Oxidative phosphorylation in mitochondria isolated from Euglena gracilis

~toci*:,,:~tlC.¢ E'~ mOPH~StCA ACTA

9

BBA ~!:5132 O X I D A T I V E PHOSPHORYLATION IN MITOCHONDRIA ISOLATED FROM EUGLENA GRACILIS

D. E. BUETOW ~._~uP. J. BUCHANAN Gerontology B,.aneh, National Heart Institute, National Institutes of He~lth, J~ublic Healtt, Semite, U.S. Department o/HeaL*h, Education and Welfare, E.~thesda, Md., and the B~lg,imore City Hospitals, Baltimore, Md. (U.U.A.) (Received July 28th, t9(~4}

SUMMARY

Oxidative phosphorylation h ~ been demonstrated in mitgchondria isolated from Euglena gracilis with a variety of substrates including NADH,~, but not NADPH,v Both oxidation and phosphorylation were inhibited by DNP .... ..~ cyanide ,riCh phosphorylation being the more sensitive. The effects of amytal, roten'o~ ~: and antimycin A parallel their effects in mammafian mitochondfia with r~,teno~e the most inhibitory. The measured P:O ratios showed one less phosphory]ation per g~atom oxygen than usually observed in mammalian mitochondria. The results suggest tile absence, or the presence in limiting amount, of some component in the electron transport chain of these mitochondria.

INTRODUCTION

Oxidative phosphorylation has been observed in cell-flee sys~ ~ms isolated from bacteria and yeasO. In general, the P:O ratios measured with b ~.terial and yeast preparations are lower than those found with mitochondria from II, ~,~mmalian tis:ues. Although increasing attention has been given to such studies wittL ~acterial systems, little attention has been given to the isolation of mitochondria ant -elated o>'~dative phosphorylation studies in protozoa. BYFIELD et al. ~ obtained a mitt c aondriat fraction from Tetrahymena pyriformis and NISHI AND SCIIERBAUM3 observed oxidatJ,m phosphorylation in Tetrahymena mitocllondrial preparations with fl-hy,d.:oxybat:rate as substrate. The measured P:O ratio was low and these mitochondr; ~ w~re somewhat resistant to 2,4-dinitrophenol-induced uncoupling of phosphorylafi )~zfrom oxif~atkm. ANDRESEN AND MUSHE'I~[4 isolated mitochondria from the amoeba, ~Thaos chaos:, but did not report any biochemical measurements. KLEIN AND NEFF s ~, ..lncl the osmotic properties of mitochondria from A canthamoeha sp. to be similax to those from mammalian tissues, bl!t presented, no tin,her biochemical measu~re~r~ents, Recently, a technique for isolating fimctional mitochoudria from Euglena graci~ ;s wa.s repor ted~;q The present paper presents a study of oxidative phosphorylation ~:t~ m~tochondria Btorhim. Biophys. ~:~, 96 0:965) 9-x7

IO

D. E. BUETOW, P. J. BUCHANAN

isolated from Euglena as well as a study of the effects of various inhibitors on both oxidation and phosphorylation. MATERIALS AND METHODS

Organism and growth conditions Experimental and stock cultures of a streptomycin-bleached strain, SM-L1, of

Euglena gracilis (bacfllaris variety) were grown in the dark at room temperature to the late !ogarithmic phase of growth on a define,a medium s as describedL The use of streptomycin-bleached Euglena eliminates problems encountered with chloroplasts fo. ~,ldin the green strains as well as problems enrountered in isolating a mitochondrial fr~ction free of chloroplast contaminants.

Isolati, a of mitochondria ~¢11swere ground in a mortar and pestle for 30 sec. Mitochondfia were isolated and .,~shed twice (with centrifugation at 7ooo × g) and resuspended in sucroseEDTA-Tris buffer medium (pH 7.4) as described previouslyL Total mitochondrial protein was determined by the method of LOWRY et al. 9. Average yield was 23 mg miwchondrial protein per IO9 cells (o.6-1.o g dry weight).

Assay procedures Respiration was measured b3~ the conventional Warburg manometric method with air as gas phase, o.2 ml 5 N KOH in the center well and o.I ml z N HC10a in the sidearm to stop the reaction. Total incubation time, unless otherwise noted, was 3° rain including 6 min equilibration time. In calculating P :O ratios, the values for oxygen consumption were corrected by direct extrapolation for the 6-rain thermoequilibration period. Composition of the medium is given in the tables. Hexokinase (EC 2.7~i.x ) (Sigma, Type III) was used in excc.~s. NaF at 14.o or 6. 7 mM was used. Both concentrations yield the same results 7. Phosphate disappearance from the medium was used as a m e , a r e of phosphate esterification. Inorganic phosphate was determined by the method of FISKE ANDSUBBAROWl° with the 1:2:4-aminonaphthol sulfonic acid reagent described by KINGn.

Preparation of inMbitors Stock solutions of rotenone, 3.oo' IO-°a M in absolute ethanol, were prepared and used for further dilutions. Rotenone was added t~) the flasks in o.o2 ml of ethanol and the same amount of ethanol was added to controls. As is the case with rat-liver mitochondria 12, this amount of ethanol did not inhibit oxidation or phosphorylation. Stock solutions of antimycit~ A, 2.73. IO-a M in absolute ethanol, were made and diluted so that o.o2 ml ethanol contained the desked amounts. Sodium amytal was dissolved in a small amount of I N NaOH a~d diluted to the desired concentratioi~ with water. RESULTS

Co~,;pling of oxidation to phosphorflation The present Euglena mitochondria were capable of coupled phosphorylation but

Biochim. Biophys. Aaa, 96 (1965) 9-x7

OXIDATIVE PHOSPHG~YLATION IN EUGLENA

II

TABLE I E F F E C T O F P H O S P t l A T ~ ACCEPXOR ON RESPII~.ATIO~

Each vessel contained 6. 7 mM phosphate (pH 7.2), 6. 7 mM MgCI~, 14.o mM Nail, 5. 7 mM succinate and an amount of mitoehondfia containing 4.47 mg protein. In addition, vessel ~. contained 2 mM ATP, a n d as phosphate aece~tor. 0. 5 mg yeast hexokinase (Sigma, Type III) dissolved in o.x ml of 0.3 mM .~lr~c.'~e. Both ve~elc were made to a final volume of 3 ml ~ith suerose-TrisEDTAL Incubation, 30 rain, 30 °.

Vessel

Respiration (patom 0 per vessel)

Phosphorylation (l~molesP~ per vessel)

P :O

i. Suecinate z. Suceinate + phosphate ac,v~'ptor

~3.0 12.5

-12.9

-1.0

are n o t " t i g h t l y c o u p l e d " as a r e m a m m a l i a n m i t o c h o n d r i a '3 a n d cet.~ain p l a n t m i t o e h o n d r i a x4. T h e o x i d a t i o n o b s e r v e d w a s i n d e p e n d e n t o f t h e presenc~ o f a p h o s p h a t e a c c e p t o r s y s t e m (Table I). F u r t h e r , " t i g h t c o u p l i n g " was n o t o b s e r v e d w~,en t h e r a p i d p o l a r o g r a p h i c t e c h n i q u e o f CHA,nCn AND WH.LbXMS ~'~w a s u s e d o n E u g l e n a m i t o e h o n d r i a i s o l a t e d a s de.scribe& or as i s o l a t e d in t h e m e d i u m d e s c r i b e d by W t s ~ a c H A.~D BON~E~ ~4 for p l a n t m i t o c h o n d r i a .

Oxidative phosphorytation E u g l e n a m i t o c h o n d r i a e x h i b i t e d o x i d a t i v e p h o s p h o r y l a t i o n w[',k a v a r i e t y of s u b s t r a t e s ('['able II). T h e P : O r a t i o w a s a p p r o x . L o for l a c t a t e a n d s u c c ' q a t e ~n~I a p p r o a c h e d 2.0 for m a l a t e , g l u t a m a t e a n d a - k e t o g l u t a r a t e . P y r u v a t e alone was n o t oxidized. W h e n 0.2 m M m a l a t e was a d d e d to t h e p ) ~ u v a t e , o x i d a t i o n , n d p b o s TABLE II OXI DATlVl~ P H O S P I t O R Y L A T 1 O N

Each vessel contained complete reaction mixture including ATP and phosphate acceptor as given in Table I. Unless otherwise noted, each substrate was at 6. 7 raM. Mitoch,)ndria containing 4.39-5.69 mg protein were added. Final volume, 3 nil. Incubation, 3o rain, 3o°,

Additions

O,tygen uptake (,uatoms]mg protein)

Phosphate esterified (pmoles/mg protein)

P:O

Suceinate L-Malate L-Glutamate a-Ketoglutarate 1..actate Pyruvate L-Malate" L-Malate'* Pyruvate, L-malate ° Pyruvate, L-malate'*

2.I 7 1.78 1.31 z.I i 2.74 o

~.97 3-o4 2.o2 4.oi 2.62 --

0.9 I 1.7 I 1.54 1.9I 0.96

o

--

o.96 o.68 LI5

L58 0.84 x.91

1.64 1.24 L66

* Malato at 0.2 mM. *" Malatc at Lv mM.

Biochim. Biophys. A~t~:-90 (1965) 9-17

I2

D . E . BUETOW, P. J. BUCHANAN

phorylation with a P : O ratio of 1.24 was observed. This a m o u n t of malate alone showed too low of a respiration to be accurately measured b y the present manometric method. W h e n a higher concentration, 1.0 raM, of malate was added to the pyruvate, there was a small increase in oxidation a n d phosphorylation over the a m o u n t s observed with this concentration of malate alone, b u t the P : O ratio was the same in both cases. Under conditions given in Table II, ~-hydroxybutyrate was not oxidized a n d citrate and isocitrate were oxidized only to a small extent, i,e., o.I-o.2/~atom oxygen per rag mitochondria! protein per 3o min.

Reduced pyzidine nucleotides LEHNI~GER1~ demonstrated that exogenous NADH~ was oxidized by liver ~itochondria if cytochrome c was added and t h a t mitochondria swollen b y water reatment showed oxidative phosphorylation ~fith NADH 2 without added cytochrome ~.:.A relatively high concentration, 3-7 mM, of NAD (part of which was continuously redu~:ed b y means of ethanol and alcohol dehydrogenase) a n d o.oi M fluoride were cop,:i~'ions shown by MALEY1~ to yield phos,phorylation without pretreating the mi~,,ct~ondria. Under conditions similar to those used b y MALEY17, the present mitochondria also showed oxidative phosphorylation with externally added NADH~ TABLE III OXIDATIVE PHOSPHORYLATION

WITH REDUCED

PYRIDINE

NUCLEOTIDES

Each vessel contained complete reaction mixture including ATP and phosphate acceptor as given in Table I. In Expt. i, NADH a was added to the sidearm and tipped in after 6 rain equilibration. In Expt. 2, NADI~H2was added with mitochondria at the start of the experiment as in Table I. Mitochondria containing 2.i2 rng protein were added in Expt. i and 4.95 mg protein in Expt. 2. Final volume, 3 ml. Incubation at 3o~ for 24 nlin in Expt. I anti 3° rain in Expt. 2.

Expt. Reduced pyridine nudeotide

Respiration (~,atom 0 per vessel)

Phosphorylation (ttmo~esP~ per ves~:l)

P:O

I

2.34 1.54 o.59 0.42 3.07

0.88 0.48 0.24 o o

o.3S o.3I o.4I ----

2

NADH~ IO mM 3*3 mM L 7 mM I.o mM NADPH 2 IO mM

(Table IiI). Increasing concentrations of NAI)H,2 gave increasing oxidation and phosphorylation, but a low P : O ratio was observed and was about the same in each case where phosphorylation occurred. KAPLAN et aL as showed that ~he oxidation of Nz~,I)PH,, through the electron transport system of rat-liver mitochondria did ;,:)t lead t~ oxidative phosphorylation, I n the present case, N A D P H , was oxidized b u t no phosphorylation was observed ('fable III).

D N P inhibition LOOMIS AND LIPMANN19 showed that 5 "Io-S--2 "Io-4 M 2,4-dinitrophenol prevented phosphorylation without afflicting or slightly stimulating oxidation in prepa-

Biovhim, Biophys. Acta, 96 (I965) 9-t7

OXIDAI'IVE PHOSPF_ORYLAT1ONIN EUGLENA

.j IO0

".I/

/

o

]3

o

/

/,IM O~.At?. ~ P

9o

too

Fig. L 2,4-Dinitrophenol (DNP) inhibition of oxidation and phosphoryh~ti~n. Each vessel contained 6. 7 mM malate and complete reaction mixture including ATP and phosphate acceptor as given in Table 1 except that fluoride was used at 6. 7 raM. Mitochondria, 3.8c mg protein per vessel. Final volume, 3 ml. In.:ubation, 3o°, 3° min. rations from rabb~t kidney. I n the present system, z,4-dinitrophen~l was inhibitory to b o t h phosphorylation and oxidation (Fig. I) with malate as substra:te. However, phosphorylation was more sensitive ~han respiration. Complete inhibitit,,u of phosphorylation was noted at 50/~M z,4-dinitrophenol whereas oxidation was inhibited 37 % at this concentration. Increasing the concentration of e,4-dinitroph~nol to ro -4 M did not further inhibit respiration. The results with Euglena com[~are favorably with results sometimes observed in plant mitochondria z°. The data of HaCKErT AND HAAS21 on skunk-cabbage mitoehondria show t h a t Io -4 M 2,4-dinitror.L;~ol inhibits, the oxidation of a-ketoglutarate by 3 0 % and phosphorylation b y 9 o % . Cyanide inhibition Cyanide inhibited both oxidation and phosphorylation of Eugiena nfitochondria witil succinate as s~fl)strate (Table IV). Phosphorylation was more sensitive to cyanide iqtfibition than was respiration, particularly at a concentration of 3 IO-S M cyanide TABLE IV CYANIDE INI{IBITION

Each vessel contained 6.7 mM succinate and complete reaction mixture i,lcludmg ATP and phosphate acceptor as given in Table I except that fluoride was used at ~; 7 mM. Mitochondria, ,1.7I nag protein per vessel. Final volume, 3 ml. Incubation, 3o°, 3° min. Concentration of

Per cent inhibition

KCN (M)

Respiration

4 " I¢~-*

92

I • ~o-*

87 76 28

Ioo 97 45

30

3°

25 18 6

30 36 ~8

7" to-5 3 ~xo -~ I " I O-~

7" :o-~ 5" ~o-~ 3-Io -~

Phosphorylation

Biochim. Biophy~,, Acta, 96 (i965) 9-x7

D. E. BUETOW, P . J . BUCHANAN

I4

and above. Phosphorylation was virtually eliminated at 7" IO-a M cyanide whereas 24% of the respiration still remained. In contrast to the effect of 2,4-dinitrophenol (Fig. I), the effect of cyanide on Euglena mitochondria does not parallel results found with certain plant mitochondria. In these latter, respiration unaffected by even higher amo~mts of cyanide has sometimes been observed. For example, cyanide at 4.6. IO-4 M did not inhibit oxidation by skunk-cabbage mitochondria but did inhibit phosphorylati m by 44% (refs. 2o, 21). Arnytal inhibition Amytal acts on isolated mitochondria as an inhihitor of the aerobic oxidation of substrates linked to DPN without affecting that of succinatO ~,-°'. Respiration and phosphorylafion were measured in the presence and absence of amytal in the present mitochondria (Table V). In the control, oxidation was 1,5z/~atoms per nag mitochonTABLE V AMYTAL

INHIBITION

Each vessel contained 6.7 mM malate and complete reaction mixture including ATP and trimsphate acceptor as given in Table I. Mitochondria added, 2.29 mg protein per vessel. Final volume, 3 mL Incubation, 3o°, 3 ° rain.

(- ncentralion of ,tmvtal (raM)

Respiration (t,atoms 0 per vessel)

Phospho~Tlation (t~molesP per vessel)

o i.o

3.48 0.90

6.02 0.77

2.0

O

--

drial protein and was accompanied by a phosph:ae uptake corresponding to a P:O ratio of 1-73. Amytal at I nan reduced oxidation by 74% and phosphorytation by 87%. At 2 raM, amytal completely inhibited oxidation. The results compare well with the data of ERYS'rER et al3 "z who showed a 68% inhibition of oxidation ~4th o 9 mM amytal and complete inhibition with 1.8 mM amytal in rat-liver mitochondfia with glutamate as substrate. Rotenone inhibition Rotenone, like amytal, inhibits the aerobic oxidation of substrates linked to DPN without affecting that of succinate23, "4. The inhibition, which is independent of coupled phosphorylation 23, was localized to the electron transfer step between DPN and flavin~:~,24. ERXs'_rEI~et al. TM showed that rotenone, unlike amytal, was firmly bound to the electron transfer system and that the rotenone-sensitive "catalyst" appeared to be present at the lowest molar ratio among known components of the liver-rnitochondrial electron transport system. Fig. 2 shows that rotenone strongly inhibited the oxidation ofmalate by Euglena mitochondria. Complete inhibition was observed when the concentration of rotenone was 5" IO-7 M, Phosphorylation was more sensitive to rotenone than was respiration. With rotenone at 5" IO-8 M, respiration was inhibited by only 12% whereas phosphorylation was inhibited by 3o%. Biochim, Biophys. ,4eta, 96 (1965) 9-17

OX ,kTIVE PF, O~PHORYLATION IN EUGLENA I

~

I IIII I

I

I

I5

I

--o

tO0

z,

~

III

/

m

/

O I O "~

tO °~" IO'* KOTENONI~ , M O L A [ ~ I T Y

10"~

Fig. 2. R o t e n o n e inhibition of respirzttion. E a c h vessel contained 6 7 m M malate a n d complete reaction m i x t u r e including A T P a n d p h o s p h a t e acceptor as given, ~n Table, I. Mi~ocbondria, 2.03 tag protein per v e s s e l Final volume, 3 ml. I n c u b a t i o n , 3o °, 30 rr, i~.

Antimycin A inhibiffon Antimycin ~ inhibits succinate oxidase activity 25and blocks the respiration of phosphorylating ;nitochondria almost completely26. Its effect has been localized between cytoctzcomes b and cz in the respiratory chain ~ The "sigmoid" type of titration curw~ observed with antimycin A has been inter[,reted by Tt~ORN~ as indicating that the antimycin-sensitive "catalyst" is present i~ the mitochondria at a capacity in I;~rge excess of the capacity of the respiratory chair~ i.e., that 9o'}~ or more of the "'catalyst" may be blocked and full respiration can sti~i' proceed. II

8

I ......

-I C

O

-

:-Z

6

~

p MOLAR A N ~ Y C I N A

14

Fig. 3. A n t i m y c i n A inhibition of oxidation and phosphorylation. Eacl~ vessel contained 6. 7 mM succinate a n d complete reaction m i x t u r e including A T P a n d phosph~.te acceptor as given in Table I. Mitochondria, 4,18 m g protein per vessel. Final volume, 3 m? Incubation, 3 o°, 30 rain.

Fig. 3 shows that antimycin A inhibited respiration and t ,:aosphcrylation shown by Euglena mitochondria with succinate as substrate. A max~,~ um effect on respira, tion was observed at a concentration of 2-3/~M antimycin A ; ;~¢.wever, a low respiration was present even at the highest amount of inhibitor used. I:~ contrast, phn~phorylation was virtually eliminated at 0.9/~M inhibitor. In ft1~'~h~r expcfiments, the Biochim. B i o ~ ~,s. ~cla, 96 {I965) 9-I 7

16

D. E. BUETOW, P. J. BUCHANAN

inhibition ¢~f res0iration was observed at even lower concentrations. At o.0 9 /,M inhibitor, resviration was still inhibited by 5I % ; however, at o.o2/~M (o.oi/~g/ml), the inhibitor did not affect respiration.

DISCUSSION

Oxidative phosphorylation has been demonstrated in a particulate fraction isolated from Euglena gracilis with a variety of substrates including NADHz, but not NADPH v The present Euglena m,~tochondria did not show "tight coupling", but were capable of coupling phosphorylation to oxi ~ation. However, the P:O ratios observed were towerthan the ratios reported t'or mammalian systems, in these latter observations, Euglena is closer to bacterial and yeast oxidative phosphorylations than to mammalian x. Both oxidation and phosphorylation of Euglena mitochondria are inhibited by z,4-dinitrophenol and cyanide (Fig. I, Table IV). In both cases phosphorylation is raore sensitive than oxidation. The z,4-dinitrophenol effect is similar to that some dines found in plants~°, 21. The effects of amytal, rotenone and antimycin A parallel the action of these inhibitors in mammalian mitochondrial*, 22.*~ with rotenone the most inhibitory (i.e., in terms of concentration) followed in order by antimycin A and amytal. These results indicate that those factors sensitive to these latter inhibitors in mammalian mitechondria are present also in Euglena mitochondria. A P :O ratio of about I.O was measured in the present system with succinate as substrate as compared lto a P:O of about 2.o commonly observed in mammalian mitochondria. Addition of the supernatant obtained from the isolation of Euglena rnitochondria did not stimulate respiration of these particles and did not improve the P:O ratio when succinate was the substrate'. Further, a P:O of about z.o was measured with malate and glutamate, for example, wh~;ch again indicates one less phosphorylation than usually observed in mammalian systems. It has been suggested that the low P:O ratios shown by bacterial and yeast preparations result in part from the loss of some component in the electron transport chain~, zS, Evidence that this is the case with Euglena has been presented recently ~9,z°. PERINI et al. '''~,ao were able to show a cyanide-sensitive a-type cytochrome ("cytochrome 605") as well as a c-type ("cytochtome 556") but not a b-type cytochrome in mitochondrial fractions from both green and bleached Euglena. A b-type cytochrome ("cytocbrome 561") associated with chloroplasts in Euglena zl was found only in those Euglena strains capable of producing functional chloroplasts z°, which the present strain cannot do sz. The absence of a b-type cytochrome in mitochondria would result m the loss of one phosphorylation during the oxidation of various substrates as observed in the present experiments. However, the present Euglena mitochondria are ~ntimycin A-sensitive and this inhibitor is known to function in the region of cytoch 9me b in marnmalian mitochondriaa3, '~4. Thus, it is also feasible that a b-type cyto, :irome (or even some other component of the electron transport chain), is present but in limiting amount, in these mitochondria. A detailed spectral and chemical analysis of the cy!ochromes present in isolated Euglena mitochondria i~ necessary to resolve the question. Phylogenetically, Euglena holds a key taxonomic position, transitory between plants and animals (ref. 32, p. 29). Further studies to delineate the electron t~ansport Biochim. B~ophys. Acta, 96 (I965) 9--~7

OXIDATIVE PF"~)SPHORYLATION IN EUGLENA p a t h w a y a s ~,ell a s a s s o c i a t e d m e c h a n i s m s p r o v e usefifl.

17 of phosphoryl~ti

~tz o f E u g l e n a

should

ACKNOWLEDGEMENT

The authors th~nk Dr. D. R. ~oANADIfor many helpful suggestions. REFERENCES r A. F. BRODIE, in S. P. COLOWZCKAND N. O. KAPLAN, Methods in E~zvmology, Vol. 6, Academic F r e ~ , New York, I963~ p. 284. 2 J. E. BYFIELD, S.-C. C~IOU AND O. H. SCHERBAUM, Biochem. Biophy+. Res. Commun., 9 (t962) 226. 3 A. z~ISHI AND Q~ H. SCHERBAU*I, Biochim. Biophys. Acta, 65 (1962i~ 2t19. 4 N. ANDRESEN AND C. x*V.MUSHETT, Compt. Rend. Tray. Lab. Cav~*s~ev'g,33 (1963) 265. 5 R. L. KLEIN AND F.. J. NEFF, Expa. Cell. Res., 19 (196o) 133. 6 D. E. BUETOW AXD P, J. BUC.~tANAN,J . Cell Biol., I9 (1963) to A. 7 D. E. BIJETOW *-~NDP. J. BUCHANAN, Exptl. Cell Res., 36 (I964) 2o;~ 8 D. E. BUETOW AND G. M. PADILLA, J. Proto:ooL, 1o (1963) 121. 9 O. H. LOWRV, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1953)

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Biochim. Bio ~Jys. Acta, t~,6 (1965) 9-17