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30 W. G. Overend, A. R. Peacocke, and M. Stacey, Trans. Faraday Soc., 50, 303, 1954.31 J. M. Gulland, D. 0. Jordan, and H. F. W. Taylor, J. Chem. Soc., p. 1131, 1947.32 W. A. Lee and A. R. Peacocke, J. Chem. Soc., p. 3361, 1951.33 G. Goldstein and K. G. Stern, J. Polymer Sci., 5, 687, 1950.34 T. Miyaji and V. E. Price, Proc. Soc. Exptl. Biol. Med., 75, 311, 1950.36 N. B. Kurnick, J. Am. Chem. Soc., 76, 417, 1954.3v S. Zamenhof, H. E. Alexander, and G. Leidy, J. Exptl. Med., 98, 373, 1953.37 S. Zamenhof and E. Chargaff, J. Biol. Chem., 186, 207, 1950.'I S. Zamenhof, in W. D. McElroy and B. Glass (eds.), Phosphorus .Mfetabolism, 2 (Baltimore:

Johns Hopkins Press, 1952), p. 301.39 L. F. Cavalieri and A. Angelos, J. Am. Chem. Soc., 72, 4686, 1950.40 L. F. Cavalieri, A. Angelos, and M. E. Balis, J. Am. Chem. Soc., 73, 4902, 1951.41 S. Zamenhof, G. Griboff, and N. Marullo, Biochim. et biophys. acta, 13, 459, 1954.42 0. Bailly and J. Gaume, Bull. soc. chim. biot., 3, 1396, 1936.43 M. Friedkin, J. Biol. Chem., 184, 449, 1950.44 S. Katz and J. D. Ferry, J. Am. Chem. Soc., 75, 1589, 1953.

' C. Tamm and E. Chargaff, J. Biol. Chem., 203, 689, 1953.

THE UNCOUPLING OF RESPIRATION AND PHOSPHORYLATION BYTHYROID HORMONES*

BY FREDERIC L. HoCHt AND FRITZ LIPMANN

BIOCHEMICAL RESEARCH LABORATORY, MASSACHUSETTS GENERAL HOSPITAL, AND DEPARTMENT OF

BIOLOGICAL CHEMISTRY, HARVARD MEDICAL SCHOOL, BOSTON, MASSACHUSETTS

Communicated July 30, 1954

The demonstration of the uncoupling of respiration of respiration and phosphor-ylation by nitrophenols' introduced a new approach to the understanding of inter-cellular energy regulation by exposing relatively easily accessible experimentalmeans of interrupting the link between energy supply and energy utilization.Furthermore, with mitochondria, halophenols such as di- or tribromo or iodo-phenols were as active as the nitro compounds, as was first observed with arabaciaeggs by Krahl and Clowes.5 This brought particularly to our attention the possibil-ity of defining the activity of the physiological halophenol, the thyroid hormone, aslikewise due to a dissociation of respiration and phosphorylation.

Efforts therefore were started with the hope of obtaining uncoupling effects withthyroxine. We have reported relatively briefly on the results of these rather ex-tensive studies,' 3, 4 mainly because the picture which developed with these ex-periments, although quite indicative, remained for some time inconclusive. It isthe purpose of this paper to report in greater detail on our results, particularly themore recent ones.Loomis made the obvious attempt to duplicate the nitro, halophenol effect on

mitochondria suspensions with thyroxine, using rabbit kidney preparations. How-ever, as in unpublished and published experiments, by many others, cf. e.g.,6 no reli-able effect could be observed. It is well known, however, that the easily and consist-ently obtained increase in respiratory rate with tissue slices taken from thyrotoxicanimals (Barker7) could never be reliably produced by an in vitro addition of thyrox-ine to normal slices. This indicated that cell membranes were relatively impermeable

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to thyroxine and that the mitochondrial membrane shown in the electron micro-graph was similarly impermeable. In view of this experience with tissue slices, itwas therefore decided to test mitochondria preparations taken from thyrotoxic rats.

METHODS

Rat Experiments. Mitochondria were prepared from the liver of a freshly killedyoung male rat (Hisaw strain, Harvard Biological Laboratories) by the method ofHogeboom, Schneider, and Pallade,8 as modified by Kennedy and Lehninger.9The chilled liver was expressed through a fine grill in a stainless-steel tissue press, thepulp suspended in cold 8.5 per cent sucrose, and treated in a Potter glass homoge-nizer. The volume was adjusted to 100 ml. with 8.5 per cent sucrose, and centrifuga-tion was performed three times at 600 g for 5 minutes each to remove easily sedi-mented structures.To the homogenate supernatant fluid 10 ml. of 1.5 N KCl containing 0.1 N

NaHCO3 were added, the suspension allowed to stand 10 minutes, and centrifuga-tion at 3,000 g continued for 20 minutes. The sedimented mitochondria werewashed in 0.15 N KCl containing 0.01 N NaHCO3. This was repeated, and thelast sediment was suspended in 0.15 N KCl to give a dry weight of approximately10 mg. of mitochondria per 0.5 ml.; the Qo. values given here are based upon suchweights.The reaction mixtures for measurement of oxidation and phosphorylation

contained 2 ,M ATP (Pabst); 1 AM DPN (Pabst); 0.02 MM cytochrome c (Sigma);10 1MM MgCl; 40 MM phosphate; 1.2 mM glycylglycine; and 50 UM substrate.The pH was adjusted to 7.4 with KOH. The side arm of the Warburg vessels con-tained 0.1 ml. of purified yeast hexokinase, step 3a,10 and 0.1 ml. of 1 M glucose(acceptor system). The total volume, after the addition of 0.5 ml. of mitochondrialsuspension, was 3.0 ml.The contents of the main chamber of each Warburg vessel were duplicated in a

10-ml. Erlenmeyer flask; these represented the "zero-time" samples, All flaskswere equilibrated at 300 for 5 minutes immediately after addition of the mito-chondria to the reaction mixture. The Warburg systems were sealed, and respira-tion was measured for 10 minutes without acceptor system (Qo2-A). The Erlen-meyer flasks then received 6 per cent TCA; the acceptor system was concomitantlytipped into the main compartment of the Warburg flasks and respiration measuredfor another 20 minutes (Qo,+A). The reaction was stopped with 6 per cent TCA.The contents of all flasks were adjusted to 12-ml. volume with 6 per cent TCA andcentrifuged. Aliquots from the zero-time flasks and the Warburg flasks wereanalyzed for content of inorganic phosphate.1" The difference (-Pi) was calculatedand the P/O ratio derived from the respiration during the presence of acceptor sys-tem: -Pi MuM per uA oxygen uptake.

Experiments with Hamsters.-Young male Syrian hamsters (Golden NuggetHamstery, Maynard, Massachusetts), weighing between 85 and 120 gm.,were used. Three or four hamsters were killed by stunning and decapitation, andtheir livers were treated as in the rat experiments.

Mitochondria were derived from the homogenate by one of two methods. Salinepreparations were made as described. Sucrose preparations were prepared by cen-

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trifuging the homogenate suspension at about 8,000 g in a Servall centrifuge in arefrigerated room at 20 for 20 minutes. The pellet was washed in 8.5 per cent su-crose and centrifugation repeated at 8,000 g for 10 minutes. The final pellet wassuspended in 8.5 per cent sucrose to give a dry weight of approximately 10 mg.of mitochondria per 0.5 ml. Qo2 values were based on dry weight.

Preparation of Thyroxine Solutions. For the in vivo experiments, DL-thyroxine(Hoffmann-La Roche) was dissolved in a minimum amount of 2 N N'aOH anddiluted with sterile distilled water to a concentration of 4 mg. per 0.5 ml.; the pHwas approximately 8. The solution for injection into control animals contained asimilar amount of NaOH and was similarly diluted. Male rats were subcutaneouslyinjected with 0.5 ml. of freshly prepared thyroxine or NaOH solution in the ventralaspect of the thigh and received 4 mg. of thyroxine on the first day and 8 mg. on thesecond, third, and fourth days. They were sacrificed on the fifth day.For the in vitro experiments, thyroxine and other phenolic compounds were dis-

solved in 0.2 ml. of freshly prepared 0.02 N NaOH per milligram and diluted withdistilled water; the pH was approximately 8. Control solutions contained asimilar dilution of NaOH; the pH was approximately 10. When these solutionsof thyroxine were in contact with the reaction mixture (buffered at pH 7.4), a pre-cipitate was formed.The solutions of thyroxine and other compounds were added to mitochondria by

one of the following methods: (a) Preincubation: once-washed sucrose mitochon-dria or twice-washed saline mitochondria were suspended in cold 8.5 per centsucrose or 0.15 N KCl with thyroxine and shaken in Lusteroid centrifuge tubes for7 minutes in a water bath at 170. The mitochondria were then sedimented bycentrifugation, resuspended in sucrose or KCl solution, and assayed for respirationand phosphorylation. (b) Direct incubation: the thyroxine solution was placed inthe main chamber of the Warburg vessel; the mitochondrial suspension was addedto the reaction mixture and the resultant suspension aliquoted into the Warburgvessels. Respiration and phosphorylation were measured at 250 after a period of 5minutes for equilibration. (c) Without incubation: thyroxine solution was tippedin with the acceptor system, allowing no time for incubation of mitochondria withthe hormone before measurement of phosphorylation began.The acceptor system was added by tipping in either immediately or after 10

minutes to allow observation of acceptor-less respiration (Qo,- A)Distribution of I'3l-labeled Sodium L-Thyroxine or Sodium L-Triiodothyronine

(Abbott Laboratories) between Mitochondria and Suspending Medium.-Mitochondriawere prepared in saline or sucrose media as described and suspended in a solutioncontaining all ingredients of the reaction mixture excepting the substrate and ac-ceptor system. The suspension was weighed on an analytical balance, and 0.2 ml.aliquots were removed and weighed in tared metal-dish planchettes. The sus-pension was then centrifuged at 17,000 rpm for 5 minutes at 20. The supernatantfluid was decanted and weighed. The sedimented materials were resuspended anddissolved in 0.01 N KOH after the mixture had been weighed. Aliquots of ap-proximately 0.2 ml. were then removed from the supernatant fluid and from themitochondrial solution and weighed in tared planchettes. All samples on plan-chettes were dried under an infrared lamp after addition of one drop of 6 N KOHand their radioactivity estimated (Tracer Lab Autoscaler). All aliquots were

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taken in triplicate and mean values used for calculation of total counts in eachfraction.

Reproducibility of Qo, Determinations and P1O Ratios.-Table 1 shows the resultsof eight duplicate runs. The Qo, values with rat liver mitochondria and glutamate

TABLE 1

NORMAL RUN IN EIGHT DUPLICATES, RAT LIVERMITOCHONDRIA WITH GLUTAMATE AS SUBSTRATE

Q02 P/0 Q02 P/O

57.8 2.26 54.9 2.3151.0 2.64 56.9 2.2058.0 2.14 50.7 2.5049.8 2.6357.5 2.17 Av. 54.8 2.36

varied by 16 per cent between 49.8 and 58. The P/O ratios ranged from 2.14to 2.63. It is concluded that P/0 differences have to be of the order of 30 per centto be significant.

EXPERIMENTAL RESULTS

Inhibition of the Phosphate Acceptor Effect on Respiration with Liver Mitochondriaof Thyrotoxic Razs.-Niemeyer et al.2 12 used for these experiments rats that had re-ceived injections of 1 mg. of thyroxine on four consecutive days. The liver mito-condria suspension was prepared on the fifth day in a manner described by Craneet al."3 The results, which have been briefly reported, are summarized in Table 2.

TABLE 2ACCEPTOR EFFECT ON NORMAL COMPARED WITH THYROTOxiC LIVER MITOCHONDRIA

Q02WITHOUT WITHACCEPTOR ACCEPTOR a P/OSYSTEM SYSTEM* (PER CENT) RATIO

Normal (8)t 19 27 +42 2Thyrotoxic (9) 28 29 + 4 2

* Glucose and yeast hexokinase.t Number in parentheses gives number of experiments.

From this series of experiments a consistent, although subtle, difference betweenthe thyrotoxic and the normal preparations appeared in average values for therespiration of acceptor-free systems. The thyrotoxic preparations, when comparedto normal, showed a 47 per cent increase in respiratory rate (first column of Table2). Significantly, the respiratory increase of the thyrotoxic mitochondria mani-fested itself only in the absence of acceptor. The addition of the acceptor system,glucose-hexokinase, wiped out the difference by increasing normal respiration to thelevel which the thyrotoxic preparation reached without acceptor.The Qo, data showed, therefore, that the thyrotoxic preparation already respired

maximally without phosphate acceptor (compare the second column in Table 2).This absence of the acceptor effect was interpreted to indicate uncoupling betweenphosphorylation and respiration in the thyrotoxic preparation. Actually, it was

during this comparison of normal and thyrotoxic mitochondria that we first becameaware of the remarkable respiratory activation due to the removal of energy-rich

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phosphate from the system.2 12 Similar observations have been reported fromother laboratories.14The fact that in the thyrotoxic liver mitochondria the acceptor effect was prac-

tically wiped out, while the P/O ratios in the same preparation did not show a de-crease, indicated to us that the acceptor response should represent a finer indicatorfor uncoupling than the effect on P/O ratios. It is considered to show a disturb-ance in the initially reversible balance between electron transfer and phosphate-bond generation, a disturbance, however, not strong enough to show up in the lesssensitive test for P/O ratio. Such an interpretation is supported by experimentswith lower concentrations of dinitrophenol. It may be seen in Table 3 that 10-1M

TABLE 3INHIBITION OF ACCEPTOR EFFECT BY Low CONCENTRATIONS, OF DINITROPHENOL

DINITROPHENOL WITHOUT WITH INHIBITION INHIBITIONM/I, ACCEPTOR ACCEPTOR (PER CENT) P/O (PER CENT)

0 16 30 .. 2.210-5 27 31 72 1.7 232 X 10-5 31 36 70 1.1 50

causes the (Qo2-A) to rise nearly to the acceptor value, while the P/O ratio is onlyslightly affected, and even at the higher concentration the acceptor effect is stillmore pronounced than the reduction in P/0 ratio.Under optimal conditions, the respiratory block in the absence of a phosphate

acceptor may be remarkable. As shown in Table 4, occasionally preparations are

TABLE 4NORNIAL RESPIRATORY STIMULATION BY ACCEPTOR SYSTEM

Q02WITHOUT WITH ACCELERATION

SUBSTRATE: ACCEPTOR ACCEPTOR FACTOR

Hlvdroxvhutvrat~l 4.0 26.4 6.6Hydroxyhutyrate 15.4 24.0 1.6Glutamate 9.4 55.8 5.9a-Ketoglutaratv 15.-3 51 .2 3.3Gluconate 24.1 71.0 2.9

encountered which are practically completely blocked for hydrogen transport if noescape mechanism for the generated phosphatebond is provided. Similar observations were HYDROGEN PHOSPHATEmade by Lardy et al. 14 The situation is illus-

DN ACCEPTOR

trated by the scheme of Figure 1, indicating very ~pgenerally the interdependence of the flow of elec-troiis from high to low potential with the conver-sion of inorganic phosphate to energy-Iich phos- I-phate ('-P). The block of hydrogen transfer, if--P is not removed and thus allowed to revert, -may then be interpreted as indicating reversi-bility in a "leakproof" system. On the other p 26hand, the fact that quite frequently in the absence Xof phosphate acceptor system high respiratory OXYGENrates are found, emphasized the difficulty in FIG. I

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obtaining undamaged preparations which do not show a slight uncoupling.The incomplete uncoupling, indicated by a persistence of normal P/O ratios in

spite of an abolished acceptor effect, may be the normal event in milder thyrotoxi-cosis, resulting in a respiration which is not controlled so rigorously by the demandof energy as it is in the normal. The economical value of the acceptor effect as theregulation of output by demand is rather obvious, and its partial breakdown will besufficient to explain the wasteful respiration in the hyperthyroid animal.

Depression of P/O Ratio in Liver Mitochondria of Thyrotoxic Rats.-Our efforts torelate thyroxine action to uncoupling of phosphorylation had thus been partiallysuccessful. Meanwhile, Martius and Hess had become interested in this problem,and they were able to obtain more radical effects by using higher concentrations ofthyroxine in vivo."5 Furthermore, by an in vitro procedure involving preincuba-tion of mitochondria with thyroxine under slightly damaging conditions, uncou-pling with thyroxine was similarly obtained. In their experiments the respiratoryincorporation of radioactive phosphate into ATP was usually used to measure phos-phorylation, following the method of Friedkin and Lehninger.16 Using the dosageformula of Martius and Hess, we were able to repeat and amplify their results.In a series of rat experiments, as shown in Table 5, a depression of P/O ratios was

TABLE 5DEPRESSION OF P/O RATIO WITH LIVER MITOCHONDRIA OF THYROTOXIC RATS*

Qo2WITHOUT WITH

SUBSTRATE TREATMENT ACCEPTOR ACCEPTOR P/Oa-Ketoglutarate Normal (8)t 21.2 34.6 1.69

Control injected (4) 19.8 32.8 1.66Thyrotoxic (4) 27.4 29.7 0.80

Glutamate Control injected (4) 18.0 38.6 1.78Thyrotoxic (4) 27.4 33.6 1.06

ft-Hydroxybutyrate Control injected (4) 19.3 31.5 1.42Thyrotoxic (3) 25.0 27.1 0.19

* Thyroxine injected: first day, 4 mg.; second, third, and fourth days, 8 mg.; assayed on fifth day.t Number in parentheses gives number of experiments.

observed"7 with various substrates and most pronouncedly with #3-hydroxybutyrateas substrate. With hydroxybutyrate as substrate, phosphorylation was practicallycompletely suppressed in the thyrotoxic mitochondria,- demonstrating that thy-roxine, like dinitrophenol, could disrupt energy transformations over the wholepotential range of respiratory electron transfer. It may be noticed, furthermore,in Table 5 that, in confirmation of Niemeyer et al.,2 12 the acceptor effect was nearlyabolished with all substrates used. It must be added here that, even with thishigh dosage, only occasionally was a series of animals encountered in which theeffects were as pronounced as reported in Table 5.In Vitro Effects of Thyroxine on Hamster Liver Mitochondria.-In spite of now

being increasingly successful in obtaining uncoupling with preparations from in vivotreated rats, we still had not succeeded in doing so with in vitro application ofthyroxine to our rat liver mitochondria. Suspecting a dependence of success on

particular properties of the mitochondria, possibly peculiar to the rat strain usedby Martius and Hess, we turned to another animal and tried the hamster. Usinganalogously prepared hamster mitochondria, it appeared soon that with hamsterliver mitochondria, on incubation with thyroxine, in vitro effects were obtainedregularly and unfailingly.

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The effect of various concentrations of thyroxine is shown in Table 6. Glutamatewas used as substrate. The P/O ratio is increasingly reduced with thyroxine con-centrations between 10-l and 10-4 M. This concentration range is similar to thatfor dinitrophenol (cf. Table 3).

In Table 7 experiments are shown with a constant concentration of thyroxine,10-4 M, and various substrates. The depression of P/0 ratios is more or less pro-

TABLE 6IN VITRO EFFECTS OF VARYING CONCENTRATIONS OF L-THYROXINE WITH

HAMSTER LIVER MITOCHONDRIA*L-THYROXINE Q2PControl 57.7 2.725 X 10-6M 54.7 2.69

10-5M 49.2 2.395 X 10-5M 37.5 0.41

10-4M 26.8 0* Temperature, 250; substrate, glutamate 50 uM.

nounced, depending on the substrate. The effect is least with a-ketoglutarate.This is not too surprising, since, as shown by Hunter,18 using this substrate with dini-trophenol, a considerable residue of phosphorylation remains. Hydroxybutyrateand succinate as substrates, however, give complete uncoupling.

In Tables 6 and 7 there appear peculiar differences in the in vitro effect of thy-roxine on respiratory rates with different substrates, which must be pointed out,although we have no ready explanation for them. With hydroxybutyrate, thenormally relatively low Qo2 is consistently increased with thyroxine. With gluta-mate, ketoglutarate and succinate, thyroxine, however, more or less inhibits respira-tion. This seems peculiar to the in vitro application and does not apply to themitochondria from thyrotoxic animals, even if the P/O ratios are low (cf. Table 6).In relation to the different permeability characteristics for thyroxine, it is sig-nificant that, in contrast to our rat preparations, the washed and isolated hamsterliver mitochondria are deficient in DPN and cytochrome c. This deficiencymanifested itself particularly in the presence of thyroxine and has been noted forDPN with other uncoupling agents."9 In Table 8 the effect of diphosphopyridine-

TABLE 7IN VITRO EFFECTS OF THYROXINE WITH LIVER MITOCHONDRIA OF NORMAL HAMSTERS

AND VARIOUS SUBSTRATESQo2 P/

THYROXINE THYROXINESUBSTRATE 10O4 M - 10'4 M -

Glutamate 49.8 55.8 0.73 2.56a-Ketoglutarate 46.8 51.2 1.55 2.65P-Hydroxybutyrate 37.4 24.0 0 2.19Succinate 57.4 70.5 0 1.79Octanoate 39.3 56.4 0.47 1.73

nucleotide (DPN) addition on normal and thyroxine-treated hamster preparationsis shown. The respiration is stimulated also in the control by pyridine nucleotide,but the effect is much stronger in the presence of thyroxine, indicating the additionaldamage apparently caused through thyroxine and probably partly due to the de-pression of phosphorylation. When DPN is added, however, thyroxine actuallyincreases QO,, parallel with a decreased P/O ratio. In Table 9 analogous experi-

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ments with the omission and addition of cytochrome c are shown. The cytochromeeffect is similar but not as marked as that of the pyridine nucleotide. In all hamsterexperiments, therefore, DPN and cytochrome c were added to the suspending me-dium. (These cofactors had also been added to the rat preparations because of thefindings of Drabkin20 that the cytochrome c content of rat liver changed with thy-roid activity.) The need for a supplementation with pyridine nucleotide and cy-tochrome c indicates a leakiness of the hamster mitochondria, which, on the otherhand, explains the consistent thyroxine effects, because it permits penetration ofthe thyroid hormone into the mitochondria. If, however, the suspension is main-

TABLE 8EFFECT OF ADDED DPN ON HAMSTER LIVER MITOCHONDRIA

ADDITIONS °°2 Pi/

3.3 x 10-4M DPN + - + -

CONTROL 18.6 11.6 1.93 2.46

104 M DL THYROXINE 24.3 3.7 0 0

SUBSTRATE fsHYDROXYBUTYRATE

TABLE 9EFFECT OF ADDED CYTOCHROME C ON HAMSTER MITOCHONDRIA

ADDITIONS o02 P/O

10-5 M CYTOCHROME C + - + -

CONTROL 20.2 16.1 2.32 2.40

IOM DL THYROXINE 26.5 10.7 0.11 0

SUBSTRATE -6 HYDROXYBUTYRATE

tained throughout in the more protective sucrose medium, the thyroxine effect prac-tically vanishes with glutamate and is weaker even with hydroxybutyrate. Acomparison of the effects in saline and sucrose, respectively, is shown in Table 10.As shown in the lower part of Table 10, a metabolic effect in the sucrose mediummay be obtained only by preincubation with thyroxine. These results further con-firm a dependence of the thyroxine effect on permeability conditions.Comparison of the Effects of Thyroxine and Triiodothyronine.-Through the work

in particular of Gross and Pitt-Rivers,2' triiodothyronine was shown to be three tofive times as active as thyroxine in animal experiments. It was therefore of interest

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to test the relative activity of these compounds inl the ill vitro system. As showni illTable I 1, with hydroxybutyrate as the substrate, triiodo- and tetraiodothyronineinhibit at similar concentration levels, with the triiodo-compound slightly less ac-tive, however, in this in vitro system. It may be noted that the respiratory effectwith triiodothyronine is greater than with thyroxine; the triiodo-compound may beless damaging to the preparation. As shown, perhaps somewhat more clearly, inFigure 2, with glutamate as substrate, the inhibition curves for tri- and tetraiodo-thyronine are rather parallel, although the triiodo-compound again appears less

TABLE 10DEPENDENCE OF THYROXIN EFFECT ON MITOCHONDRIA PREPARATION AND SUBSTRATE

TYPE OF APPLICATION OF NO.OFPREPARATION SUBSTRATE THYROXIN IO4M/L EXP Q03 PlO

SALINE GLUTAMATE NONE 15 55.0 2.47

PRE-INCUBATED 8 42.9 1.17DIRECT 7 42.1 0.90

fi-HYDROXY- NONE 14 21.4 2.16BUTYRATE PRE - INCUBATED 7 24.0 0.47

DIRECT 7 27.2 0.51

SUCROSE GLUTAMATE NONE 7 51.5 2.60PRE-INCUBATED 3 40.3 Q48

DIRECT 4 43.3 2.29

a-HYDROXY - NONE 6 24.2 2.16BUTYRATE DIRECT 6 27.7 1.15

TABLE 11IN VITRO EFFECTS OF THYROXINE AND TRIIODOTHYRONINE ON NORMAL HAMSTER LIVER MITOCHON-

DRIA *P/OL-THY- TRIIODO-

CONCENTRATION CONTROL ROXINE THYRONINE

10-4M 2.25 0.59 1.345 x 10-'M 2.21 0.49 1.61

10-5AI 1.78 1.89 2.08* Glucose-hexokinase is used as a phosphate "acceptor" system.

Qo2L-THY- TRIIODO-

CONTROL ROXINE THYRONINE

17.1 24.1 28.922.1 29.3 32.425.2 20.5 22.8

effective. In the whole animal the triiodothyronine, although more active, seemsto show a more transient activity than the tetraiodo-compound,22 indicating thatthe outer cell wall may be more permeable to the triiodo- than to tetraiodothyro-nine. With easier permeation a greater over-all activity could be reconciled with asomewhat lesser effect on the mitochondrial system. This would be in parallelwith the general experience with halophenols5 that a higher halogen content runsparallel with greater activity. A further illustration of the importance of the perme-ability factor with iodinated phenols is presented by comparison of free diiodopara-

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3.0

2.0K_

I .0H

v 2 4 6 8 10CONCENTRATION: Mx IO-

FIG. 2

TABLE 12EFFECT OF BUTYi-3,5-DIIODO-4-HYDROXYBENZOATE ON MITOCHONDRIA

BUTYL ESTER (M)- 10-5 3 X 10 -5

11 22 24with acceptor* 27 24 27

P/O* Glucose pluse hexokinase.

3.2 1.1 0.3

10-4

26240

TABLE 13DISTRIBUTION OF I'31-LABELED THYROXINE BETWEEN MITOCHONDRIA AND SUSPENDING

MEDIA*

PREPARATION OFANIMAL MITOCHONDRIA

Hamster Saline

Frozen and thawedSucrose

Saline

Protamine-desoxyribo-nucleic acid precipi-tate, pH 7.4

ADDITIONS

L-Thyroxine, 10-4 ML-Thyroxine, 2 X 10-6MTriiodothyronine, 10-4ML-Thyroxine, 10-4 ML-Thyroxine, 10-4 MTriiodothyronine, 10-4ML-Thyroxine, 10-4 MTriiodothyronine, 10-4M

Thyroxine, 10-4 l

P/O(PER

PER CENT OF TOTAL CENTCOUNTS OF

MITO- CON-CHONDRIA SUPERNATE TROL)

83.7 16.4 6580.1 19.990.8 10.062.1 33.4 7241.6 54.1t 10077.6 21.345.0 56.1 673.0 25.3 8

71.0 29.0* Three to four per cent mitochondrial suspension in a mixture containing 0.04 M glycylglycine, 0.0007 M

ATP, 0.013 M phosphate, 0.025 M KC1, 10-5 M cytochrome c, and 3.3 X 10-4 M DPN; pH = 7.4. Immediatelyspun down at 20 at 17,000 rpm for 5 minutes.

t Cloudy supernatant fluid.

p

THE/N VITRO EFFECTS OF L-- THYROX/INEAND OF TRI/ODOTHYRON/NE COVHAAMSTER L / VER MITOCHONDRIA

SUBSTRATE: GLUTAMATE

K \ TRIIODOTHYRON INE

L- THYROX I NE

I I

Q02QO2

Rat

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hydroxybenzoate, with its butyl ester, the latter being also known as a thyroxineanalogue.23, 24

In Table 12 uncoupling is shown with the butyl ester at 10-6 M. However, asshown in Figure 3, the free acid uncouples rather poorly if at all. There is, there-fore, at least a thousand fold increase in activity observed with esterification, pre-sumably due to the greater ease of penetration of mitochondria by the ester.

Distribution of Thyroxine and Triiodothyronine between Medium and Mito-chondria. Since thyroxine and also triiodothyronine show tendencies of coprecipi-tation, it was important to estimate the actual concentration of thyroxine inequilibrium with the mitochondria suspension. For this purpose data on theequilibration of radioactive thyroxine and triiodothyronine with mitochondria wereobtained. In Table 13 the results of such experiments are shown (cf. "Methods").Although somewhat erratically, the major part of thyroxine attaches itself to themitochondria; 80-90 per cent of the thyroxine may be carried down by the corpus-cular material. From the last column of Table 13 it may be seen, furthermore,that there appears to be no correlation between the metabolic effect and the ab-sorption of thyroxine on the mitochon-dria. As much thyroxine was carried EFFECT OF FREE Di-IODO-p-HYDROXYdown by rat mitochondria which did not BENZOATEshow any appreciable uncoupling as with °the uncoupled hamster particles. These z

80

data show the difficulty of assessing ex- 'r10fVLi.actly the concentration of thyroxine in- ° 60-side the mitochondria. ,40 ,

40-

COMMENTS2 0 -

For evaluation of the situation in theliving animal the experiments with prepa- '

4

rations from thyroxine-treated animals 2 4 6 8 10 x 0 Mseem more significant than those obtained 3,5 Di-ODO-a -HYDROXY BENZOICACID

in vitro. While the essential parallel be- FIG. 3tween the in vivo and in vitro data makesit a safe conclusion that in both cases we are dealing with the same effect, the ratherhigh concentrations of thyroxine needed in the in vitro experiments indicate thatthe actual concentration inside the mitochondria is probably considerably smallerthan the concentration in the medium. This is illustrated by the results on thedistribution of radioactive iodinated compounds between mitochondria and super-natant. Thyroxine deposits on the mitochondria independent, however, of meta-bolic action. On the other hand, the fact that mitochondria suspensions taken fromthyroxine-treated animals remain uncoupled after having undergone isolation andextensive washing makes it apparent that once thyroxine has entered the mito-chondria it is bound very tightly and not easily removed.One of the most interesting phenomena which have come to light during these

studies is the reciprocal relationship between respiratory rate and phosphate-bondrelease. As a regulatory feature of energy turnover, this gearing of output by de-mand is impressive. Experimentally, a disturbance of this regulation appears to bea more sensitive indicator for uncoupling than the measurement of P/O ratios.

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This is understandable if we realize that the acceptor system, hexokinase-glucose, isapplied in considerable excess and will therefore extract a maximum of phosphatebonds, even from a slightly uncoupled system. But a disturbance of the regulationof the energy output by demand will be sufficient to explain much of the sympto-matology of at least milder forms of thyrotoxicosis. Therefore, the mere absenceof the acceptor effect in the thyroxine-treated animal is significant.On the other hand, theoretically it is important that in vitro experiments as well

as in vivo experiments have shown that the coupling mechanism may be completelydisrupted by thyroxine. It is well known that three or more phosphate bonds maybe formed for every passage of a single pair of electrons from substrate to oxygen.

This has been interpreted to imply a series of independent conversion mechanismson successive oxido-reduction levels during the electron passage. In view of thisscheme an interference by thyroxine of one specific step has been an attractivepostulate.1'- 1 However, a reduction of P/O ratios to zero level excludes such a

mechanism.25Indeed, in view of this potentially all-or-none effect of most uncoupling reagents,

including thyroxine, it appears likely that an extensive pooling occurs betweenthe various oxido-reduction levels, which, through an eventual transphosphoryla-tion step, connects with the adenylic acid system.

In order to describe through the uncoupling effect the normal as well as the toxicaction of thyroxine, some difficulties seem to arise. The symptomatology on theenzyme level fits very well with the toxic manifestation of thyroxine. On the otherhand, the effect of thyroxine on the athyroid animal has in some manner to be rec-

onciled with the uncoupling action. There can be no doubt that in this phase ofactivity the thyroid hormone promotes growth and increases the rates of synthesesas shown, e.g., by acceleration of amino acid incorporation. It may, however, beargued that, for an orderly synthetic activity, balance is needed between hydro-genation and condensation reactions. Considering, for example, fatty acid synthe-sis, this would become impossible if all substrate hydrogen were forced into conver-

sion to phosphate-bond energy. No hydrogen, then, would remain available forthe two hydrogenation reactions which have to follow every condensation step.It may thus be visualized that the hormone regulates the system in such a manner

as to allow enough hydrogen to escape transformation to balance properly hydro-genation and condensation against each other. In this manner a loosening of a

tight coupling would be beneficial and would indeed allow synthetic reaction toproceed more smoothly.

* This investigation was supported in part by research grant from the National Cancer In-stitute, of the National Institutes of Health, Public Health Service; the Life Insurance MedicalResearch Fund; and the Rockefeller Foundation.

t Research Fellow of the Jane Coffin Childs Memorial Fund for Medical Research. Presentaddress: Biophysics Research Laboratory, Department of Medicine, Harvard Medical School,and Peter Bent Brigham Hospital, Boston, Massachusetts.

1 W. F. Loomis and F. Lipmann, J. Biol. Chem., 173, 807, 1948.

2 H. Niemeyer, R. K. Crane, E. P. Kennedy, and F. Lipmann, Feaeration Proc., 10, 229,1951.8

F. L. Hoch and F. Lipmann, Federation Proc., 12, 218, 1953.IF. Lipmann and C. H. duToit, Science, 113, 474, 1951.

6 G. H. A. Krahl and M. E. Clowes, J. Cellular Comp. Phydsiol., 11, 21, 1938.6J. D. Judah and H. G. Williams-Ashman, Biochem. J., 48, 33, 1951.

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7S. B. Barker, Physiol. Rev., 31, 205, 1951.8 G. H. Hogeboom, W. C. Schneider, and G. E. Pallade, J. Biol. Chem., 172, 619, 1948.9 E. P. Kennedy and A. L. Lehninger, J. Biol. Chem., 179, 957, 1949.

10 L. Berger, M. W. Slein, S. P. Colowick, and C. F. Cori, J. Gen. Physiol., 29, 379, 1946.1 C. H. Fiske and Y. Subbarow, J. Biol. Chem., 66, 375, 1925.12 H. Niemeyer, R. K. Crane, E. P. Kennedy, and F. Lipmann, Bol. Soc. biol. Sanliago, Chile,

10, 54, 1953.13 R. K. Crane and F. Lipmann, J. Biol. Chem., 201, 235, 1953.14 H. A. Lardy and H. Wellman, J. Biol. Chem., 195, 215, 1952.lo C. Martius and B. Hess, Arch. Biochem. and Biophys., 33, 486, 1951.16 M. Friedkin and A. L. Lehninger, J. Biol. Chem., 178, 611, 1949.17 G. F. Maley and H. A. Lardy, J. Biol. Chem., 204, 435, 1953.18 F. E. Hunter, in W. D. McElroy and B. Glass (eds.), Phosphorus Metabolism, 1 (Baltimore:

Johns Hopkins Press, 1951), 321.1"F. M. Huennekens and D. E. Green, Arch. Biochem., 27, 428, 1950.10 D. L. Drabkin, J. Biol. Chem., 182, 335, 1950.21 J. Gross and R. Pitt-Rivers, Lancet, 1, 593, 1952.22 S. P. Asper, Jr., H. A. Selenkow, and C. A. Plamondon, Bull. Johns Hopkins Hosp., 93, 164,

1953.23 J. H. Wilkinson, Biochem. J., 54, 485, 1953.24 C. H. duToit, F. L. Hoch, E. Wright, and F. Lipmann, I6 Congrhs international de biochimie

(Paris, July 21-27, 1952), p. 50, "R6sumes des communications."25Recently, through in vitro experiments with thyroxine, an antagonism between magnesium

and thyroxine has come to light. It was first observed by J. Bain (J. Pharmacol. Exptl. Therap.110, 2, 1954) and confirmed in our laboratory by Harting and Mudd (unpublished). The concen-trations of magnesium required to overcome the thyroxine effect are about fifty fold that of thy-roxine. This seems not compatible with the formation of a chelate. The phenomenon deservesfurther attention.

26 C. H. duToit, in W. D. McElroy and B. Glass (eds.), Phosphorus Metabolism, 2 (Baltimore:Johns Hopkins Press, 1952), 597.

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