RESPIRATORY ENZYMES IN OXIDATIVE PHOSPHORYLATION

VI. THE EFFECTS OF ADENOSINE DIPHOSPHATE ON AZIDE-TREATED MITOCHONDRIA*

BY BRITTON CHANCE AND G. R. WILLIAMSt

(From the Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania)

(Received for publication, August 1, 1955)

Papers I to IV of this series (l-4) have dealt with changes in the oxida- tion-reduction levels of the components of the respiratory chain upon initia- tion of oxidative phosphorylation in “tightly coupled” preparations of liver mitochondria. Changes in the oxidation-reduction levels of DPNH,l ffavo- protein, and cytochromes b and c were readily recorded (3), while those due to cytochromes a and cz3 were of small magnitude and difficult to detect. Addition of a low concentration of azide, which does not appreciably in- hibit respiration or phosphorylation, greatly magnifies the steady state changes of cytochromes ~3, a, and c caused by ADP addition. Thus cross- over points (1) in the respiratory chain can be accurately located. It is also possible to “titrate” these components with ADP and to record the speed with which the ADP-cytochrome interaction occurs. Such experi- ments give insight on the chemical nature of intramitochondrial DPNH and on possible intermediate reaction steps between ADP and the electron transport system.

Preparations-The preparations and reaction media are described in Paper II (2).

Methods-Both the split beam and double beam recording spectropho- tometers, as well as the platinum electrode technique, are described briefly in Papers II and I, respectively (2, 3).

Results

Correlation of Oxygen and Cytochrome Kinetics-Whereas the reduction of cytochrome a on the transition from State 4 to 3 (see Paper HI (1)) causes a very small change in the steady state level of cytochrome a, ad-

* The support of part of this work by the National Science Foundation and by the Office of Naval Research is gratefully acknowledged.

t Present address, C. H. Best Institute, Toronto. 1 The abbreviations used in this paper are ADP = adenosine diphosphate, Pi =

inorganic phosphate, ATP = adenosine triphosphate, DPNH = reduced diphospho- pyridine nucleotide, DNP = 2,4-dinitrophenol.

477

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473 RESPIRATORY ENZYMES. VI

dition of azide makes this spectroscopic change much larger, and a typical record obtained in the presence of this inhibitor is given in Fig. 1, A. The upper trace records the increased respirat)ion caused by the initiation of oxidative phosphorylation upon ADP addition and its cessation when the added ADP is phosphorylated. The lower trace of Fig. 1, A records, as an upward deflection, the reduction of cytochrome a during oxidative phos- phorylation, followed by its reoxidation when the respiration slackens. It is apparent that the kinetics of the change of the steady state of cytochrome a are consistent with the changes of respiratory rate; in fact, the changes in slope of the oxygen trace correspond closely to the amplitude of the changes in the cytoehrome a.

The spectroscopic changes that accompany the addition of azide to mitochondria in State 4 (Paper III (1)) are illustrated by Fig. 1, B : azide addition causes partial reduction of cytochromes a, c, and a3, as indicated by the prominent absorption peaks at 605, 550, and 445 rnp. A small shoulder on the long wave-length side of the cytochrome c band is caused by a reduction of cytochrome b and the trough at 465 to 490 rnp is caused by the reduction of oxidized flavoprotein. If the respiration is increased by the addit’ion of ADP (State 4 to 3 transition), the reduction of cyto- chromes a, c, and a3 increases considerably, as is indicated by the increased heights of their absorption bands. On the other hand, the cytochrome b band is less distinct, and that of flavoprotein is definitely smaller, as will be discussed later.

E$ect of Azide upon Respiration and Phosphorylation-When a compo- nent of the respiratory chain has a high activity relative to the rate-limit- ing step, its steady state level can be drastically altered without a propor- tional change of the rate of electron transfer through the chain. This is the case with cytochrome a3, which reacts with oxygen very rapidly (see Paper IV, Table IV (4)) compared with its turnover number (see Paper II (2)). With glutamate as a substrate as in Fig. 1, A, the azide concentra- tion used causes only about 10 per cent inhibition of respirat,ion. If the respiration rate is increased somewhat by using succinate as a substrate, the inhibition is somewhat greater (see Table I). In either case there is a considerable range of azide concentrations within which large changes of the steady state levels of cytochromes a and a3 are observed in the State 4 to 3 transition with only small inhibition of respiration. It should be noted that the respiration in the quiescent State 4 is not measurably in- hibited by the higher azide concentrations, while the respiration in the active State 3 is affected (see “Discussion”).

Table I also includes some values of the ADP: 0 ratio in the presence of azide, and it is seen that no decrease occurs up to 276 PM. This result is in accord with that of Loomis and Lipmann, who used a 5-fold greater

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R. CHANCE AND G. R. WILLIAMS 479

azide concentration for the uncoupling of phosphorylation (5). Slater (6) found that about the same azide concentration was necessary, but, in con- trast to the results of this paper and of Loomis and Lipmann, found that

state 4 plus azide

state 4 plus azide and ADP

420 440 460 480 500 520 540 560 580 600 620

2mp'

FIG. 1. A, correlation between changes in respiration rate caused by ADP addi- tion and changes in the steady state level of cytochrome a. The upper trace is re- corded by means of a vibrating platinum micro electrode and the lower trace is recorded by means of a double beam spectrophotometer. Guinea pig liver mito- chondria, glutamate as substrate, 184 fiM azide, isotonic medium, 25” (Experiment 38813). B, the effects of azide and azide plus ADP upon the extent of reduction of cytochromes. The base-line in both traces corresponds to mitochondria in State 4; p-hydroxybutyrate as substrate, rat liver mitochondria, 400 pM azide, 1 mM ADP for the lower trace, isotonic medium (Experiment 466f-5).

the respiration of the muscle sarcosomes was much more resistant to azide than that of the liver mitochondria.

Since azide is believed to inhibit respiration by combining with the oxi- dized form of cytochrome ~1x3, it might have interfered with the formation of any high energy intermediate attached to the oxidized form of the en-

02 + u3” x I - u3”’ - I + 02- (1)

zyme and thereby lowered the ADP : 0 value. Such a decrease is not ob-

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480 RESPIRATORY ENZYMES. VI

served at these aside concentrations and this result is in accord with other data (Paper III (1)) that lead to the conclusion that the reduced and not the oxidized form is bound in the --I compound (Paper V (7)). It is also unlikely that the reduced form of cytochrome a3 is affected by the inhibitor I, since Lehninger (8) has been unable to demonstrate a decrease of the P:O value in the presence of CO. It may be that phosphorylation dots not occur in the cytochrome a3-oxygen reaction.

Afinity for ADP-The ADP concentration required to give maximal res- piration in State 3 has been computed from the platinum electrode record- ings described in Paper I (3) and has been termed the ADP affinity of the respiratory chain. The same quantity can be evaluated on an entirely different basis by measuring the extent of reduction of cytochrome a in State 3 in the presence of various initial ADP concentrations. A typical

TABLE I

Effect of Azide upon Respiration Rate and Phosphorylation

Guinea pig liver mitochondria, succinate as substrate, 26” (Experiment 37813).

0 92 276 390 ___~

Respiration rate in State 3. 2.0 1.9 0.90 0.40 <‘ “ “ “ 4.................... 0.11 0.11 0.26 0.10

Phosphorylative activity 1 2.0 1 2.15 j 2.10 1

series of measurements is illustrated by Fig. 2, which shows how both the extent of reduction and the duration of the “cycle” increase with ADP con- centration. It should be noted how sensitively this spectroscopic test re- sponds to ADP (see “Discussion”).

In a cycle such as that shown in Fig. 2, ADP is acting as a substrate and the simple equation derived for the Michaelis theory may be applied with reasonable accuracy.

P 1 _=- e

1 + 5 2

(2)

where P/e is the fractional reduction of cytochrome a, x is the substrate or ADP concentration, and K, is the affinity.

In Fig. 3, A, we have plotted the increment of reduction of cytochromes a and c as a function of ADP concentration and obtained half maximal effects with about 60 pM ADP for this experiment. A summary of other

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5. CHANCE AND G. R. WILLIAMS 481

data for cytochromes u3, a, and c is given in Table II. These values are somewhat larger than those computed from the ADP concentralion re- quired to give half maximal respiration (20 to 30 pM ADP (3) or 10 pM

ADP (9)). It is also possible that the ADP affinity is not, identical for the various members of the respiratory chain.

Cytochrome a plus 184~M Azide 605-63Omy log IO/I .$003

FIG. 2. The effect of increasing concentrations of ADP upon the extent and dura- tion of the reduction of cytochrome a in azide-treated mitochondria. Experimental conditions as in Fig. 1, A.

85pM Azide

: ,Y 100 300 500

8 .- h 0

[ADPbM

A FIG. 3. A, comparison of the ADP titration of cytochromes a and c in the presence

of 85 PM azide. Guinea pig liver mitochondria, succinate as substrate, isotonic me- dium, 10” (Experiment 382b). B, titrations with ADP in the presence of low and high azide concentrations. Rat liver mitochondria, P-hydroxybutyrate as substrate, isotonic medium, 25” (Experiment 459).

A decrease in the rate of electron transport decreases the amount of ADP required for half maximal spectroscopic effects; for example, Table II shows that, in the case of cytochrome u3, only 23 NM ADP are required at 10”. If excess azide is used, a similar reduction of the ADP concentration is observed (Fig. 3, B), but, under these conditions, the titration curve is no longer hyperbolic (10). At 800 PM azide, the amount of cytochrome a reduced upon ADP addition increases linearly with the ADP concentration

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482 RESPIRATORY ENZYMES. VI

and then breaks sharply at 34 PM ADP, as if a component of the respiratory chain were being titrated.2 As shown by the spectrophotometric assays of Paper II, Table II (Z), the cytochrome a concentration is equivalent to about 2 pM, a quantity insufficient to explain this result. In fact, DPNH is the only known component present in sufficient amounts (about 38 pM

DPNH in State 4) to account for this result. Thus the simplest hypothesis is that the ADP utilization by the azide-inhibited respiratory chain is caused by the over-all reaction (6)

DPNH N I + Pi + ADP t- DPNH + ATP + I (3)

As illustrated by Equation 5 (see below), one must also consider that an appreciable amount of the hypothetical X - I compound may accumu- late in State 4 and may contribute to the titration. The close agreement

TABLE II

Summary of Results on ADP Titrations of Cytochromes as, a, and c

Succinate or glutamate as substrate.

Cytochrome aa Cytochrome a Cytochrome G

Gluta- mate Succinate Glutamate Succinate “&:“,- Succinate

rrK,“p~. ........................ 23 40 44 40 76 -70 Temperature, “6 .................. 10 26 26 26 26 26 Azide, PM. ....................... 68 85 184 85 184 85 Experiment No .................. 385 379 388b 379 388 379

of the titration values with the DPNH content suggests that this contribu- tion is small. It has not yet been possible to devise an experiment in which DPNH - I and X - I can be titrated separately.

If we are observing a titration of a component of the phosphorylation system, then variations in the amount of mitochondria used in the titration should vary the ADP titration value at high azide concentrations. Table III shows that larger amounts of mitochondria require a larger ADP titra- tion to give half maximal effect over the rather narrow range of the experi- mental data. In no experiments do our titrations fall to the very low value of lo-’ M obtained by Slater and Holton for the a-ketoglutarate-cytochrome c system (9), and it must be concluded that their value applies only to their preparations of sarcosomes which have much less reducible DPN (11).

2 The ADP utilization in the 4 second interval required to reach maximal reduction of cytochrome a (see Fig. 1) is relatively small. For the conditions of Fig. 3, B, ADP utilization due to respiration during the 4 second interval is roughly 8 pM at 34 pM

ADP.

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B. CHANCE AND G. R. WILLIAMS 483

Lehningers has observed a DNP-sensitive P32 uptake by cyanide-inhibited mitochondria to which ADP is added, and in a particular experiment there was 0.06 mpmole of P uptake per 0.005 mg. of mitochondrial protein, this value being near the maximal one observed in several experiments. In order to compare his result with our titration data, we convert his value to the higher protein concentration used in the experiment of Fig. 3, B (4 mg. per cc.). Lehninger’s P32 uptake is then equivalent to the formation of 48 PM ATP32, as compared with 38 PM DPNH - 1 computed to be present from spectrophotometric data, and an end-point of the ADP titration of 34 PM (Fig. 4). This agreement is as good as could be expected in view of the variabilities of mitochondrial preparation. The concordance of these results confirms our rather different experiments (Paper III (l)), on the basis of which we have concluded that mitochondria in State 4 contain DPNH in an inhibited form. These new correlat,ions indicate that the hypothetical compound (DPNH - I) could convert an equivalent amount

TABLE III

Effect of Mitochondrial Concentrations upon Apparent Aflnity of Cytochrome a for ADP

800 pM azide (Experiment 459).

Approximat,e protein concentration, mg. per cc. ADP for half maximal effect, PM..

of ADP to ATP without the need for electron transport. Thus the ADP affinity as obtained from spectroscopic data appears to contain two com- ponents: (a) a stoichiometric reaction with high energy compounds present in the mitochondria in the resting state and (b) a “Michaelis” affinity which depends upon the rate of electron transport in the respiratory chain.

Reaction Kinetics-The kinetics of the State 4 to 3 transition are of con- siderable importance in evaluating (a) whether intermediate chemical re- actions or permeability barriers intervene between ADP and its reaction partners and (b) whether ADP reacts with the different phosphorylation sites at the same or different speeds.

Table IV shows that the kinetics of oxidation of DPNH and cytochrome b upon addition of ADP to the mitochondria in State 4 are not significantly speeded up by an increase in the ADP concentration. Since these two sub- stances are at least three components down the respiratory chain from the terminal oxidase, the maximal speed with which they can be oxidized may be set by the capabilities of the cytochrome chain, and not by the speed of the ,4DP reaction itself. On the other hand, the speeds of reduction of

3 Dr. A. L. Lehninger, personal communication.

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484 RESPIRATORY ENZYMES. VI

the cytochromes, especially c and a, in the azide-treated system are suf- ficiently rapid to be used for a study of the kinetics of the interaction of ADP with the respiratory chain. As a control experiment, me have com-

TABLE IV

Kinetics of State 4 to 3 Transition

Rat liver mitochondria, p-hydroxybutyrate as substrate, 5” (Experiment 306).

ADP, FM

component 96 ) 290 / 1600 1 3200

I Half time

Reduced nvridine nucleotide..

/ ;; / ;; i ;; 1 ;;

Cytochrome b

I II % 763Osec. 445 -455p

I log zo/l=o.oo2 T

Cytoc hrome a3

17 22 17 16 1 17 1 22 1 17 1 16

1.2mM

0 IO 20 30sec 0 IO 20 30sec 55 2 -541 my &

Cytochrome a Cytochrome c

FIG. 4. Direct recordings of the kinetics of cytochrome reduction caused by the addition of ADP to the azide-inhibited mitochondria. Experimental conditions as in Fig. 3, A.

pared the speed of reduction of the cytochromes caused by the addition of a sufficient excess of azide (13 mM) to stop respiration rapidly with that caused by the addition of an excess of ADP. The former reaction is found

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B. CHANCE AND G. R. WILLIAMS 485

to be about 9 times more rapid and this result shows that the kinetics of the latter reaction are not set by the rate of transfer of reducing equivalents along the cytochrome chain, but by the rate of the ADP interaction itself.

Fig. 4 presents direct recordings of the kinetics of reduction of these three cytochrome components measured at appropriate pairs of wave- lengths with the double beam spectrophotometer (1). On the basis of half times, the reaction with cytochrome a is most rapid. This result is borne out by studies over a wide range of ADP concentrations (see Fig. 5) in which the initial slopes of reaction kinetics, such as those il- lustrated by Fig. 4, are measured. The initial rate of reaction with cyto- chrome a is more rapid than that with cytochrome c, and in both cases the speed of reaction ceases to follow the required linear increase of rate

0/ 200 600 1000

FIG. 5. Rate of reduction of cytochrome a or c as a function of ADP concentra- tion. Experimental conditions as in Fig. 3, A.

with increasing ADP concentration, as if a rate-limiting reaction were in- terposed between the respiratory chain and ADP. The fact that the kinetics of cytochrome a are more rapid than those of cytochrome c suggests independently that cytochrome a may be bound as cytochrome a - I compound.

In order to determine whether this rate limitation is due to a chemical reaction or to impermeability of the mitochondria to added ADP, the re- action kinetics of Fig. 4 have been investigated with hypotonically treated mitochondria. It is found that the kinetics of reduction of cytochrome a upon ADP addition to mitochondria that are freely permeable to DPNH (cf. Paper III (1)) are no faster than those measured with mitochondria that are impermeable to DPNH. This result is illustrated by the two kinetic traces of Fig. 6, one in isotonic medium, the other in hypotonic medium (0.10 OSM). Thus the break in the kinetic curves of Fig. 5 is due

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486 RESPIRATORY ENZYMES. VI

to one or more chemical reactions interposed between the electron transport chain and the phosphate acceptor. For this reason, the transfer reactions

i, ’ iso &“dS Isotonic Medium 6

Hypotonic Medium

A B FIG. 6. That the rate of reduction of cytochrome a caused by addition of 800 FM

ADP to the azide-inhibited mitochondria is not dependent upon the permeability of the mitochondria (Experiment 479a) is illustrated. Note that the sensitivity used for record B is about twice that used for record A and that the half times of the reactions are about the same. Note that reduction of cytochrome a is registered as a downward deflection in this figure. Rat liver mitochondria, succinate as sub- strate, media, 0.25 and 0.1 OSM, and azide concentrations, respectively, 120 and 40 PM in records A and B, 10” (Experiment 479a).

TABLE V

E$ect of ADP upon Steady State Levels of Aside-Treated Guinea Pig Liver Mitochondria

Succinate as substrate (Experiment 378).

I Per cent reduction of components

Azide state No.

.~ __... 8-i

0 4 0 3

92 4 92 3

83

12 24

a t b fP DPNH ~-____ -___ ___. ..-~-

-0 20 37 46 100 19 18 18 36 73 19 90 71 100 71 78 53 69

Heavy rule indicates crossover point. fp = flavoprotein.

might be written as follows, for example, for cytochrome c:

cn N I + x -- cn + x - I (4)

X N I + Pi + ATP -A ADP + X + I (5)

E$ect of A&de upon Crossover Point-In Paper III (1) me have discussed how the crossover point in the steady state changes caused by the State 3

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B. CHANCE AND G. R. WILLIAMS 487

to 4 transition identifies a pair of respiratory enzymes involved in oxidative phosphorylation. In the uninhibited preparation, the crossover point lies between cytochromes a and c, as is indicated by the heavy rule in Table Q, the preparation consisting of guinea pig liver mitochondria with succinate as a substrate. In the presence of 92 PM azide, the State 3 to 4 transition gives a crossover point further down the chain, between cytochromes c and b. This effect is also indicated by the spectra of Fig. 1, B, as we have pointed out previously. With greater inhibition, it is possible to cause re- duction of flavoprotein upon addition of ADP, but still obtain oxidation of DPNH, giving a crossover point between these two components. At even greater inhibition, DPNH and the other components fail to show an oxida- tion upon addition of ADP. As yet, no crossover point between flavopro- tein and cytochrome b has been demonstrated.

DISCUSSION

Our finding that respiration in the active state of mitochondria (State 3) is appreciably inhibited by higher azide concentrations (cf. Table I) with- out any appreciable effect upon the resting or State 4 respiration may afford an explanation for the selective effects of azide upon activity and resting respiration, as for example in nerve fibers (12). This effect is in agreement with the hypothesis that phosphate acceptor is generated upon conduction of electrical impulses and accelerates respiration in a small number of mitochondria that are located near the axon surfaces or nodes (13). This activity respiration is azide-sensitive in the manner indicated by Table I, but the azide levels required for nerve are lower than those of Table I (0.1 to 0.2 mM for 90 to 100 per cent inhibition (12)).

The azide-inhibited respiratory chain is a relatively sensitive and specific indicator of ADP. For example, 10e6 M ADP gives an optical density change in terms of cytochrome a reduction that can be recorded with an accuracy of better than 10 per cent. Thus the sensitivity is of the order of 1OW M in a 1 cc. volume which corresponds to 1O-g mole ADP. Inter- estingly enough, this sensitivity is comparable to that of the firefly luciferase system for ATP (14) when a similar type of photoelectric circuit is used.

It is found that somewhat less ADP is required to give half maxima1 rate of electron transport, as measured in terms of oxygen consumption, than to give half maximal spectroscopic effects, as measured in the State 4 to 3 transition. It is not unreasonable that this is so, for maximal respira- tion may be achieved when only the rate-limiting component of the respira- tory chain has been saturated with ADP. Other components, present in excess, need not have reacted completely with ADP in order to give maxi- mal rate of electron transport. It has not yet been possible to demonstrate conclusively just what the differences in the titration values for the various

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488 RESPIRATORY ENZYMES. VI

cytochromes may be, although the data of Table II are suggestive. On the other hand, differences of the reaction kinetics of cytochromes a and c with ADP are clear-cut.

The fact that increasing concentrations of ADP do not cause linearly increasing rates of change of the steady state oxidation-reduction levels of any of the components of the respiratory chain gives direct evidence for intermediates between ADP and the electron transport system. The na- ture of these intermediates is not known, but several possibilities can be eliminated. For example, similar non-linearities are observed when un- coupling agents such as dibromophenol or dicoumarol is used. Since phos- phate is not required for the latter reactions, it follows that these un- coupling agents act in place of ADP + Pi in a reaction similar to that of Equation 5. Thus, in the reaction with an uncoupling agent or with ADP + Pi, the hypothetical X - I compound serves as an adequate ex- planation for the reaction kinetics (15). Other evidence for intermediates in the phosphorylation reaction comes from isotopic studies, but in this case it appears that these intermediates are in the reaction of ADP with phosphate compounds.*

SUMMARY

In the presence of appropriate low concentrations of azide as an inhibitor of the respiratory chain of liver mitochondria, the transition from the rest- ing State 4 to the active State 3 caused by addition of ADP shows cross- over points (1) between cytochromes b and c and between flavoprotein and DPNH. These crossover points identify pairs of respiratory enzymes involved in oxidative phosphorylation.

The ADP concentration required for half maximal spectroscopic effects appears to consist of two factors: one related to a Michaelis affinity for ADP and dependent upon the rate of electron transport and t.he other a Gtration of an endogenous high energy compound in the resting mitochon- dria. Since the latter titration value closely approximates the measured content of the intramitochondrial DPNH, it is suggested that] DPNH in the resting mitochondria is an inhibited form.

The kinetics of reaction of ADP with the respiratory chain, measured in terms of the speed of the State 4 to 3 transition upon adding ADP, are first order with respect t.o ADP concentration only for lower values of concen- tration. At higher concentrations, considerable deviations occur that lead to the conclusion that intermediate reaction steps intervene between ADP and the respiratory chain. In our experiments impermeability to ADP is largely ruled out by observing the same experimental results in hypotoni- tally treated mitochondria.

4 Dr. Mildred Cohn, personal communication,

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B. CHANCE AND G. R. WILLIAMS 489

BIBLIOGRAPHY

1. Chance, B., and Williams, G. R., J. Biol. Chem., 217,409 (1955). 2. Chance, B., and Williams, G. R., J. Biol. Chem., 217,395 (1955). 3. Chance, B., and Williams, G. It., J. Biol. Chem., 217,383 (1955). 4. Chance, B., and Williams, G. R., J. Biol. Chem., 217,429 (1955). 5. Loomis, W. F., and Lipmann, F., J. Biol. Chem., 179,503 (1949). 6. Slater, E. C., Biochem. J., 69, 392 (1955). 7. Chance, B., and Williams, G. R., J. Bid. Chem., 217, 439 (1955). 8. Lehninger, A. L., in McElroy, W. D., and Glass, B., Phosphorus metabolism,

Baltimore, 1, 351 (1951). 9. Slater, E. C., and Holton, F. A., Biochem. J., 66,530 (1953).

10. Chance, B., and Williams, G. R., Science, 121,621 (1955). 11. Holton, F. A., Biochem. J., 61, 46 (1955). 12. Brink, F., Jr., Bronk, D. W., Carlson, F. D., and Connelly, C. M., Cold Spring

Harbor Symposia Quant. Biol., 17, 60 (1952). 13. Gasser, H., Cold Spring Harbor Symposia Quad. Biol., 17,32 (1952). 14. Strehler, B. L., and Totter, J. R., Arch. Biochem. and Biophys., 40, 28 (1952). 15. Chance, B., and Williams, G. R., Resume des communications, 38 Congres In-

ternational de Biochimie, Brussels, 300 (1955).

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Britton Chance and G. R. WilliamsMITOCHONDRIA

DIPHOSPHATE ON AZIDE-TREATED THE EFFECTS OF ADENOSINE

OXIDATIVE PHOSPHORYLATION: VI. RESPIRATORY ENZYMES IN

1956, 221:477-490.J. Biol. Chem. 

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