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Felice AzzoneLars Ernster, Gustav Dallner and Giovanni Energy TransferAmytal on Mitochondrial Electron and Differential Effects of Rotenone andARTICLE:1963, 238:1124-1131.J. Biol. Chem. http://www.jbc.org/content/238/3/1124.citationAccess the most updated version of this article at ...  
Felice AzzoneLars Ernster, Gustav Dallner and Giovanni Energy TransferAmytal on Mitochondrial Electron and Differential Effects of Rotenone andARTICLE:1963, 238:1124-1131.J. Biol. Chem. http://www.jbc.org/content/238/3/1124.citationAccess the most updated version of this article at .SitesJBC AffinityFind articles, minireviews, Reflections and Classics on similar topics on the Alerts: When a correction for this article is posted• When this article is cited• to choose from all of JBCs e-mail alertsClick here http://www.jbc.org/content/238/3/1124.citation.full.html#ref-list-1This article cites 0 references, 0 of which can be accessed free at by guest on October 17, 2013http://www.jbc.org/Downloaded from THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 238, No. 3, March 1963 Printed in U.S.A. Differential Effects of Rotenone and Amytal on Mitochondrial Electron and Energy Transfer * LARS ERNSTER, GUSTAV DALLNER, AND GIOVANNI FELICE AZZONE From the Wenner-Gren Institute, University of Stockholm, Stockholm, Sweden, and the Unit for the Study of Physiopathology, Institute of General Pathology, University of Padova, Padova, Italy (Received for publication, April 2, 1962) Rotenone, a plant product with wide commercial use as a fish poison, has been shown by Fukami and Tomizawa (1, 2) and by Lindahl and oberg (3, 4) to act on isolated mitochondria as powerful inhibitor of the aerobic oxidation of substrates linked to diphosphopyridine nucleotide without affecting that of suc- cinate. The inhibition, which was independent of the presence of coupled phosphorylation (3), could be localized to the elec- tron transfer step between diphosphopyridine nucleotide and flavin (3, 4). These effects of rotenone resembled those pre- viously described with Amytal (5-7). By undertaking a fur- ther comparison of the effects of the two compounds, in the pres- ent study, we found that rotenone duplicates all of the known effects of Amytal on electron transport. In striking contrast, however, is its lack of effect on a number of Amytal-sensitive energy transfer reactions, such as the dinitrophenol-induced adenosine triphosphatase (8, 9) and the inorganic orthophos- phate-adenosine triphosphate exchange (10) reactions. Data re- lating to these observations are presented in this paper, and some of their implications are discussed. It is also shown that rote- none, unlike Amytal, is firmly bound to the electron transfer system, thus rendering possible a “titration” of the rotenone- sensitive catalyst. Calculations derived from such titrations indicate that the rotenone-sensitive catalyst is present at the low- est molar ratio among known components of the liver-mitochon- drial electron transport system, and probably constitutes the rate-limiting catalyst during the aerobic oxidation of mitochon- drial DPNH in the presence of phosphate and phosphate accep- tor. EXPERIMENTAL PROCEDURE Preparative Procedures-Rat liver mitochondria were prepared as described previously by Ernster and Low (11) and washed twice with 0.25 M sucrose. Mitochondrial protein constituted approximately 20 mg per g of liver, wet weight, as determined by the biuret method (12). Submitochondrial particles with DPNH oxidase activity (13) were prepared by adopting the procedure described by Kielley and Kielley (14) for preparation of ATPase. Mitochondria from 40 g of liver were suspended in 55 ml of 0.003 M phosphate buffer, pH 7.5, and treated for 2 minutes in the cold with an Ultra Turrax blender (24,000 r.p.m.). Large particles were re- moved by centrifugation in a model L Spinco ultracentrifuge, No. 40 rotor, at 20,000 x g for 10 minutes. The supernatant * This work has been supported by grants from the Swedish Cancer Society. solution was recentrifuged at 105,000 X g for 30 minutes, and the sediment was resuspended in 0.003 M phosphate buffer, pH 7.5. The suspension was used in the DPNH oxidase assay. Pretreatment of mitochondria with rotenone or Amytal was made by adding 4 X lo-’ M rotenone or 0.002 M Amytal (final concentrations) to suspensions of mitochondria in 0.25 M sucrose (the suspension contained mitochondria from 100 mg of liver, wet weight, per ml). The additions of rotenone and &4mytal were made in 0.2 ml of ethanol per 10 ml of suspension, and a control containing ethanol only was run. The samples were al- lowed to stand for 10 minutes at O”, and then centrifuged at 8000 X g for 15 minutes. The mitochondria were resuspended in 0.25 M sucrose and recentrifuged. Submitochondrial particles were pretreated by adding 1OV M rotenone or 3 X lo+ M anti- mycin A (final concentrations), both in 0.2 ml of ethanol, to lo-ml aliquots of particle suspension in 0.003 M phosphate buffer, pH 7.5 (the suspension contained particles from 1 g of liver per ml). A control was again run with ethanol. Recentrifugation was car- ried out at 105,000 x g for 30 minutes, and the surface of the pellet was rinsed carefully with phosphate buffer. d4ateriuZs-Rotenone, &HZ206 (for structural formula, see The Merck Index), manufactured by S. B. Penick and Company, New York, was a kind gift of Dr. K. E. oberg, Uppsala. Stock solutions of rotenone, 0.5 to 1 X 1O-3 M in absolute ethanol, were prepared and used for further dilutions. Rotenone was added to the samples in 0.02 ml of ethanol. The same amount of ethanol was added to the samples incubated without rotenone. This amount of ethanol had no effect on the reactions studied (cf. Table I). Solutions of sodium Amytal were prepared in water. Oligomycin was generously supplied by Professor Henry 9. Lardy, and coenzyme Qo, by Professor D. E. Green, Madison, Wisconsin. Coenzyme Q9 (Merck), vitamins K1, Kz, and K3 (Hoffmann-La Roche), and all other reagents were commercial products. Assay Procedures-The composition of the medium for meas- uring respiration and phosphorylation was as follows (unless otherwise specified): 0.025 M P i32, pH 7.5, 0.008 M MgClz, 0.01 or 0.02 M substrate, 0.001 M ATP, 0.03 M glucose and hexokinase in excess, 0.05 M KCl, 0.05 M sucrose, and mitochondria were added to each vessel as described in the legends for the figures and tables. Respiration was measured by the conventional Warburg method, with air as gas phase and 0.2 ml of 2 M KOH in the center well. Phosphate uptake was determined by the isotope distri- bution method recommended by Lindberg and Ernster (15). In calculating P:O ratios the values for oxygen consumption 1124 March 1963 L. Ernster, G. Dallner, and G. F. Axxone 1125 were corrected by direct extrapolation for the 5-minute period of thermoequilibration. Reduction of acetoacetate by succinate or by pyruvate plus malate (16) was assayed at 30” in a system containing, in a final volume of 2 ml, 0.01 M succinate or 0.01 M pyruvate + 0.01 M L-malate, 0.0042 M acetoacetate (prepared and standardized according to the method of Krebs and Eggleston (17)), 0.05 M KCI, 0.008 M MgClz glycylglycine buffer, pH 7.5, and 0.05 M sucrose. The samples were fixed with 1 ml of 1.5 M perchloric acid and then centrifuged, and acetoacetate was determined by the method of Walker (18). Pi-ATP exchange was measured according to the procedure of L6w et al. (10) with minor modifications (see legend of Fig. 2). ATPase activity was determined according to the method of Lijw (9). DPNH oxidase activity of the submitochondrial particles was determined by following the oxidation of DPNH at 340 rnp in a Beckman DK2 spectrophotometer. The assay system con- tained 1.33 X 1O-4 M DPNH and 0.05 M phosphate buffer, pH 7.5, in a final volume of 3 ml. The temperature was 22”. RESULTS E$ects of Rotenone on Electron Transfer Oxidation of Pyridine Nucleotide-linked Substrates and Suc- cinate-Rotenone was found to inhibit respiration with gluta- mate, but not with succinate, as substrate (Table I), in agreement with earlier reports (l-3). With the amount of mitochondria used, 5 x 10-S M rotenone inhibited glutamate oxidation by ap- proximately two-thirds without appreciably lowering the P : 0 ratio. Under the same conditions, succinate oxidation was not affected by a rotenone concentration of lo+ M; the slight de- crease in the P: 0 ratio with succinate at higher rotenone concen- trations may be attributed to an inhibition of the further oxida- tion of fumarate, formed during succinate oxidation (cj. also Table V). Rotenone also inhibited the oxidation of other pyridine nucleo- tide-linked substrates, such as pyruvate, malate, a-ketoglutarate, and ,&hydroxybutyrate. As was found previously with Amytal, the inhibitory effect of rotenone was not relieved by an uncou- pling concentration of 2,4-dinitrophenol (19, 20), and only to a low extent by added DPN and cytochrome c (21). Oxidation of Extramitochondrial DPNH-Lehninger (22, 23) first demonstrated that liver mitochondria catalyze two types of oxidation of exogenous DPNH: one through the phosphorylating respiratory chain, and another, nonphosphorylating reaction that requires externally added cytochrome c and supposedly takes place at the surface of the mitochondria. The two path- ways were found later to differ also with respect to their response to Amytal (6, 21, 24) and antimycin A (25-27), which inhibit only the “internal,” phosphorylating pathway but not the “ex- ternal,” nonphosphorylating one. Data in Table II compare the effects of rotenone and Amytal on the oxidation of exogenous DPNH. Continsous reduction of added DPN was achieved by means of ethanol and alcohol dehydrogenase. A relatively high concentration of DPN, 5 mM, was used, and 0.01 M fluoride was included in the reaction mix- ture, since these conditions have been shown by Maley (28) to yield maximal phosphorylation. Respiration and phosphoryla- tion were measured in the absence and presence of added cyto- chrome c. In the absence of cytochrome c, the respiration was TABLE I Effect of rotenone on respiration and phosphorylation Each vessel contained 0.01 M L-glutamate, succinate, or a-keto- glutarate, or 0.01 mM pyruvate + 0.01 mM L-malate, or 0.02 mM DL-@-hydroxybutyrate; 0.025 M Pi32, pH 7.5; 0.008 M MgClz; 0.001 M ATP; 0.03 M glucose; 150 Kunitz-MacDonald units of yeast hexo- kinase; 0.05 M KCl; 0.05 M sucrose; and an amount of mitochondria containing 3.0 mg of protein. When indicated, 10e4 M 2,4-dini- trophenol, or 0.0015 M DPN and 1.6 X 1OV M cytochrome c, were added. Final volume, 2 ml. Incubation took place at 30” with air as gas phase. Time measured, 17 minutes (Experiment 1) or 20 minutes (Experiment 2). Additions L-Glutamate L-Glutamate L-Glutamate L-Glutamate L-Glutamate Succinate Succinate Succinate Succinate Succinate Pyruvate, L-malate Pyruvate, L-malate r-Ketoglutarate r-Ketoglutarate oL-&Hydroxybutyrate or+Hydroxybutyrate L-Glutamate L-Glutamate L-Glutamate, 2,4-dinitrophenol L-Glutamate, DPN, cyto- chrome c Rotenone M 0* 5 x 10-S 10-7 2 x 10-T 10-e 0* 5 x 10-B 10-T 2 x lo-’ 10-C 0 4 x 10-r 0 4 x 10-T 0 4 x 10-T 0 4 x 10-T 4 x 10-7 4 x 10-7 .espir- ation ZtOWZS( 8.7 3.1 0.7 0.6 0.3 11.9 12.8 12.0 11.7 12.1 8.5 0 9.2 1.7 4.7 0 10.7 0 0 1.0 .moles P 22.9 7.5 21.4 22.5 19.9 21.0 18.9 * When 0.02 ml of ethanol was added, the respiration (micro- atoms of oxygen) and phosphorylation (micromoles of phosphate) were, respectively, 9.2 and 23.5 with L-glutamate, and 11.4 and 23.0 with succinate. TABLE II Comparison of efects of rotenone and Amytal on oxidation of extramitochondrial DPNH The vessels contained 0.0125 M Pi, 0.005 M MgClz, 0.002 M ATP, 0.03 M glucose, 150 Kunitz-MacDonald units of hexokinase, 0.01 M KCl, 0.1 M sucrose, 0.005 M DPN, 0.042 M ethanol, 0.2 mg of crystal- line alcohol dehydrogenase, 0.01 M NaF, and, when indicated, 1.6 X 10e5 M cytochrome c, 0.002 M Amytal, and 4 X 10-T M ro- tenone. Final volume, 2 ml. Mitochondria containing 4.0 mg of protein were added to each vessel. Incubation took place at 30” for 30 minutes. Additions None. ............... Amytal .............. Rotenone ........... Phosphoryl- ation Without cytochrome c - I With cytochrome c Respiration Respiration Phosphoryl- ation #atoms 0 pmoles P pawns ~moles P 2.3 4.1 5.9 5.7 0.4 0.5 3.4 1.1 0.2 0.2 3.4 1.6 1126 Diferential E$ects of Rotenone and Amytal Vol. 238, No. 3 TABLE III Bypass of rotenone-sensitive site of respiratory chain by vitanlin KS Experimental conditions were as described in Table I; the sub- strate was L-glutamate. Mitochondria contained 3.86 mg of pro- tein per vessel. The final concentrations of the additions were: rotenone, 4 X 10-T &I; antimycin A, 1.8 X 10-C M; Dicumarol, lo-6 u; each quinone, 10-j M. Incubation took place at 30” for 20 minutes. 2 - - - Additions None Rotenone Vitamin KS Rotenone, vit.amin KS 1,4-Naphthoquinone Rotenone, 1,4-naphthoquinone 1,4-Benzoquinone Rotenone, 1,4-benzoquinone 2.Methyl-1,4-benzoquinone Rotenone, 2-methyl-l,4-benzoqui- none uatoms 0 12.0 0.4 15.1 12.6 10.3 1.7 12.5 0.3 12.6 0.9 None 8.3 Rotenone 0.5 Dicumarol 8.7 Rotenone, vitamin Ka 10.6 Rotenone, vitamin Ka, Dicumarol 2.4 Rotenone, vitamin Kz, antimycin A 1.9 Rotenone, vitamin K1 0:5 Rotenone, vitamin Kz 0.5 Rotenone, coenzyme QO 2.0 Rotenone, coenzyme Q9 0.1 L 1 pyruvate+malate d 8 3 2- -0 L \ \ succinate B 31- 0 0 0" s i I- 0.5 Rotenone, MxlO 1 Phos- ,horyl ation moles P 30.6 2.6 33.9 2.2 21.3 1.7 26.5 2.6 2.7 1.6 30.2 2.4 28.0 2.2 19.7 2.4 16.9 1.9 13.7 1.3 P:O FIG. 1. Effect of rotenone on reduction of acetoacetate by suc- cinate and by pyruvate + malate. Each vessel contained 0.0042 M acetoacetate, 0.02 M succinate or 0.01 M pyruvate + 0.01 M L- malate, 0.008 M MgCL, 0.02 M glycylglycine buffer, pH 7.5, 0.05 M KCl, 0.05 M sucrose, and an amount of mitochondria containing 3.9 mg of protein in a final volume of 2 ml. Incubation took place at 30” for 15 minutes. relatively slow, and was accompanied by a phosphate uptake corresponding to a P: 0 ratio of approximately 1.8. Added cytochrome c substantially enhanced the respiration but induced only a slight increase in the net phosphate uptake; this increase in phosphate uptake probably was due to phosphorylation cou- pled to the oxidation of reduced cytochrome c by oxygen, as in- dicated by the studies of Maley (28). Both 2 mM Amytal and 4 x 10-T rotenone inhibited virtually completely the respiration and phosphorylation occurring in the absence of added cyto- chrome c but, left the cytochrome c-induced increase in these activities unaffected. Hence it can be concluded that rotenone, in the same way as Amytal, inhibits only the “internal,” phos- phorylating type of DPNH oxidation, and not its “external,” nonphosphorylating c0unterpart.l Bypass of Rotenone-sensitive Site by Vitamin K3--Conover and Ernster (13, 29, 30) have demonstrated that the inhibition of respiration in rat liver mitochondria by Amytal can be overcome by the addition of a catalytic amount of vitamin KS. A similar effect, as shown in Table III, occurred also in the case of the rotenone inhibition. As with Amytal, the vitamin KS effect, was not, duplicated by vitamin Kl or KP, or, to any significant extent, by identical concentrations of 1,4-naphthoquinone, p-benzoqui- none, 2-methylbenzoquinone, 5,6-dimethoxy-2-methylbenzo- quinone (coenzyme Qo), or higher coenzyme Q homologues. The vitamin KS-mediated respiration was inhibited by Dicuma- rol and antimycin A, and was connected with a phosphorylation of 1 P:O unit lower than that observed with the original system. These findings are consistent with the conclusion that vitamin K) induces a bypass of the rotenone-sensitive site of the respira- tory chain by way of the Dicumarol-sensitive flavoenzyme called DT diaphorase (cj. 30), and that the electrons mediated by vit,a- min KP enter the terminal respiratory chain at the level of cyto- chrome b. Succinate-linked Acetoacetate Reduction-It was recently re- ported from this laboratory that rat liver mitochondria catalyze an endergonic reduction of acetoacetate by succinate (13, 16, 31, 32). The reaction was shown to involve a backflow of electrons through the Amytal-sensitive site of the respiratory chain and to proceed at the expense of high energy bonds generated in the terminal oxidation of succinate. This reaction is also inhibited by rotenone (Fig. 1). Replacement of succinate by pyruvate + malate, which allowed a direct dismutation via DPN, resulted in an acetoacetate reduction that was insensitive to rotenone. These findings, which are analogous to those reported with Amytal (13, 16, 31, 32), further substantiate the concept that the succinate-linked acetoacetate reduction in isolated liver 1 By the use of ethanol, alcohol dehydrogenase, and a relatively low (0.75 mM) concentration of DPN or, alternatively, a relatively high concentration (8 mM) of DPNH, Ernster et al. (6) found a very low phosphate uptake in the absence of cvtochrome c. which was greatly increased by the addition of cytochrome c. Amytal, 2 mM. inhibited the respiration only partiallv in both the absence and presence of added cytochrome c and abolished the accompany- ing phosphorylation almost completely; similar findings were now made with rotenone. Since the same concentration of Amytal did not alter significantly the P:O ratio obtained with succinate, Ernster et al. (6) concluded that the inhibition of the cytochrome c-induced phosphorylation by Amytal could not be due to a true uncoupling effect, but rather meant that this whoswhorvlation ac- tually-originated-from the “internal,” phospioryiating pathway. Why, under the conditions used by Ernster et al. (6), externally added cytochrome c promoted the phosphorylation coupled to DPNH oxidation by the “internal” pathway, or why, in the ex- periments of Maley (28), a similar effect could be achieved by in- creasing the concentration of external, oxidized, DPN, or by fluoride, are questions that cannot readily be answered at this moment. They would seem to deserve future attention. March 1963 L. Ernster, G. Dallner, and G. F. Axzone 1127 I 2 6 6 8 Rotenone, M x lo’, or Amytal, Mx103 FIG. 2. Comparison of effects of rotenone and Amytal on Pi- ATP exchange. The assay system contained 0.01 M Pi*‘, pH 7.5, 0.01 M ATP, 0.008 M MgClz, 0.05 M KCl, 0.05 M sucrose, and an amount of mitochondria containing 4.12 mg of protein in a final volume of 2 ml. Incubation was performed at 30”. Aliquots were removed after 5, 10, and 15 minutes, and the exchange rate was calculated by the method of Boyer, Luchsinger, and Falcone (34). The rates were virtually constant throughout the period measured. a ‘-.~P.NP,Amytal -.._ --._ -. “. I , 2 L 6 8 Rotenone, M x 10’ P i-ATP exchange by rotenone leveled off with increasing amounts of rotenone in a manner similar to, but even weaker than, that observed previously with antimycin A and cyanide (10, 34). ATPase ReuctionsAmytal was previously found to stimu- late slightly the endogenous ATPase activity of rat liver mito- chondria, and to exert an inhibition of the ATPase activity elic- ited by 10e4 M dinitrophenol (8). A strong inhibition of the dinitrophenol-induced ATPase by Amytal was demonstrated by Low (9) to occur in the presence of an appropriate concentra- tion of atebrin or chlorpromazine. These effects of Amytal are compared with those found with rotenone in Fig. 3. As can be seen, none of the effects of Amytal on the various ATPase re- actions was duplicated by rotenone. In fact, rotenone appears to be the first respiratory inhibitor so far investigated that leaves mitochondrial ATPase reactions totally unaffected. P:O Ratio with Succinate-It has been a general observation (35-37) that Amytal decreases the P : 0 ratio with succinate. It is seen in Table IV that rotenone, whenadded in an amount sufficient to inhibit DPN-linked electron transfer, lowered the P:O ratio to an extent that may be attributed to the inhibition of DPN-linked oxidations. Once this value was reached (as at 8 X 10-S M rotenone in the present experiment), the P:O ratio did not decrease further even if the concentration of rotenone was increased 25-fold or more. On the other hand, Amytal, b 2 L 6 8 or Amytal, M x lo3 FIG. 3. Comparison of effects of rotenone and Amytal on ATPase activity under various conditions. The assay system contained 0.005 M ATP, 0.005 M Tris buffer, pH 7.5, 0.125 M sucrose, and, when indicated, lo-+’ M dinitrophenol, 1.2 X 10e4 M chlorpromazine, and 1.7 X 10W3 M atebrin in a final volume of 2 ml. bation took place at 30” for 20 minutes. The amount of mitochondria added to each vessel contained 0.91 mg of protein. Incu- DNP denotes 2,4-dintrophenol. mitochondria proceeds via the respiratory chain, and not, as has been argued (33), by a direct dismutation by way of DPN. added either alone or in combination with rotenone, induced a progressive decrease in the P : 0 ratio. E$ects of Rotenone and Amytal on Energy Transfer Reactions Kinetics of Rotenone Inhibition Pi-ATP Exchange Reaction-Fig. 2 compares the effects of Binding of Rotenone to Mitochondria-Lindahl and ijberg (3) rotenone and Amytal on the rate of the Pi-ATP exchange. In have observed that the inhibition by rotenone is not reversed confirmation of earlier results (lo), 2 rnM Amytal inhibited the when the respiration of gill filaments from rotenone-poisoned P i-ATP exchange by approximately 40 %, and 8 mu Amytal, by fishes is measured in a rotenone-free medium. This finding almost 90 %. On the other hand, rotenone induced only a slight suggested that rotenone might be firmly bound to the mito- inhibition of the exchange, even when added in large excess of chondria. This would be the opposite of the case of Amytal, the amount required for complete inhibition of DPN-linked elec- which is readily removed from the mitochondria by washing.2 tron transport (cf. Table I). The extent of inhibition of the 2 0. Jalling, unpublished observation. 1128 Diflerential E$ects of Rotenone and Amytal Vol. 238, No. 3 As shown in Table V, pretreatment of mitochondria with rote- none and subsequent washing (for details of pretreatment, see “Experimental Procedure”) indeed resulted in a persisting inhibi- tion of the respiration, whereas the Amytal-pretreated mito- chondria respired at a rate equal to that of the washed control. It may be noted that the respiration of both the washed control and the Amytal-pretreated sample was lower than that of the original mitochondria, and was stimulated by added DPN. Apparently, the pretreatment procedure had caused a loss of DPN from the mitochondria. It is significant that, in spite of this fact, added DPN did not appreciably restore the respiration of the rotenone-pretreated sample. This observation, which is in accordance with previous findings with Amytal (21), shows that DPNH generated by intramitochondrial dehydrogenases re- acts preferentially with the “internal” type of DPNH-cyto- chrome c reductase even after the respiration had been rendered dependent on added DPN. TABLE IV Comparison of effects of rotenone and Amytal on P:O ratio with succinate as substrate Experimental conditions were as described in Table I; the sub- strate was 0.2 M succinate. Mitochondria contained 5.48 (Ex- periment 1) or 3.66 (Experiment 2) mg of protein per vessel. Incubation took place at 30” for 20 minutes. Additions None...................................... 8 X 10-s M rotenone. . 4 X 10-T M rotenone.. 2 X lO+ M rotenone....................... 2 X 10+&l Amytal.. _. .__ _. .___. ._ 4 X 10-3M Amytal.. _, .___._. _. ._.. 6 X lO+MAmytal.. ..__......_._........ 4 X 10-T M rotenone + 2 X 1OW M Amytal.. 4 X 10-T M rotenone f 4 X lop3 M Amytal. 4 X 10-T M rotenone + 6 X lop3 M Amytal. TABLE V - I E -- - P: 0 ratio ixpe:ment :xperiment 2 2.3 2.0 1.9 1.7 1.8 1.7 1.9 1.7 1.7 1.6 1.6 1.5 1.2 1.4 1.8 1.6 1.5 1.5 1.0 1.1 Effect of pretreatment with Totenone and Amytal on respiration of mitochondria Experimental conditions were as described in Table I; the sub- strate was 0.01 M L-glutamate; when indicated, 0.0015 M DPN was added. Mitochondria were pretreated, when indicated- with 0.002 M Amytal or 4 X 1OW M rotenone as described in “Ex- perimental Procedure.” The amounts of mitochondria in the vessels were 3.66, 4.08, 3.81, and 5.27 mg of protein, respectively, for the samples that were untreated, washed, treated with Amytal, and treated with rotenone. Incubation t,ook place at 30” for 10 minutes. Treatment Oxygen consumed Without DPN With DPN patoms None ._ ._................... 4.0 Rewashed. 1.7 Treated with Amytal, rewashed .I 1.6 Treated with rotenone, rewashed.. / 0.0 3.5 3.9 0.5 Rotenone, m~moles/sample FIG. 4. Titration of rotenone-sensitive catalyst. Experimen- tal conditions were as described in Table I, except that the sub- strate was 0.01 M pyruvate + 0.001 M L-malate. The amount of mitochondria added to each vessel contained 3.1 (A) or 6.2 (B) mg of protein. Time measured, 20 minutes. Titration of Rotenone-sensitive Factor-Fig. 4 shows a titration of the “rotenone-sensitive factor,” carried out by measuring the oxygen consumption (with pyruvate + malate as substrate) in the presence of varying amounts of rotenone and two different amounts of mitochondria (A and B). As could be anticipated, the respiratory values as functions of the amount of rotenone described straight lines and these were parallel for the two amounts of mitochondria used. From the intersection of the lines with the abscissa, the minimal amount of rotenone per unit of mitochondrial protein required for complete suppression of respiration can be est.imated. This value, which may be taken as an expression of the amount of the rotenone-sensitive factor in the mitochondria, was 24.8 mpmoles of rotenone per g of protein (Fig. 4A), and 23.9 mfimoles of rotenone per g of protein (Fig. 4B). It is also possible from Fig. 4 to deduce the amounts of rotenone required between no inhibition (dotted verti- cal lines) and complete inhibition; this value is 0.068 mpmole in A, and 0.134 mpmole in B. If it is assumed that the rotenone- sensitive factor is an electron-transferring catalyst, the apparent “turnover number” of this catalyst can be calculated by dividing the rates of oxygen consumption in the two samples by the above values. This gives 6000 2-electron equivalents per mole per minute for A, and 5900 2-electron equivalents per mole per min- ute for B. With five preparations of mitochondria, and with pyruvate + malate or glutamate as substrate, the amount of the rotenone-sensitive factor varied between 24.7 and 28.0 mpmoles per g of protein, and its apparent turnover number ranged be- tween 5400 and 7500 2-electron equivalents per mole per minute. Comparison with Antimycin A and Oligomycin-In Fig. 5, rotenone and two other inhibitors, antimycin A and olgomycin (38, 39), are compared with regard to the above parameters (for previous titrations with these two compounds, see (3942)). It can be seen that the amount of inhibitor required for complete (or, in the case of oligomycin, maximaP) inhibition was approxi- mately twice as high with antimycin A, and about 8 to 9 times as high with oligomycin, as with rotenone. Another striking 3 Oligomycin inhibits only that part of the respiration that is obligatorily coupled to phosphorylation (13, 39). March 1963 L. Emster, G. Dallner, and G. F. Axzone 1129 difference is that the ratio, TABLE VI amount of inhibitor required from no to maximal inhibition total amount of inhibitor required for maximal inhibit.ion is rather large with antimycin A (cf. also (40-42)) and oligomy- tin, whereas it is relatively small with rotenone. It appears, in other words, that whereas the rotenone-sensitive factor is present only in small excess of the total respiratory capacity, the anti- mycin A- and oligomycin-sensitive factors occur in large excess. An estimate of the turnover numbers was not possible from these data, because the experimental points were insufficient to deter- mine the precise slopes of the lines. Effect of rotenone and antimycin A on submitochondrial DPNH oxidase The assay system contained 4 X 1OW M DPNH, 0.05 M phosphate buffer, pH 7.4, and enzyme preparation, containing 1.65 mg of protein, in a final volume of 3 ml. The reaction was started by the addition of the enzyme and followed at 340 rnp for 1 to 3 min- utes in a Beckman DK2 spectrophotometer, with glass cuvettes of l-cm light path. The temperature was 22”. In the pretreat- ment experiments, the enzyme preparation was treated with 1 X 10m6 M rotenone or 3 X 10m6 M antimycin A, as described in “Ex- perimental Procedure.” Effect of Rotenone on Submitochondrial DPNH Oxidase Submitochondrial preparations of DPNH oxidase of both the phosphorylating (43-45) and nonphosphorylating (45-47) types have been reported to possess a sensitivity to Amytal and anti- mycin A. A nonphosphorylating DPNH oxidase from rat liver mitochondria, prepared after mechanical disruption of the mito- chondria by the method of Kielley and Kielley (14), has recently been studied in this laboratory in relation to these inhibitors (13, 48). The preparation exhibited an 80 to 90% sensitivity toward both Amytal and antimycin A. Data reported in Table VI compare the effect of rotenone in this preparation with the effect of antimycin A. It can be seen that lOms M rotenone gave maximal inhibition of the DPNH oxidase activity-an inhibition of approximately 80%-and that this concentration, as in the case of the intact mitochondria, was lower than the concentration of antimycin A needed for the same maximal inhibition. Preparation Inhibitor added in test Untreated None Rotenone, 3.3 X 1OW M Rotenone, 6.7 X 10-9 M Rotenone, 10-s M Rotenone, 1.3 X 10-s M Antimycin A, 1.8 X 10-S 1 Antimycin A, 3.7 X 10-E n Antimycin A, 5.5 X 10-s n Pretreated with rotenone Pretreated with antimycin A None 50 None 46 None 50 The data in Table VI also show that pretreatment of the sub- mitochondrial particles with either rotenone or antimycin A (for details of the pretreatment, see “Experimental Procedure”) resulted in a persistent inhibition of the respiration just as in the case of the intact mitochondria. An attempt was made to re- store the activity of the two preparations by combining them, since it was thought that the inhibited rotenone- and antimycin- sensitive factors of the respective preparations might be replaced by their uninhibited counterparts. Such a utilization of the anti- mycin-sensitive factor of an uninhibited Keilin and Hartree heart muscle preparation by an inhibited one has been demon- strated previously by Thorn (42). In the present case, however, no reactivation occurred with the combined rotenone- and anti- mycin A-inhibited preparations. Pretreated with rotenone and with antimycin At - * Micromoles of DPNH oxidized per minute per g of protein. t This assay was performed with a mixture of a rotenone-pre- treated and an antimycin A-pretreated preparation, each added in one-half the amount previously used. DISCUSSION From the data reported in the present paper and from those already published by Fukami and Tomizawa (1, 2) and by Lin- dahl and oberg (3, 4), the pattern of action of rotenone on elec- tron transport seems to be identical with that of Amytal, and this identity suggests a common site of action of the two agents. However, there is an important difference between rotenone and Amytal with regard to their effects on certain energy transfer reactions. In sharp contrast with Amytal, rotenone inhibits the mitochondrial Pi-ATP exchange reaction to only a slight extent, and leaves the dinitrophenol-induced ATPase reaction and the phosphorylations accompanying the aerobic oxidation of suc- cinate completely unaffected. These reactions are inhibited by Amytal even though the concentrations of Amytal required for these effects, as a rule, are higher than those needed for DPN- flavin-linked electron transport. That rotenone lacked similar effects was established by using amounts of rotenone in large (20- to loo-fold) excess of those needed to block DPN-flavin electron transport. In addition, Amytal has been shown to in- hibit the ATP-dependent contraction of swollen mitochondria (49) and the relaxation of muscle fibers induced by the sarcotubu- lar ATPase (50, 51). Also in these cases, the effect of Amytal is not duplicated by rotenone, as has been established by prelimi- nary experiments in this laboratory. Evidently, rotenone blocks DPN-flavin-linked electron transfer in a more specific manner 10 20 30 40 50 60 160 240 320 Inhibitor, mpmoles/g protein FIG. 5. Comparison of titration curves with rotenone, antimy- tin A, and oligomycin. Experimental conditions were as de- scribed in Table I; the substrate was 0.01 M n-glutamate. The amount of mitochondria added to each vessel contained 3.68 mg of protein. Time measured, 20 minutes. ZE: acti- vity* 214 212 202 42 41 213 179 27 Diferential Efects of Rotenone and Amytal Vol. 238, No. 3 than does Amytal, which inhibits also a number of energy trans- fer reactions. The inhibition of the Pi-ATP eschange and dinitrophenol-in- duced ATPase reactions by Amytal has been taken as evidence for the involvement of an actual electron shuttle between DPN and flavin in these reactions (10). The flavin theory of oxidative phosphorylation (10, 52), involving a phosphorylated form of the reduced flavin as primary high energy intermediate, was partly based on the above interpretation of the Amytal effect. However, the present findings that the Pi-ATP exchange and the dinitrophenol-induced ATPase are unaffected when the DPN- flavin electron shuttle is completely inhibited by rotenone necessi- tate a different interpretation of the Amytal inhibition. Indeed, this conclusion is not in disagreement with the revised version of the flavin theory recently suggested by Ernster (16), in which an unknown radical other than phosphate is the partner of the reduced flavin in forming the primary high energy intermediate. This revised mechanism no longer requires an electron shuttle between DPN and flavin as an obligatory partial reaction of the Pi-ATP exchange and dinitrophenol-induced ATPase reactions. Calculations of the minimal amount of rotenone required for complete inhibition of DPN-flavin-linked electron transport have given values ranging between 24 and 28 mpmoles per g of mito- chondrial protein. This value is lower than the corresponding values for antimycin A and oligomycin (Fig. 5) and is, to our knowledge, the lowest value ever reported for an inhibitor of mitochondrial electron transport. In addition, the above value is considerably lower than the reported mitochondrial contents of various electron transfer catalysts, including pyridine nucleo- tides (53-57), flavins (53), quinones (58, 59), and cytochromes (53, 57). Significantly, the amount of rotenone required for complete inhibition of DPN-flavin-linked electron transfer is 10 to 20 times less than that fraction of the mitochondrial flavin that, according to Chance and Williams (53), becomes reduced in the presence of a DPN-linked substrate when respiration is blocked by antimycin A or anaerobiosis. Thus, if one assumes that the rotenone-sensitive factor is an electron-transferring catalyst, and that at least 1 molecule of rotenone is needed to block 1 molecule of the catalyst, it follows that this catalyst can- not be identical with the DPNH dehydrogenase flavoprotein. Furthermore, the apparent turnover number of the rotenone- sensitive catalyst is of the order of 5000 to 8000 2-electron equiva- lents per mole per minute, which, again, is very different from the turnover number of 1.3 million per mole of flavin per minute reported for the solubilized DPNH dehydrogenase (60). Another point of interest arises from the findings reported in Fig. 4, according to which the extent of inhibition of respiration by a given amount of rotenone was almost directly proportional to the total amount of rotenone added. In other words, only a small amount of rotenone was needed to obtain an observable inhibition in relation to the amount required for complete in- hibition. This situation is in striking contrast to that found with antimycin A, which gave a “sigmoid” type of titration curve, in agreement with previous findings of Ackermann and Potter (40) and Potter and Reif (41) and of Thorn (42). The oligomycin titration curve was also of the sigmoid type. The titration curve with antimycin A may be interpreted, in accord- ance with Thorn (42), as indicating that the antimycin-sensitive catalyst is present in the mitochondria at a capacity that is in a large excess of the capacity of the respiratory chain, i.e. that 90 % or more of the catalyst may be blocked and full respiration can still proceed. By the same reasoning, then, it may be concluded that the rotenone-sensitive site is needed at nearly full capacity during maximal respiratory activity in the presence of phosphate and phosphate acceptor and, thus, that the rotenone-sensitive catalyst probably constitutes the rate-limiting factor of DPN- linked mitochondrial respiration under conditions of maximal respiration and phosphorylation. SUM!MARY The effects of the fish poison, rotenone, on the respiration, phosphorylation, and related reactions of rat liver mitochondria have been investigated and compared with those of Amytal. In agreement with previous data in the literature, rotenone inhibits the aerobic oxidation of pyridine nucleotide-linked substrates but not that of succinate. The inhibition is not relieved by 2,4- dinitrophenol or by added diphosphopyridine nucleotide and cytochrome c, but is overcome by added vitamin K3, which ac- tivates a bypass of the rotenone-sensitive site. Rotenone also inhibits the endergonic reduction of acetoacetate by succinate, but not the dismutative reduction of acetoacetate by pyruvate + malate. Oxidation of extramitochondrial DPNH is likewise inhibited by rotenone, yet only partially in the presence of added cytochrome c. The DPNH oxidase activity of submitochondrial particles is also highly sensitive t,o rotenone. All these effects are similar to those of Amytal. In contrast to ilmytal, rotenone leaves unaffected the mito- chondrial inorganic orthophosphate-adenosine triphosphate ex- change, and the resting as well as the 2,4-dinitrophenol-induced adenosine triphosphatase reactions, the latter in both the ab- sence and presence of atebrin and chlorpromazine. Unlike Amytal, rotenone also does not lower the P:O ratio with succi- nate as substrate. It is concluded that rotenone blocks diphos- phopyridine nucleotide-flavin-linked electron transport in a more specific manner than does Amytal, which inhibits also a number of energy transfer reactions. Rotenone also differs from Amytal in that it is firmly bound to mitochondria and to submitochondrial particles. The extent of inhibition of respiration by rotenone is dependent on the amount, rather than the concentration, of rotenone added. The amount of rotenone required for complete inhibition of respiration is 24 to 28 mpmoles per g of mitochondrial protein, and the apparent turnover number of the rotenone-sensitive catalyst is 6000 to 8000 a-electron equivalents per minute per mole of catalyst. It is concluded that the rotenone-sensitive catalyst occurs at the lowest molar ratio among known components of the liver mito- chondrial electron transport system, and probably constitutes the rate-limiting catalyst during maximal respiratory activity. Acknozuledgment-The skillful technical assistance of Miss Kerstin Eriksson is gratefully acknowledged. REFERENCES 1. FUKAMI, J., Botyu-Kagaku, 21, 122 (1956); Chem. Abstr., 61, 9068 (1957). 2. FUKAMI, J., AND TOMIZAWA, C., Botyu-Kagaku, 21,129 (1956); Chem. Abstr., 61,9068 (1957). 3. LINDAHL. P. E.. AND OBERG, K. E.. Exptl. Cell Research, 23, 228 (1961). ’ _ 4. ~BERG, K. E., Exptl. Cell Research, 24, 163 (1961). 5. JALLING. 0.. ERNSTER. L., AND LINDBERG, O., Acta Chem. 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Differential Effects of Rotenone and Amytal on Mitochondrial

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