by 3 mm ATP, 2mm MgCl., and 1 mg/ml BSA. The pH readings are those taken at the end of the contraction period.

FIG. 6. The effect of high pH in accelerating swelling and increasing the BSA requirement for contraction. Con-traction initiated with 1 m-m ATP, 1 mM MgCl, with (solid line) and without (dashed line) 1 mg/ml BSA.

257

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.61

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NIn00

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PLANT PHYSIOLOGY

dIria suggests 2 mlajor tests: Is there ani increase inwxater conteilt of the miiitochondria? \Vtill ATP +-Mg reverse the decrease in GD, extruding water? AinexperinIent suimmarizing the results obtailned from in-vestigatinig these questions is shown in figure 2.Blovine serum albumiin3 xvas inclu(led witlh the ad-(litiolls of ATI' and Mg to bind any fatty acids (U-factor) whiclh ulight be released (lurinig the swellingperiod ( 17, 38).

The addition of ATP + Mg prevenlted imiost ofthe decrease in OD when added initially anid largelyreversed the (lecrease when a(lded to swollen mito-chondria. There was a close correspondlenice betweelnthe fillal 01) of the suspensions and the water coni-tent of miiitochondria recovered from thenii. If themllitocholndria iliaiintainied in ATP are takeni to rep-resenit 100 % contraction, and those fully swollen torepreseilt zero conitraction, theni the addition of ATPto swolleni mitochonidria gave 72 % contraction byphotometric illeasurenlent and 75 % by gravimetric.Tt xwas coilclt(led that light scatteriing iieasureillentsgive a goo(l lildicatioin of the level of water ul)takeaind extrusioll.

Figure 3 shows the resl)oilsesxwhell the mitochon-dria suspleIlded in 0.4 Mr sucrose were illjected intocuvettes with KCl coiiceltrationis varviilg from isoos-iliolar (0.2 i\ ) to buffer alone. As would be pre-licted froim the work of Tedeschi aind Harris (32. 33),there \\as a rapid osmotic a(ljtlstln1eilt lprior to the tiilethe first readiilg was in]ade (30 sec) which resultedin lox\'er initial ( 'Ds witli lower KCI conlcenltratiolls.Following this a(ij ustulent, al a(ldditiollal slow spon-taneous swelling ensued, beillg greater ill extent withiilcreasillg KCI.

Very likely the elletration of salt is reslionsiblefor the greater sxvelliilg wilich occurred in the svstenlxvith isoosiilolar KCI (3, 31). \Vlhere OIlly bulffer(0.02 xT Tris) xas preseilt. however, the iliitial osmllo-tic adjustuleint (swelling) wxas large aldfl tile addi-tional subsequient swellinlg probal)iv represeIlts fur-tiler stretching of mitociloln(drial illeillbraiies wvlicll ap-proach tlheir lilmlits of extensibility. The relativelvlow level of ATP-induced reversal of tile total svell-iilg (initial rapid osmotic adjustnleilt pltus subsequentslow adjustillent) in tile hypotollic iiledia is probablya reflectioni of damiiage to the conltractile meclallaisinias a result of the greater extent of swelliig, an(l theestablishmlenlt of equilibriumii at a lox exterlial os-motic pressure.

Tedeschi amId Harris (33) poimlt out that 110ost ofthe difficulties arisiing froill refractive index chanigescaan be avoided by using imledia of ilearly coilstantcOilIlipOsitiOll. It wvas (lecide(l to uise 0.2 mx KCI (ap-proximlately isoosillotic with the 0.4 Mi sucrose use(lill isolatioin) ill all subsequent experimnenits. Theral)id Osillotic a(ijustmemlts would be avoided. aild al-though complete expulsion of xxater gained (lutrillgspoiltatleous swelling could inot be expected. there xxas.

3Abbreviations used: BSA, bovine serum albunlini.CCP, carboniyl cyanide it-chlorophenyl hydrazone.

iio reasoni to thinlk that the functioning of the COil-tractile mleclhaniism \as iml)aired.

In viewx of the fact that the contractile mechaniismis thouglht to be closely associated with the highenergy interme(liate system of oxidativ-e phosphorvla-tioIn ( 15, 24, 26), it wxas imiiportanit to know how swell-ing in KCI affected oxidative p)hosphorylation. \sShOWvn in tal)le 1, a pretreatmilenlt in KCI which wouldallow spontanieous swelling did not uncouple the miito-chondria. Howe-er, coml)ared xvith the ap)rol)riateconitrol (addition of ATP + MIg + BSA to mllain-tain contraction), there wvas somiie decline in substrateoxidationi. The higher level of oxidation cannot lIeattributedl to mcainteinanice of contraction, for BS .\alone wxas equally effective. anid it was established iiisel)arate exl)eriments that BSAX (lid nioL plrevent spwi-taneous swelling or initiate contraction. "'he no11-l)ortant l)oint is that the spontaneous .swellin-g leaxcthe coupling mleclhaniism functional.

In other experimiients not (letailed here, mlitoclhonl(Iria xvere transferred to sucrose of (lifferinig osmolar-ity and changes in OD and water content were meas-ured. Except at very loxv sucrose concentrations(0.05 Mi or less), there xvas I1O significalit spmo-taneous swelling following the initial rapidl osmoticadjustmenit. Betxx een 0.5 NM acnd 0.005 M1.sucrose,the xvater conitenit of the miiitochondrial plellets in1-creased 25 %.

Swvelling and contraction inl phosphate and Trishbuffers were compared (fig 4 ) Phosphate (20 in xiaccelerated the rate of swxellin- and inhihited tile rateof cointractioni. In aniothier exlperiment, BS.\ (I vngml) was fouind to slightly retard the swellinig rate.Ethanol (1 %) did not affect swelling, btit sliglhtl\inhibited the ATP-induced conitraction rate.

Figure 5 slloxs levels of swelling and colntractionat xvaryiilg pH. lMinimial swxelling rates occutr at )d6.5, anid maximiial contractioll rates at pH /.2 to 7 4.The accelerated sxelling above pH 6.5 might be re-lated to release of fatty acids (U-factor), as the (Ie-pendence uponl BSA for conitraction iilcreases wvithpH (fig 6).

Table 1. Effect of Prctrcatmcint iM TIvis-Buffcred xt /10I Sidbsc(peiwi Oxidative I'llosP'lh orWla toi1

\Vashecl mitoclhonidrial pellets \\ ere susped(le(d ill theswelling mediunm (0.2 m KCI + 0.02 mi Tris p)H 7.5)cointaining 5 mnm ATP, 3 mis MNgCl., and 2 mg/mlBi .\as iIl(licate(l. After 20 miniutes preincubation at 30° ali-quots were transferred to \Narburg vessels and(I oxidlatiVephosphorylation measure(I wvith the pyruvate-malatemledium. The sucrose \,as omitted, and the vesselincreased by 40 ninr KCI and 4 mnm Tris added wvith theulitochondria.

Preinicubatiolladditives

N oneC:TIP, MIg, BSA.kA'l0, MIgBSA

Qo. (N)790119011801120

P/02.32.42.42.4

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STONER AND HANSON-SWELLING IN CORN MITOCHONDRIA

For further work, it was decided to continue usingpH 7.5 where both swelling and contraction would berapid. Bovine serum albumin was routinely added atthe time contraction was initiated.

Electron Microscopy. MAfitochondria from an ex-

periment like that of figure 2 were examined withthe electron microscope. WVe were not skilled enoughin electron microscopy to follow structural changes infine detail, but gross morphological changes in theinner membrane were readily aplparent (fig 7). Mi-tochondria held in sucrose or ATP show dense cris-tae or involutions of the inner nmembrane. The mito-chondria in 0.4 M sucrose appear to have an innermembranie which is partially plasmolyzed, while theless dense outer membranie seems unaffected (fig 7a).This observation is in accord with suggestions thatthe outer membrane is quite permeable ( 1, 30, 36).The mitochoiidria held contracted in KCl byATP showlittle or none of the plasnmolysis but the inner mem-

brane is very electron-deinse, and the cristae are large(fig 7b).

Upon swelling in KCl, the large cristae seem to bewithdrawn into narrow tubulles, which present circularprofiles in cross section (fig 7c). In median sectionsof the mitochondria, a peril)heral location for thesetubules is shown. When the swollen mitochondria are

contracted by ATP(fig 7d), the large, dense cristae re-

appear in most mitochondria, but there seem to befewer mitochondria where the cristae occupy the en-

tire volume delineated by the outer membrane.Characteristics of A TP-Induced Contractioni.

The optimal concentration of ATP for contrac-tion was 1 to 3 mNIi. Above 3 imzs ATP, the finallevel of contraction was somiiewhat reduced. TheATP-induced contraction was accompanied by a

release of inorganic phosphate (fig 8). Thecontracting mitochondria thuis exhibited an adenos-ime triphosphatase activity. Over the first 4 min-utes when contraction was most rapid, the rateaveraged 39 ,umoles Pi/mg N per hour. Subse-quently, the rate slowed to 26 unioles/mg N per hour,and was maintained at this level throughout the incu-bation, even after contraction vas essentially com-

plete.It was found that ADP (with Mg and BSA)

would gradually induce contraction after a delay ofabout a minute. However, whlen hexokinase and glu-cose were added with the ADP, there Nwas no contrac-

tion. This result was interpreted to mealn that ade-nylate kinase, knowni to be associate(d with plantmitochondria (7), produce(d sufficient ATP to activ-ate contraction, but that in the presence of a hexokini-ase-glucose trap the ATP did niot accumiiulate to ade-quate levels. The additioni ot AMN2P (with MIg and 1BSA) did not effect contraction:, neithler did M\g nor

BSA, alone or in conmbinationi.Oligomycin strongly inhibited ATP-induced coIn-

traction (a value is included with table III), but oli-gomycin had no effect on swelling rates. This re-

sult is similar to that obtained by Packer et al. (24)with heart mitochondria. When ATP was added

initially to maintain the contracted state, oligomycininitiated swelling and inhibited the release of Pi (fig9). Oligomycin is thought to interfere at a terminalstep with the reversible transfer of Pi between ATPand a phosphorylated intermediate of oxidative phos-phorylation (14). Hence, it can be deduced thatmaintenance of contraction in KCl requires a continu-ous input of energy into the high energy intermediatesystem via a process which releases Pi from ATP.

The effect of oligomycin was of sufficient im-portance that we made observations on the effective-ness of the compotund as an inhibitor of oxidativephosphorylation and adenosine triphosphatase (tableII). At about 0.04 ug/ml (1.3 m,umole oligomycin/mg mitochondrial N), oxidative phosphorylation waspractically eliminated and 0 consumption was re-duced by half. Beyond this point additional oligomy-cin had no effect, suggesting that all of the terminalphosphate transfer system was already blocked. It istempting to deduce that only half of the electrontransfer is couipled to phosphorylation, but this wouldbe inconsistent with the good P/O ratios obtained inthe controls. There is an alternative possibility:some coupled respiration might be shifted to an oligo-mycin insensitive process such as the salt uptake knownlto occur in these mitochondria (9) or to maintenanceof contraction (see later).

The adenosine triphosphatase study was in KCluinder conditions similar to those of figure 9, andthus not directly comparable with that of oxidativephosphorvlation with its complex medium containingsncrose, substrates and cofactors. About 0.05 ug oli-gonmycin/ml (3.9 nm/umoles/mg N) were required be-fore the mitochondria showed evidence of saturation(table II). However, the concentrations of oligo-mycin neede(d for half maximal inhibition are similar;0.81 and 0.90 mumoles/mg N for oxidative phos-phorylation and adenosine triphosphatase. About 20to 25 % of the adenosine triphosphatase activity meas-tired in the contraction experiments is oligomycin in-sensitive.

The contraction mnaintained by the ATP-hydro-lyzing system can be uncoupled by 10-5 M CCP (fig

Table II. Inihibitioni of Oxidative Phosphorylktion andAdenosine Triphosphatase by Oligomnycin,

All treatments contained 2 % ethanol.

Adenosinetriphosphatase

Oligomycin ,trnoles Pi/hrjug/ml Qo., (N) P/O per mg N

None 2150 2.6 44001 2900? 1840 2.3 210 04 1080 0.40.05 140.10 1080 0.2 120.20 1130 0.1 120.40 1150 0.21.00 102.00 1290 0.1

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PLANT PT YSIOLOGY

1)) w-ith ino significaint chanige in l'i released. Inother work it wvas folnnd that CCP added with oligo-mycin produced Ino chanige over oligomycin alone(i.e.. as in fig 9) in(licatinig that lncouplinig is sub-

seqtueiit to the oligomycin-sensitive site in AT'P-powered contraction.

The effectiveness of CC I' oni oxidative plhlo-phorylation and( a(lenosinie tripho()sphatase is shown iII

/1.d. a.

.;iO..'.NI}.

e kf . * . NSyR..e

i i'>TNF'.W. .

{i K;

dU~~~~~~~~~~~~~~~~~~~~~~~~~~AIl

Fi. 7 ElectrInm iiiicrograplis of iiiitochondria in various stages of swelling anid contraction. Treatments as infigure 2: a ) Initial state in 0.4 -m sucrose; b) Maintained in contracte(l state c ) Swollen state cd) Contracted with ATPafter swelling. Magnification X 30,000. 0. Outer membranie fronm vhich the inner miienmbranie has been wvithdrawi-n byIJlasmnolysis leaving a large intermlemlbrane space. T, Median section of a sx-ollen miiitoclhond(Irion showing peripheraltubular cristae.

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STONER AND IIANSON-SWELLING IN CORN .lTOCNDHORI A 261

figure 11. 'T'he oxidation rate of pyruvate-malate ismaintainied until phosphorylation is almiost completelyunicoupled. The adenosine triphosphatase is pro-moted by concentratiolns of CCP which uncoulple.With CCP concentrations in excess of 10-- bothadenosine tri,phosphatase and electroni transfer de-clinie to lower levels, but are not eliminated. NXstimulation of adenosine triphosphatase occurs if 0.5 M\sucrose is added to the KCl + Tris medium. Thisprotective action of sucrose max account for the fail-ure of some investigators to filnd a DNP-stimulatedadenosinle tril)hosl)hatase (27. 35). We finid a DNX-stimulated adenosine triphosphatase in KCl (29).

The effect of CCP on swelling rates is shown infigure 12. At high concentrations (10-5 At), the un-coupler accelerates swelling, but does not alter thefinal swollen volume. The same is true for DNP(29). Since the concentrations required are abovethose needed for uncoupling, it is dubious that theacceleration of swelling is directly related to un-coupling.

Figure 13 depicts the effectiveness of CCP as afunction of concentration on contraction with andwithout BSA. The BSA milust bind somile CCP forits effectiveniess is sharply reduced in the presence ofBSA. Concentrations of CCP which uncouple oxi-dative phosplhorylation (nmaking due allowance forthe BSA binding) and increase adenosine triphos-phatase begin to inhibit contraction, but there is aninexplicable reversal of this trend at those concentra-tionls where respiration and adenosinie triphosphatasebegin to decline (cf. fig 11). Still higher concentra-tions are inhibitory.

The Mg requirement for ATP-induced contractionis illustrated in figure 14. The endogenous AMg isapparently adequate to permit some contraction, butaddition of Mg is promotive. Although not shownl.the addition of Mg alone is without effect.

Suibstrate-Induced Conttractiont. Figure 14 alsoshows a surprising discovery made at this time. Thegeneral requiremiient for substrate oxidation to pro-duce large amplitude swelling in animal mitochon-dria (2, 15) suggestedl that the addition of succinatemight produce a fturther incremlent of swellinig in cornimitochondria. The opposite effect was realized: themitochondria contracted rapidly (half-timze of about30 seconds versus 3 to 4 minutes for ATP). Therewas no apparent requirement for Mg, and furtherwork fully verified that exogenous Mg was neitherneeded nor promotive.

In contrast to the lack of response to Mg was theeffect of phosphate, which inhibited contraction withsuccinate much more than with ATP (fig 15). In-vestigation of this phenomenon revealed that additiolnof ADP in the presence of a hexokinase trap wouldpartially reverse the phosphate inhibition (fig 16).Presumably, the inhibitory action of phosphate isthrotigh the oxidative phosphorylation mechanism andan acceptor system for the phosphate reverses the in-hibition. It nmust be emphasized that substrate aloneis all that is required for contraction. The respira-

tioni does nIOt hiave to b)e coup)led to phosphOrylat iolas wvith miany animlal preparations (21. 26). In otherexl)erimellts. it was found that the effects of suc-cinate and A'T'P are not additive; both lead to thesaimie level of contraction, the succinate very rapidly.The phosphate inhibitionvwas stuidied with MAg andoligomycin as variables (fig 17). 'The inhibition wasnot materially altered, indicating that the entry ofphosphate intO the sensitive site does not requireexogenous Mg. nor is it blocked by oligomycin.

Contractioni was studied with other substrates.Malate serves as uvell as sucecinate. particularly ifMAD is. added. Pyruvate was ineffective alone, butquite active if a malate or succinate "sparker" wasadded. Very rapid contractioni was obtained withNADH (1 nmI).

One oddity appeared in studying contraction withNAD. used as a control for NADH. While 1 mmNAD would not give contraction (1 mm NADHwould), 5 mm NAD gave good contraction, althoughthere was a slight delay (fig 18). The subsequentaddition of nmalate wvith low levels of NAD caused arapid contraction to a level conditioned by the amountof NAD added. The contraction caused by 5 mmNAD could be completely bloicked by cyanide (fig19). Cyanide did not block ATP-powered contrac-tion. It was decided that the high contcentrationi ofMIAD probably activated the oxidation of ein(logenioussubstrate, pro(lucing the noted contractioni. 1'i;sumably, the corn mitochondria we work wvith aresufficiently depleted of N'AD) that eni(logeinous sub-strate oxidation of this type is of little conisequelnce inthe other experimlenits we report here.

Cyanide xvas also effective in blocking contractionwith other substrates. Furthermore. if mitochondriawere held coiitracted N-ith succinate. the a(ldition ofcyanide permitted rapid swelling (fig 20). Note alsoin figure 20 that addition of Pi cauises the rapid es-tablishment of a nlew. lowver equilibriuml.

Table 1II lists the percentage inhibition of conl-traction obtainied xvith other inhibitors of electrontranlsport. Rotenone was ineffective xvith succinatebut effective xvith niialate wvhich is in accord with itsspecific site of action in blocking NADH oxidation(5). The specificity of rotenone was verified in the\Varburg apparatus: .At 0.01 inv\t rotenone, the oxida-tion rate of nmalate + pyruvate decreased [e.g., Qo.(N) from 1470-650], while the oxidation of succinatealone increased (740-1200). No investigation wasmade of this respiratory increase, but it was not dueto uncotupling as the P/O valutes were not significantlyaltered. The response may be related to the observa-tion that succinate is poorly oxidized by these cornmitochondria unless pyruvate is added (9). presum-ably due to accumulation of inhibitory concentrationsof oxalacetate (25). Rotenone would be expectedto inhibit oxalacetate production by virtue of itsability to block oxidation of NADH produced duringthe conversion of malate to oxalacetate.

Oligomycin was ineffective with substrate-pow-ered contraction in contrast to its effectiveness withATP-powered contraction (table III).

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262 PLANT PHYSIOLOGY

.6

.10 / / ~~~~~~~~~~~~~~oligomycin.0FXoSOD /// ~~~ ~~15OD

52 1

0 S~~~~~~~~~~1p

W. 5 | 0:7

0 \0 20 ( 30_ 2 0 10 20 30

w W 0~~~~~~~~~~~~~~~1

0~~~~~~~~

oA- 40 2 0 4- 0 0 3

0

2 ~~~~~~ATPase4 0

0 - ~~~~~~~~0Wo --A~~~~~~~~~~~~~~~d

MlNUTES

LOG MOLARITY CCP

-MINUTES

LOG IUOLARITY CCP

MINUTES

hMINUTES

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STOINE'R AND HANSON-SWELLING 1N CORIN MITOCHONDRIA

Table III. Inihibition of Substrate-Powered ContractionPercentage inhibition calculated from change in OD

4 minutes after adding substrate. Ethanol was added tocontrol where used with inhibitor.

Substrate

SuccInateSuccinateMalateSuccinateMIalateSuccinateMalateSuccinateATP + Mg

Inhibitor

A.ntiniycinMalonateMalonateAmytalAmytal

RotenoneRotenoneOligomycinOligomycin

0.01 nmi

8 mM8 illM

5 nmi

5 mM0.01 IllnI

0.01 mM1 jug/ml1 ,Ukg/ml

The action of CCP was again most peculiar. Atconcentrations which uncouple the mitochondria with-out affecting respiration (fig 7), there is an in-crease in contraction rate (fig 21). Beyond 10-6 M

CCP, however, there is a strong inhibition. Figure21 also shows that BSA acts to increase the concen-

tration of CCP needed to produce a response. Sincethe shape of the curves is not changed, it is likely thatBSA binids CCP, reducing the effective concentratioln.

Substrate (succinate or pyruvate + succinate)a(dded initially was just as effective as ATP + Mgin p)reventing swelling (fig 20). However, this was

only true at pH 7.0 to 7.5 (fig 22). With higherpLH, contraction could not be maintained, and at p1HI8.5 swelling was almost as rapid as if no substratehad been added. We have not yet investigated this

phenomiienon in detail, but it would appear most im-portanit for uiniderstanding substrate-powered contrac-tion. ATP-powered contractioln was not studiedabove pH 8.0, but the results reported in figures 5 and6 suggest that a similar response could be expected.

Swelling in Sucrose. The failure to obtain swell-ing in sucrose was of interest, and some experimentswere conducted to see if swelling could be initiated.It was learned that if mitochondria were allowed toage in sucrose at room temperature, the addition ofsticcinate would bring about rapid swelling. At firstit was thotuglht that this response was related to therespiration-dependent swelling in animal mitochon-dria, but fturther work revealed that the swelling wasinsensitive to antimycin A, and that a variety of di-carboxylic acids or phosphate would produce the sameresult. Citrate and EDTA were particularly effec-tive, presumnably because they are effective chelatingagents. A time-course study with citrate is shown infigure 23. The degree of swelling is proportional tothe time of aging in the cuvette. WVhen citrate isadded initially, no swelling is produced. We con-clude that the stability of mitochondria in sucrose isdependent on some cation-stabilized membrane struc-ture. \\Vith aginig this structure wxeakeins to the poinitwhere chlelating agents can remiiove the catioins, theorganization of the strutctuire is destroyed, anld pernme-ability is greatly increase(l. It is possible that thistime-of-addition effect of citrate is throuiglh the typeof lipid l)eroxidation studied by Hunter et al. (11).Lynn et al. (20) have recently shown that the perme-ability of liver mitochondria ml-emibranes cani be strik-inigly altered by small anmouniits of Ca or fatty acids.

FIG. 8. The release of Pi accompalnyinig ATP induced contraction. Contraction initiated with 3 mM ATP, 2nim MgCl, and 1 mg/ml BSA. Aliquots withdrawn at different times for Pi analysis.

FIG. 9, 10. The release of ATP-maintained contraction by oligomycin and CCP, and the resultant effect on

adenosine triphosphatase. Concentrations: 3 mM ATP, 2 mM MgCI,, 1 mg/ml BSA initially; 2 ,ug/ml oligomycinand 10-', M CCP a(dded.

FIG. 11. The effect of CCP on oxidative phosphorylation and adenosine triphosphatase. The adenosine triphos-phatase studies were made with a separate lot of mitochondria in 0.2 M KCI + 0.02 M Tris (pH 7.5) 0.5 M sucrose.

FIG. 12. Swelling rate of corn mitochondria as a function of CCP concentration.FIG. 13. ATP-induced contraction of corn mitochondria as a function of CCP concentration. The CCP was

added initially, and contraction induced at 20 minutes with 3 m.t ATP + 2 IllM MgCl., -+ 1 mg/ml BSA.FIG. 14. The Mg requirement for ATP induced contraction, and the rapid contraction produced by succinate alone.

Mitochondria were allowed to swell for 20 minutes in the KCl-Tris medium (not shown), then contracted with 1 mMATP + 1 mg/mi BSA 1 mm MgCl, or with 8 mm succinate + 1 mg/ml BSA.

FIG. 15. The inhibition of succinate- and ATP-powered contraction by phosphate. Contraction initiated after 20minutes swelling in the KCl-Tris medium with 8 mM succinate or 3 mM ATP + 2 mm MgC12, both with 1 mg/mlBSA.

FIG. 16. The partial relief of phosphate inhibition of succinate-powered contraction by ADP acceptor plus a hexo-kinase trap. Concentrations: glucose, 5 mM; hexokinase 5 KM units/ml; MgCl2, 1 mM; succinate, 8 mM; BSA, 1 mg/ml; ADP, 1 mM; Pi, 5 mM.

FIG. 17. The failure of Mg and oligomycin to alter Pi inhibitioni of succinate-powered contraction. After 20 min-utes swelling in the KCl-Tris medium contraction was initiated with 8 miM succinate + 1 mg/ml BSA. Concentra-tions of additives: 1 mM MgCl,; 1 jug/ml oligomycin (added initially) 2 mmI (upper experiment) and 1 mM(lower experiment) potassium phosphate, pH 7.5.

FIG. 18. Contraction obtained with various concentrations of NAD, anid the enhanced contraction resulting fromsubsequent addition of 8 mm malate.

FIG. 19. Inhibition of NAD contraction by cyanide, and the ineffectiveness of cyanide in preventing ATP-poweredcontraction. Concentrations: NAD, 5 niim; KCN, 5 mM; ATP, 3 mI; MgCl,, 2 mm; 1. mg/ml BSA was added witlthe NAD.

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PLANT PHYSIOLOGY

Ihex- isuialize respirationl to result in an exchange ofHF- for C+'. releasing Ca'- to react with acidic phos-pliolipids.

Discussion

Cornl iimitochondria clearlI show a spontaneousswelling ini KCl, hut not sucrose. Only after agingand treatmenit with a chelating agent cani the niito-chonidria swell in sucrose. The swelling is assumedto l)e accomiipanied by penetrationi of the suspendingsolute, KCI in the experimlenits to be discussed here.Spontanieous swelling must be differentiated from the

,Succinateftpyruv te

.6 .10

add~~~~~~~~~~~~~J

~~~~~~~~~~~~~~~~~No

0

~~~~~~~0 _

0N

0

apidl osmllotic' adijustnielit. svhiclh we have minimize(dhb uisinig miiedia isoosImiotic with the isolation imiedium.Spontaneous swelliing is slowxT alnd appears to reflectthe osmilotic adjuistmllenit wN-hiclh follows soltute p)enietrKl-tion anid the relaxastion of sonic conltractile mileclhanlismii.Water einters with relaxation and(1 is expelled oni re-establishment of thle conitracted state (fig 2), a proc-ess which reqtires ellergy from hydrolysis of ATP oroxidationi of substrate. Spontaneous swelling is niotin. itself deleterious to subsequent oxidative phos-phorylation, although there does appear to be someinhibition of electroni transport if swellilng occurs inthe absence of BSA (table I).

-7 - 6 -5 -4 0 10 20 30LOG MOLARITY CCP MINUTES

substrate 2eg/ '\ q_ _DDNP CCP_- r

spontaneous

ATP

oligomycin Mg

ADP

24

controcted- state

p

MINUTES

FIG. 20. Induction o1f swelling by phosphate and cyanilec, aii(l the additional s elling indluce(i by the uncoupler(;CP in cyanide inhibited mitochondria. Mlitochondria maintained in cintracted state w-ith 8 iuls each of succinateandl pvruvate; swelling induced with 5 mni l otassium phosphate or 1 mni KCN + 10-'; m CCP.

FIG. 21. The effect of CCP on contraction initiated by 8 mM-,\ succinate 4- 1 mg/ nil BSA. Previous swelling wasfor 20 minutes in the KCl-Tris medium.

FIG. 22. The inability of succiniate-pyruvate (8 miM eachl) to maintaiin contraction above pH 7.5. Medium was0.2 M KCI -L- 0.02 Tris-maleate buffer with the pH aCijuste(d as iIlicated.

FIG. 23. Citrate-induced swelling of mitoch-ondria maintaine(l in the unnswollen .;tate s' ith 0.4 \i suicrose. Citrate(1 m-m adjusted to pH 7.5 uvitli KOH) added at tinme intervalk ii-dicated.

FIG. 24. A scheme to explain the relationship between pxhdatvx j'husdjrylation, adenosine triphosphatase, veliivzand mntraction.

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STONEIR AND HANSON SWEILLING IN CORN MITOCHIONDRIA

TI'he electron micrographs indicate that the mecha-inismil is associated with the innier meml)rane. Micro-graphs of liver mitochondIria also show the changesto be associated with the innier membrane and matrix(20).

There are 2 principal distinctions to be miiade be-tween the behaviour of corn imitochonidria anid mostanimal preparationis: No swelling agents are nieeded,and contraction can be obtained with substrate alone(i.e., nonphosphorylating respiration).

'With respect to spontaneous swelling, we can onlysuggest that the corn mitochondria must be very per-meable compared with most animal preparations.For instance, corn mitochondria oxidize exogenousDPNH very rapidly (data to be published separately)which is a characteristic of many plant preparations(8) but not of tightly coupled animal mitochondria(16).

The only report we know concerniing nonphos-phorylating substrate-induced contraction in animalmitochondria is the recent work of Lynn et al. (20).In this case, rat liver mitochondria were allowed toswell in succinate. Upon the addition of EDTA,there was a rapid contraction which was antimycin-sensitive, oligomycin-insensitive. These are similarto the results we have obtained, and it is of interestthat our isolation and washing media include EDTA.The differences exhibited by corn and liver mito-chondria may be a reflection of isolation procedures,rather than of intrinsic properties.

It is generally realized that there must he someconnection between the high energ- intermediatesystem of oxidative phosphorylationi and contrac-tion. A schematic representation of the relationshipas we deduce it to occur in corni mitochondria isshown in figure 24. It is a miiodified type I schemewhich we have used and explained previously (9, 29).The scheme makes no attempt to depict how highenergy intermediate formation is linked to electrontransfer.

The scheme attril)utes contraction to a mechanismassociated with the nonphosphorylated high energy in-termediate. It is assumed that the degree of contrac-tion is a funietion of the relative amount of intermedi-ate in this state. In the absence of substrate or ATP,spontaneous hydrolysis of the intermediate resultsin swelling. The addition of phosphate acceleratesthe breakdown of the intermeediate hy phosphorolysis.The oxidation of suibstrate directly reestablishes theintermediate (and expulsioni of water follows, pre-sumably at a slower rate), but in the presence ofphosphate there is a rapid colnversion to the phos-)horylated intermediate. In the presence of a trap.the phosphate is transferred to ADP. regenerating theintermediate for recoupling. This accounts for thesparing-action of a trapping system oni the phosphateinhibition of contraction (fig 16).

The production of the nonphosphorylated highenergy intermediate fromii ATP is indicated to beaccompanied by a stoichiometric yield of phosphate.The inhibitory effects of added phosphate on ATP-

powered contraction (fig 15) couild be attributed tomnass action. The site of oligomycin action in block-ing ATP-powered contraction and adenosiine triphos-phatase is the same as that depicted by Packer et al.(24) and Lardy et al (14); i.e., in the transfer ofphosphate between ATP and the phosphorylated inter-mediate. 0Oligomycin cannot act in preventing phos-phorylation of the intermediate, as is thought to betrue for liver initochondria (28), for it does not pre-vent the phosphate inhibition of contraction (fig 17).The respiration in the presence of oligomycin (tableII) can be accounted for by assuming that there is anaccelerated spontaneous hydrolysis of the nonphos-phorylated high energy intermediate when the normalADP-acceptor system is blocked: this short circuitwould recycle the intermediate providing for someelectron flow.

The continued release of Pi from ATP after con-traction is accomplished is assumed to result from con-tinued spontaneous hydrolysis of the nonphosphoryl-ated intermediate (fig 8, 24).

There is one set of observations which is notreadily explained by the scheme. The uncoupling ac-tion of CCP (or DNP) is generally considered to bein the hydrolysis of the nonphosphorylated highenergy intermediate (34), which would account forthe CCP-stimulated adlenosine triphosphatase (figI1). If this intermediate is closely linked to the con-tracted state, acceleration of swelling with CCPshould be obtained with the uncoupling concentrationsof 10- M (fig 11). However, CCP concentrations10 to 100-fold higher are needed to accelerate swelling(fig 12) andl inhibit contraction (fig 13 21). Ina physiological sense, concentrations of uncotupler areneeded vllich begin to suppress respiration. The sig-nificance of this is nlot clear to us, although a reason-able postulate would be that the inhibited respirationiis a reflectioni of a loss of membrane semipermeability.

The oligomycin-inhibited, CCP-stiniulated adenios-ime triphosphatase (table II and fig 11) must reflectthe loss of Pi from ATP through the intermeediatesystemii. The fact that the CCP-stimulated adenoosinietriphospphatase is not found in the presence of highlevels of sucrose could be due to a limitation by su-crose on the entry of ATP into the mitochondrialadenosine triphosphatase sites. This effect of su-crose was noted earlier by Johnson and Lardy (13)for several Krebs cycle acids, and has been confirmedin our laboratory.

At the present time, wve can go no fturther in cor-relating and explaining the results. Tlhere is obvi-ouslv some very important connection between high-energy intermediates and membrane fuInctioIn, but itwill not be resolved until the biochemistry of the in-termediates is described. Correlative studies are alsoneeded on ion transport accompanying swelling andcontraction, for it is by no means certain that thesquiggle used to designate the nolphosphorylated highenergy intermediate (fig 24) really represents a co-valent bond. The charge separation theory of Mit-chell (22) could be invoked to explain contraction as

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PLANT P11'Sll IA)GV

a.ssociated with remioald of 11 and ()11 ions froi ahydrophobic adenosiine triphosphatase center.

Literature Cited

1. BRIERLEY, G. P. 1964. Membrane permeability andion translocation in mitochoindria. Abstr. 4th Ann.Meeting Am. Soc. Cell. Biol. p) 13A.

2. CHAPPELL, J. B. AND G. D. GRTFVITLLE. 1963. Theinifluence of the conliosition of the suspendingmedium on the properties of mitochondria. Symp.Biochemii. Soc. (Camnbridge) 2.3: 39-65.

3. CILELAND, K. W. 1952. Permneability of isolate(Irat heart sarcosomes. Nature 170: 497-99.

4. DILLEY, R. A. 1964. ight-induce(l potassium ef-flux from spinach chloroplasts. Biocheml. Biophys.Res. Commun. 17: 716-22.

5. ERNs .IER, L., C;. DAI.I NER, AND G. F. AZZONE.1963. Differential effects of rotenone anid amytaloni mitchondrial electroni andc energy transfer. J.Biol. Chem. 238: 1124-31.

6. 1 AKE, C. H. AND Y. SUT713ARONV. 1925. Thle colori-metric determination of p)hosplioris. J. Biol.Chem. 66: 375-400.

7. FRITZ, G. AND A. W. NAYILOR. 1956. Phosphoryl-atioin accompanying suicciniate oxidation by imito-chondria from cauliflower buds and mung beanseedlings. Physiol. Planitartum 9: 247-56.

8. HACKETT, D. P. 1959. Respiratory mechanisms inhigher planlts. \Nnn. Rev. Plantt PhYsiol. 10: 113-46.

9. Hoi;( s, T. K. ANT) J. TB. HT\NT!ON. 1956. Calciumaccumulation by nlaize niito-chondria. PlantPllysiol. 40: 101-09.

10. HTONDA S. T. ANDx:.\. . MIF NSTFR. 1960. Optic-allymeasuire(d and lack-el volume of lupine mito-chondria. Arch. Biochimi,. Biooihv.s. 88: 118-27.

1 1. HIUNTITR, F. F. JR., A. SCoTT, P. F. HOFFSTEN, J. M.GFDICKr, T. WTETNSTEIN, .\\N') .\. SCHNEIDER. 1964.Studies on the mleclaniisiim of swx elling, lysis, anddisintegration of isolated liver initochondria ex-pose(l to mixtures of oxidize(d and(l reduiced( gluta-tione. J. Biol. Chemz. 239: 614-21.

12. JACKSON, K. L. AND N. PACE. 1956. Some per-mleability properties of isolated rat liver cell mito-choindria. J. Gen. Physiol. 40: 47-71.

13. JOHNSON, D. AND H. A. LARDY. 1958. Substrate-selective inihibitioin of imiitoclioid(lrial oxidations byenhanced tonicity Nature 181 701-02.

14. LARDY, H. A., J. IL. CONNELLY, AND D. JOHNSON.1964. Inhibition of phosphoryl transfer in mito-chondIria by oligomycin and aulrovertin. Biochemii-istry 3: 1961-68.

15. LFEHNINGER, A. L. 1962. Water uptake and ex-trlSiOn by mitochondria in relation to oxidativep)llosphorylation. Physiol. Rex,. 42 467-517.

1,. IJ:11 NINGER, A. 1. 1964. The Mitochondrion. Ben-jamin, New York.

17. I.FININGNFR, A. L. AND 1 L. F. REMIMFRT. 1959. Anienlogenous uncoupling and swelliing agent in livermitochlouidria and its enzymic formation. J. Biol.Chemll. 234: 2459-64.

18. LYONS, J. M. AND H. K. PRATT. 1964. An effect(of ethylene on swelling of isolated mitochondria.Arch. Biochem. Biophys. 104: 318-24.

19. LYONS, J. M., T. A. WHEATON, AND H. K. PRATT.1964. Relationship between the physical nature of

of iiitochoiidrial membranes and cliilling sensitivityin plants. Plant Physiol. 39: 262-68.

20. LYNN, W. S. JR., S. FORTNEY, ANI) R. H. BROwN.1964. Role of EDTA and metals in mitochon(drialcontraction. J. Cell. Biol. 23: 9-19.

21. MACFARLANE, M. G. AND) A. G. SPlENCER. 1953.Changes in the water, sodiumii and potassina coni-tent of rat-liver mitochondria durinlg metabolisml.Biochem. J. 54: 569-75.

22. M\ITCHELL, P. 1961. Couplinig of plhosphorylationto electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nature 191: 144-48.

23. MOoRE, D. H. AND P. M. GRINLEY. 1957. Prob-lems in methacrylate embedding for electron nmicro-scopy. J. Biochem. Biophys. Cytol. 3: 255-60.

24. PACKER, L., R. H. MARCHANT, AND Y. M+UKOHI.RITA.1963. Coupling of energy-linked functions in niito-chondria and chloroplasts to the control of memii-branie structure. In: Energy Linked Funct ons ofMitochondria. B. Chance, ed. Acadenmic Press.p 51-73.

25. PARDEE, A. B. AND V. R. POTTER. 1948. Inthlibition1of sticcinic dehydrogenase by oxalacetate. J. Bio].Chem. 176: 1085-94.

26. PRICE, C. A., A. FONNESU, AND R. E. DAVIES. 1956.Movement of water and ions in mitochondria. Bio-chem. J. 64: 754-68.

27. REID, H. B. JR., A. C. GENTILE, AND R. A{. KLEIN.1964. Adenosine triphosphatase activity of cauli-flower mitochondria. Plant Physiol. 39: 1020-23.

28. RoSSI, C. S. AND A. L. LEHNINGER. 1964. Stoi-chiometry of respiratory stimulation, accumulationof Ca"+ and phosphate, and oxidative phosphoryl-atioIn in rat liver mitochondria. J. Biol. Chenm.239: 3971-80.

29. STONER, C. D., T. K. HODGES, AND J. B. HANSON.1964. Chloramphenicol as an inhibitor of enlergy-linked processes in maize mitochondria. Nature203: 258-61.

30. TEDESCHI, H. 1959. The structure of the mito-chondrial membrane: Inferences from permeabilitxproperties. J. Biophys. Biochem. Cytol. 6: 241-52.

31. TEDESCHI, H. 1961. Osmotic reversal of niiito-chondrial swelling. Biochem. Biophys. Acta 46:159-69.

32. TEDESCHI, H. AND D. L. HARRIS. 1955. The osIm-otic behavior and permeability to nonelectrolytes ofmitochondria. Arch. Biochem. Biophys. 58: 52-67.

33. TEDESCHI, H. AND D. L. HARRIS. 1958. Some ob-servations on the photometric estimation of mito-chondrial volume. Biochem. Biophys. Acta 28:392-402.

34. ITTGNAIS, P. V. 1963. Nonphosphorylate(l inter-mediates in oxidative phosphorylationi. Biochinii.Biophys. Acta 78: 404-19.

35. WFDDING., R. T. AND M. K. BLACK. 1962. Re-sponse of oxidation and coupled phosphorylation inplant mitochondria to 2,4-dichlorophenoxy-aceticacid. Plant Physiol. 37: 364-70.

3f). WERKHEISER, WT. C. AND W. BARTLEY. 1957. I[hestudy of steady-state concenitrations of internalsolutes of mitochondria by rapid centrifugal trans-fer to a fixation medium. Biochem. J. 66: 79-91.

37. WVARD, R. T. 1958. Prevention of polymerizationdamage in methacrylate embedding media. J. His-tochem. Cytochem. 6: 398.

38. WOJTEZAK, L. V. AND A. L. LEHNINGER. 1961.Formation and disappearance of an endogenous un-coupling factor during swelling and contraction ofmitochondria. Biochim. Biophys. Acta 51: 442-56.

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