OXIDATIVE PHOSPHORYLATION BY AN ENZYME COMPLEX FROM EXTRACTS OF MITOCHONDRIA

I. THE SPAN ,‘-HYDROXYBUTYRATE TO OXYGEN*

BY CECIL COOPERt AND ALBERT L. LEHNINGER

(From the Department of Physiological Chemistry, School of Medicine, The Johns Hopkins University, Baltimore, Maryland)

(Received for publication, August 1, 1955)

It has been rather generally observed that oxidative phosphorylation with high efficiency may be demonstrated with mitochondria isolated from animal tissues only if they possess relatively intact morphology (1, 2). Disruption of mitochondrial structure by procedures such as freezing and thawing, grinding, and treatment with various dispersing agents such as bile salts and alcohol-water mixtures, etc., has been found to lead to loss of the phosphorylating activity, although the enzymes involved in electron transport may survive such treatment. Thus the preparations of Keilin and Hartree (3) are highly active so far as succinoxidase and cyto- chrome oxidase activities are concerned but show no coupled phosphoryla- tion (4). Furthermore, little success has attended efforts to restore the phosphorylating activity by addition of cofactors. Largely because of the apparent dependence of oxidative phosphorylation on more or less intact mitochondrial structure, little direct information concerning the enzymatic mechanisms involved in the coupling process has become available.

Following a systematic study of various means of disrupting mito- chondrial structure, in which a very sensitive isotopic method for detecting phosphorylation even at extremely low P: 0 ratios was used, we have found it possible to demonstrate the occurrence of oxidative phosphorylation in lipoprotein-rich fractions of relatively low particle weight separated from digitonin extracts of mitochondria. The P: 0 ratios observed with such preparations approach in magnitude those observed with intact mitochon- dria. Although these preparations evidently retain considerable organiza- tion of both the electron transport and phosphorylating enzymes, they have permitted much more direct study of various aspects of oxidative phosphorylation than has been possible in the past with intact mitochon- dria. In this communication the preparation and properties of this multi-

* Supported in part by grants from the National Institutes of Health and the Nutrition Foundation, Inc.

t Postdoctoral Fellow of the National Institutes of Health.

489

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490 OXIDATIVE PHOSPHORYLATION. I

enzyme complex and its ability to catalyze phosphorylation coupled to the oxidation of fi-hydroxybutyrate by molecular oxygen will be described.’

Papers II and III deal with the ability of this enzyme complex to carry out phosphorylations coupled to known segments of the respiratory chain (6, 7). Communications concerning the ATPase2 activity, exchange reac- tions, and some aspects of the mechanisms of oxidation and phosphoryla- tion will follow.

Methods

Preparation of Phosphorylating Enzyme Xystem-Mitochondria were iso- lated from 15 per cent homogenates of ratI liver in 0.25 M sucrose by the method of Hogeboom et al. (8). Relatively loose homogenizers and short homogenization periods were employed. Only well fed male Wistar rats (Carworth Farms) averaging about 200 gm. in body weight were used. Ordinarily batches of six livers (about 60 gm.) were carried through the entire procedure. The mitochondria were washed twice with 0.25 M su-

crose and then suspended in 0.25 M sucrose. The remaining nuclei and unbroken cells were removed at 600 X g; the mitochondria were then re- sedimented at 9000 X g. This mitochondrial pellet, derived from six rat livers, was suspended in 7.5 ml. of cold 1 .O per cent solution of recrystallized digitonin (Fisher) in HzO. The suspension was kept at 0” for 20 minutes and then centrifuged at 75,000 X g for 25 minutes in the Spinco model L ultracentrifuge. The supernatant solution was decanted in such a manner as to remove also the very loosely packed gelatinous upper layer of the sedimented material. The remainder of the sediment, designated Sedi- ment S-l, was put aside for a second extraction (see below). The mixed supernatant material was then sedimented at 100,000 X g for 25 minutes. The pellet formed was suspended in 7.5 ml. of cold 0.25 per cent digitonin solution and recentrifuged at 100,000 X g for 25 minutes. The superna- tant solution was discarded and the pellet was resuspended in 5.0 ml. of cold distilled Hz0 to yield the preparation hereafter designated as Prepara- tion P-l. This suspension was used directly in the enzyme test systems; it usually contained from 350 to 750 y of total N per ml.

Sediment S-l, which remained after the first extraction with digitonin described above, was now subjected to a second extraction; from the second

1 A preliminary communication has been published (5). 2 Abbreviations, BOH, &hydroxybutyrate; ATP and ADP for adenosine tri- and

diphosphates, respectively, and, similarly, ITP, IDP, UTP, UDP, etc., for the cor- responding tri- and diphosphates of inosine, uridine, guanosine, cytidine, and thymi- dine; DPN+ and DPNH for oxidized and reduced diphosphopyridine nucleotide, respectively; EDTA, ethylenediaminetetraacetate; DNP, 2,4-dinitrophenol; P, in- organic phosphate; Dicumarol, 3,3’-methylenebis(4.hydroxycoumarin); FeJ1 cyt c, ferrocytochrome c; Fe111 cyt c, ferricytochrome c.

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C. COOPER AND A. L. LEHNINGER 491

extract another batch of active enzyme (designated Preparation P-2) of similar enzymatic properties may be obtained. For the extraction Sedi- ment S-l was suspended in 7.5 ml. of cold 1 per cent digitonin for 20 min- utes. The procedure as described above for the separation of Preparation P-l from the digitonin extract was now repeated exactly and the final pellet was suspended in 5.0 ml. of cold distilled water to yield Preparation P-2.

Several variables concerning the yield of active enzyme complex have been studied. In brief, it has been found that digitonin concentrations below 0.7 per cent do not extract activity. Two successive extractions with 1 per cent digitonin were found far more effective than single extrac- tions with more concentrated digitonin. Concentrations of digitonin higher than 2 per cent inhibited phosphorylating activity. Preparations isolated from livers of fasted rats yielded little or no activity by the pro- cedure described; differences in extraction and sedimentation of the enzyme complex were evident in this case.

Analytical-Oxidation of /3-hydroxybutyrate was followed either by measuring the stoichiometric accumulation of acetoacetate (9) or by meas- uring oxygen uptake amperometrically with the open type oxygen electrode of Davies and Brink (10). In the latter type of measurement a 1.0 ml. reaction cell was employed which contained a stationary open type plati- num micro electrode and a KC1 bridge leading to an Ag-AgCl reference cell, arranged in such a way as to avoid any difficulty through contamination of the reaction cell by Ag+. The electrode was initially calibrated by the method of Davies and Brink (10) ; in addition each experimental determina- tion of rate of oxygen uptake was also accompanied by calibrations consist- ing of current measurements at full saturation of the medium with air and also at zero concentration of oxygen, produced by addition of dithionite at the end of the enzyme test.3 The current corresponding to the relatively very low rate of diffusion of oxygen to the electrode was determined in the presence of the enzyme reaction medium with substrate omitted; substrate was then added and measurements made over 10 to 15 minute periods. The change in 02 concentration was continuously recorded by means of a Brown recording potentiometer. In a parallel cell addition of cyanide to the same medium to inhibit cytochrome oxidase permitted separate meas- urement of the base-line diffusion rate.

Phosphate uptake was measured by the isotopic method already de- scribed (11) ; the only modifications were the complete omission of the phosphate “carrier” from the trichloroacetic acid used to deproteinize the reaction medium and the omission of the acetone treatment in the depro-

3 Personal communication from Dr. Britton Chance.

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492 OXIDATIVE PHOSPHORYLATION. I

teinization. The P : 0 ratios given are estimated to have an average error no larger than 10 per cent.

CDP and UDP were obtained from the Pabst Laboratories; GDP and IDP from the Sigma Chemical Company.

EXPERIMENTAL

Phosphorylation Coupled to Oxidation of /3-Hydroxybutyrate-When the enzyme Preparation P-l was incubated with BOH, ADP, and inorganic phosphate, oxygen was taken up and acetoacetate accumulated according t)o the equation

(1) n(-)-p-Hydroxybutyrate + $02 + acetoacetate + Hz0

Coupled to this oxidation were the disappearance of inorganic phosphate and the net formation of equivalent amounts of ATP. Some typical data taken from a large number of experiments are presented in Table I; others are shown in the form of control values in other tables. It is seen that in the presence of all the components enumerated, both oxidation and phos- phorylation occurred. In the absence of substrate, neither oxidation nor phosphorylation occurred. Omission of either ADP or phosphate caused great decreases in the rate of oxidation of BOH, demonstrating dependence of oxidation on both phosphate and phosphate acceptor, as seen in intact mitochondria (12). It is also noted that amperometric measurement of oxygen uptake agreed closely with calorimetric measurement of acetoace- tate accumulation, in accord with Equation 1.

From 92 to 99 per cent of the orthophosphate disappearing during the coupled phosphorylation could be recovered as newly formed ATP, fol- lowing chromatography of the adenine nucleotides on Dowex 1 columns (13). In a typical experiment 2.83 X lo4 c.p.m. of P32 disappeared from the inorganic phosphate pool as determined by the extraction method, and 2.59 X lo4 c.p.m. were recovered in the eluates corresponding to ATP. No activity was detected in the ADP fraction.

Of a large number of measurements of the P : 0 ratio, some were as high as 2.8 (maximum expected, 3.0 (14)) ; h owever, most values observed were between 1.5 and 2.4. Some of the deficiency may be accounted for by the ATPase activity of the enzyme complex, which will be considered in another communication. The addition either of fluoride or of yeast hexokinase plus glucose did not increase the P: 0 ratios. Similarly, EDTA was found to be without effect on the P: 0 ratio, contrary to its action on intact mitochondria (15, 11).

The data in Table I also demonstrate that it is unnecessary to supple- ment the reaction medium with Mg++, although intact mitochondria are

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C. COOPER AND A. L. LEHNINGER 493

known to require Mg++ in the medium for maximal P: 0 ratios (11). In fact, the addition of Mg* causes uncoupling of phosphorylation.

Both oxidation and phosphorylation proceed in a linear manner with time (Fig. I), even for extended periods. Actually the enzyme prepara-

TABLE I

Requirements for Oxidation and Phosphorylation

The test system contained 0.01 M DL-BOH or 0.005 M D-BOH, 0.0024 M ADP, 0.010 M P (pH 6.0, labeled with P”2), and enzyme (100 to 150 y of N) in a total volume of 3.0 ml. Omissions or additions to standard test system as shown below. Incu- bated in air at 23” for 15 minutes; enzyme tipped into main compartment at end of 5 minute equilibration period.

Expe&yent

1

6

Omissions or additions

Complete system Minus BOH

“ ADP “

PS Complete system (DL-BOH)

L‘ “ (D-BOH) “ “

Minus BOH Complete system

Minus BOH Complete system

+0.005 M MgClz +0.01 “ “

Complete system “ CL

~+%Ol~

0.47 o.oot 0.17 0.24 0.41 0.40 0.21 0.00 0.22 0.00 0.48 0.28 0.24 0.54 0.538

-AP

p+dt?

0.76 0.00 0.00 0.09 0.80 0.77 0.36 0.00 0.58 0.00 0.75 0.34 0.26

P -* 0

1.62 0.00 0.00 0.38 1.95 1.93 1.71

2.64

1.56 1.21 1.08

* The expression P:O designates the ratio of moles of P esterified per pair of elec- tron equivalents transferred to acceptor; in this case P:O * AP/A acetoacetate.

t In experiments in the absence of added BOH no net oxygen uptake is detectable with the oxygen electrode.

$ In this experiment the orthophosphate concentration was 3 X 10e5 M, all of which was taken up.

$ Microatoms of oxygen uptake measured amperometrically.

tions are relatively stable at 0”. Little, if any, activity is lost for 8 to 10 hours; at 24 hours the P: 0 ratio is still 50 per cent of that of the fresh preparation. Freezing and thawing destroy phosphorylating activity. In intact mitochondria inactivation of phosphorylation is produced by pre- incubationin phosphate-containing solutions (16). However, phosphate ap- pears to have no specific inactivating effect on the isolated enzyme complex.

The isotopic measurements of phosphorylation described were not com-

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494 OXIDATIVE PHOSPHORYLATION. I

plicated by simultaneous occurrence of the P32-ATP exchange reaction de- scribed by Boyer et al. (17). This exchange reaction occurs only when the ratio of ATP to ADP is very high; its properties and significance will be discussed in a later communication.

Most of the experimental tests have been carried out under conditions in which relatively small absolute amounts of oxidation and phosphoryla- tion were measured. However, these conditions were chosen solely for reasons of economy because of the relatively lengthy preparation of the enzyme. Actually all the measurements were well within the accurate range of the methods used for measuring oxidation and phosphorylation.

1.4

t-i g 0.6

I

=. 0.6

0.2

IO 20 30

MINUTES

FIG. 1. Rate of oxidation and phosphorylation. Test system as described in Table I. Temperature, 22’.

Xpecifiity of Substrates and Phosphate Acceptors-The isolated enzyme preparations are not capable of oxidizing a wide spectrum of substrates, in direct contrast to intact mitochondria. The data in Table II show that P-hydroxybutyrate is consistently oxidized at a high rate, by both Prepa- rations P-l and P-2. Succinate is also oxidized by both preparations, but especially well by Preparation P-2. A number of other intermediates of the tricarboxylic acid cycle were found not to be attacked. Although the enzyme particles possess a high degree of functional organization with re- spect to the action of the respiratory chain and the coupled phosphoryla- tions, they obviously do not retain the organized Krebs cycle and fatty acid cycle activity so evident in intact mitochondria.

The specificity of the phosphate acceptor has been carefully studied in view of the recent findings in a number of laboratories of the existence and biological reactivity of the 5’-di- and triphosphates of nucleosides other than adenosine. The data collected in Table III show that the 5’-diphos- phates of inosine, uridine, cytidine, and guanosine are not capable of re-

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C. COOPER AND A. L. LEHNINGER 495

placing ADP in the test system and are thus probably inactive as phosphate acceptors. When UDP or CDP was added to systems already containing ADP as phosphate acceptor, barely significant increases in the P:O ratio were seen in some but not all experiments (see Table III) ; however, the

TABLE II

Substrate Specificity of Enzyme Complex The test system contained substrates at 0.005 M, 0.01 M P (pH 6.5), 0.0024 M ADP,

and enzyme Preparation P-l (50 to 75 y of total N) in a total volume of 1.0 ml. The rates of oxygen uptake were measured amperometrically at 23’; the rates were usually linear for 10 minutes.

Experiment No. Substrate - Preparation P-l Preparation P-2

DL-BOH 18.2 22.0 Succinate 6.2 29.2 Citrate 0.0 D-Malate 0.0 DL-BOH 12.6 L-Glutamate 2.0 DL-BOH 15.2 Succinate 17.2 a-Ketoglutarate 0.0 Pyruvate 0.0 Citrate 0.0 DL-BOH 10.6 Choline 0.0 Succinate 3.6 DL-BOH 21.4 a-Ketoglutarate* 0.0 L-Malate 4.0 DL-BOH 16.0 L-Malate 0.0 Fumarate 0.0

Oxygen uptake, mmicroatoms per min.

* The test system was supplemented with 0.0007 M coenzyme A, 0.0012 M reduced glutathione, and 0.0024 M GDP.

addition of IDP or GDP produced neither an increase nor a decrease in the P: 0 ratio.

Other experiments, to be described in a later report, clearly show that the failure of IDP, GDP, UDP, and CDP to act as P acceptors is not due to an extremely high rate of enzymatic hydrolysis of either the added diphos- phate or the corresponding triphosphate.

Uncoupling of Phosphor&don 0~ Inhibitors-In Table IV are presented the effects of addition of various substances known to inhibit oxidation and

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496 OXIDATIVE PHOSPHORYLATION. I

TABLE III

Specificity of Phosphate Acceptor

The test system contained 0.01 M DL-BOH, 0.0024 M nucleoside 5’-diphosphates as indicated, 0.01 M P buffer, pH 6.0 (1.15 X IO6 c.p.m. of Paz), and enzyme (130 y of N) in a total volume of 3.0 ml. Incubated for 15 minutes at 23”.

Experiment No Nucleoside S’-diphosphate c&ox&ate formec P uptake

None ADP IDP GDP ADP + IDP

“ + GDP None ADP UDP CDP ADP + UDP

‘I

“ + CDP

@de pmole

0.24 0.00 0.59 0.92 0.24 0.00 0.23 0.00 0.59 0.99 0.60 0.97 0.17 0.00 0.47 0.76 0.17 0.00 0.18 0.00 0.49 0.94 0.34 0.58 0.30 0.65

P 6

0.00 1.56 0.00 0.00 1.68 1.62 0.00 1.62 0.00 0.00 1.92 1.71 2.17

TABLE IV

Effect of Inhibitors on Oxidation and Phosphorylation

The test system contained 0.01 M DL-BOH, 0.0024 M ADP, 0.01 M P buffer (pH 6.0), and enzvme (140 to 200 y of N) in a total volume of 3.0 ml. Inhibitors added as

Exper;xnt

indicated. Incubated for 15 minutes at 23”.

Inhibitor

None DNP (5 X lO-6 M)

Pentachlorophenol (5 X 10e5 M) KCN (0.001 M) CaCls (0.0025 M) None Antimycin A (0.05 y)

“ ‘I (1.0 ‘1) None Gramicidin (20 y) None Dicumarol (1 X 1OW M)

“ (5 x 10-e “) “ (1 X 10-S ‘1)

Arsenate (0.03 M) None Methylene blue (5 X lo+ M)

A aceto- acetate AP

p?FWle /mmle

0.22 0.58 0.22 0.00 0.22 0.00 0.00 0.00 0.22 0.55 0.39 0.68 0.09 0.10 0.00 0.00 0.41 0.72 0.53 0.00 0.32 0.46 0.31 0.19 0.30 0.00 0.30 0.00 0.29 0.19 0.34 0.58 0.30 0.00

P 0

2.64 0.00 0.00 0.00 2.50 1.74 1.11 0.00 1.76 0.00 1.44 0.61 0.00 0.00 0.66 1.71 0.00

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C. COOPER AND A. L. LEHNINGER 497

phosphorylation. As already mentioned, DNP is extremely active in un- coupling phosphorylation, as is pentachlorophenol (18). Cyanide, as ex- pected, blocked both oxidation and phosphorylation, presumably by com- bination with cytochrome ~3, which the enzyme preparation is known to contain (see below). Similarly, antimycin A was found to block electron transport completely, and with it also the coupled phosphorylation, in agreement with findings in intact mitochondria that it interferes with re- duction of cytochrome c (19). Gramicidin, Dicumarol (20), methylene blue, and arsenate also uncoupled phosphorylation. Dicumarol was ex- tremely effective; at 1 X 1W M about 50 per cent uncoupling occurred.

TABLE V

Effect of pH on Oxidation and Phosphorylation

The test system contained 0.01 M P (2.40 X lo6 c.p.m. of P”“), 0.01 M DL-BOH, 0.0024 M ADP, and enzyme (110 y of N) in a total volume of 3.0 ml. Reaction media adjusted to pH values shown; little change occurred during reaction period of 15 minutes at 23”.

I A acetoacetate I

P b

4.0 5.0 6.0 7.0 8.0 9.0

&mde pm&

0.02 0.00

0.08 0.02 0.26 0.44 0.28 0.44 0.24 0.14 0.17 0.08

0.00 0.25 1.69 1.57 0.58 0.47

The significance of the uncoupling effect of Dicumarol is discussed in more detail in Paper III (7).

It has already been pointed out that addition of Mg* causes uncoupling in the isolated enzyme complex, in contrast to the actual requirement for added Mg++ shown by intact mitochondria. In Table IV it is seen that Ca++, a very potent uncoupling agent in intact mitochondria (15, 21), has virtually no effect on phosphorylation in the isolated enzyme prepara- tions. Indeed, concentrations as high as 0.01 M have not depressed the P : 0 ratio. The effects of Mg++ and Ca* in the isolated enzyme prepara- tion are thus quite different from those observed in intact mitochondria.

Other Significant Variables-Studies have been carried out on the effect of varying the reaction conditions. Perhaps the most significant findings relate to the effects of pH and orthophosphate concentrations. In Table V is shown the effect of varying pH. It is seen that the rate of oxidation of P-hydroxybutyrate has a rather broad maximum between pH 6.0 and 8.0, most likely a resultant of the optimal pH values for the various mem-

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498 UXIDATIVE PHOSPHORYLATION. I

bers of the respiratory chain. However, the ancillary phosphorylating enzymes appear to have a rather sharp maximum at pH 6.0 to 7.0; although phosphorylation still occurs at pH 5.0 and 9.0, the P:O ratios are quite low.

The P: 0 ratio is also heavily dependent on the concentration of ortho- phosphate in the medium. Although phosphorylation occurs even at very low concentrations of orthophosphate (i.e. 2 X lo+ M), the P:O ratio be- comes maximal at about 0.01 M phosphate and upward; half maximal val- ues are seen at 0.0001 M phosphate (Table VI). For this reason phosphate concentration was generally held at high levels (0.01 M) in the various ex- periments, necessitating the use of a means of measuring uptake of phos-

TABLE VI

Effect of Phosphate Concentration

The test system contained 0.01 M DL-BOH, 0.0024 M ADP, orthophosphnte (pH 6.0) in the concentrations indicated, labeled with 2.07 X 10” c.p.m. of P32, and en- zyme (110 y of N) in a total volume of 3.0 ml. Incubated for 15 minutes at 23”.

Phosphate concentration A acetoacetate AP

M pmole pmole

0.0001 0.22 0.19 0.0005 0.25 0.25 0.001 0.32 0.37 0.005 0.37 0.61 0.01 0.48 0.75 0.025 0.48 0.76 0.05 0.44 0.74

P 0

0.86 1.00 1.16 1.65 1.56 1.58 1.68

phate independent of its concentration, such as the isotopic method used. When lower phosphate concentrations were employed, calorimetric meas- urement of phosphate disappearance was perfectly feasible; such measure- ments agreed exactly with those carried out with the isotope procedure. The rate of oxidation of BOH also increased with increase of concentration of phosphate.

Study of the effect of varying the ADP concentration revealed that rather low concentrations, probably less than 5 X 1O-4 M, sufficed to satu- rate the system. However, in order to maintain high capacity, concen- trations of 0.0024 M were generally used.

LIPNH As Substrate-Since the D( - )+hydroxybutyrate dehydrogenase of these isolated mitochondrial enzyme systems is DPN-linked, it appeared possible to employ chemically reduced DPNH as substrate instead of BOH, as had been done in earlier experiments with intact mitochondria (22). Added DPNH was found to be rapidly oxidized at the expense of

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C. COOPER AND A. L. LEHNINGER 499

oxygen by the isolated Preparation P-l enzyme. However, measurement of the phosphate uptake revealed that the P: 0 ratios were always con- siderably lower than those in experiments with BOH as substrate (see Table VII). Another striking difference observed with DPNH as sub- strate was the fact that its oxidation was only partially sensitive to anti- mycin A at concentrations which completely prevented oxidation of BOH in an otherwise identical experimental system (Table IV). Since the al- ready low P:O ratio with DPNH (about 0.50) was not significantly de- creased in the presence of antimycin A, it appears that all the observed phosphorylation is coupled to passage of electrons from ferrocytochrome

TABLE VII

Added DPNH As Substrate

The test system contained 0.01 M DL-BOH as indicated, 0.01 M P (pH 6.5, labeled with P”“), 0.0024 M ADP, and enzyme Preparation P-l (50 to 60 y of N) in a total volume of 1.0 ml. When DPNH was used as substrate, a total of 230 mpmoles was added in six equal amounts over the total time interval, which was 8.4 minutes. Oxygen uptake was measured with the oxygen electrode. In Experiment 2 the test system also contained 1 X 10e4 M cytochrome c.

T

Expehvznt Substrate

-

DPNH “ “

“

BOH DPNH

Antimycin A -AOz /

-AP I

P 6

Y mmicroaloms mpmoles

0.0 89.4 36.9 1.0 48.6 16.3 0.0 153 77.0 1.0 125 57.0 0.0 203 347 0.0 218 52.0

0.41 0.34 0.50 0.46 1.71 0.24

c to oxygen (this step is not sensitive to antimycin A (7)) and none to elec- tron transport between DPNH and cytochrome c. From these findings and certain others in Papers II and III (6, 7) it appears probable that when added DPNH is used as substrate a large fraction of its oxidation by cytochrome c may occur by the non-phosphorylating, antimycin-in- sensitive, DPN-cytochrome reductase activity (23), which is known to be present in enzyme Preparations P-l and P-2. Tentatively, it appears that the DPNH generated in situ from bound DPN by action of the /Lhydroxy- butyrate dehydrogenase reduces cytochrome c preferentially and wholly via a phosphorylating pathway involving the antimycin-insensitive com- ponent. Further properties of added DPNH as reductant of cytochrome c are detailed in Paper II (6).

Di$erence Spectrum of Respiratory Carriers-In order to determine the nature of the respiratory carriers participating in the phosphorylating elec-

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500 OXIDATIVE PHOSPHORYLATION. I

tron transport, a difference spectrum was plotted. A system composed of enzyme, ADP, and phosphate, and exposed to oxygen (to keep the carriers in the oxidized state), was used as a “base-line” for the measurements. Against this “base-line” the spectrum of an enzymatically reduced system was determined. This system contained BOH, ADP, phosphate, and cyanide to prevent reoxidation of the carriers below cytochrome ~3. The difference spectrum measured with a Beckman DU spectrophotometer equipped with a photomultiplier is seen in Fig. 2. It may be noted that typical peaks and depressions were observed (24) corresponding to the re-

0.020

l-i \H 0.010

3 0

0 0.010

0.020

400 500 600

h (m)r)

FIG. 2. Difference spectrum of enzyme complex. The optical density at different wave-lengths of a cuvette containing 0.01 M BOH, 0.005 M ADP, 0.01 M P (pH 6.5), 0.005 M KCN, and Preparation P-2 enzyme complex (250 y of total N) in a total vol- ume of 1.0 ml. (representing the reduced enzyme complex) was read against a “blank” cuvette containing the same medium without BOH and cyanide (representing the oxidized complex). The roman numerals indicate the peaks or depressions cor- responding to the reduced forms of the carriers as follows: I, y-peak of cytochrome c; II, r-peak of cytochrome a and the cyanide complex of cytochrome a~; III, reduced flavoprotein; IV, p-peak of reduced cytochromes b and c; V, a-peak of reduced cyto- chrome c; VI, a-peak of reduced cytochrome a and the cyanide complex of cyto- chrome ~3.

duced forms of flavoprotein, cytochrome c, cytochrome a, and to the cya- nide complex of cytochrome as. The difference spectrum in Fig. 2 does not show the unequivocal presence of a peak corresponding to reduced cytochrome b (24). However, such a peak has been clearly identified in difference spectra measured in the presence of antimycin A. There were no absorption peaks or depressions in the visible range not accounted for by known respiratory carriers. Non-enzymatic reduction with dithionite yielded essentially identical difference spectra.

Similar studies indicate that an absorption peak corresponding to re- duced DPN is also formed during reduction of the carriers by BOH. Since the position of this peak is anomalous, it is being studied in greater detail.

Recovery and Absolute Activity of Enzyme Complex-In a typical prepara- tion of the enzyme complex the washed mitochondria from 60.0 gm. of

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C. COOPER AND A. L. LEHNINGER 501

fresh rat liver, containing 120 mg. of total N, yielded a total of 3.1 mg. of total N as Preparation P-l and 2.7 mg. of total N as Preparation P-2. The total recovery of N from the starting mitochondria is thus only 4.8 per cent.

The recovery of “fi-hydroxybutyrate oxidase” activity (assayed mano- metrically with DL-BOH as substrate in the presence of P and ADP, but without addition of either DPN or cytochrome c) in the combined Prepa- rations P-l and P-2 is approximately 20 per cent of that present in the mitochondria. The estimation of activity of this multienzyme sequence is, of course, only a measure of the rate-limiting step, which has not been identified with certainty but is believed to be cytochrome b. The re- covery of phosphorylation activity has been approximated by comparing the phosphate uptake coupled to the oxidation of ascorbate in Preparations P-l and P-2 (Paper III) with the phosphate uptake in intact mitochondria under optimal conditions. Such measurements indicate that as much as 25 per cent of the phosphorylating activity of intact mitochondria may be recovered in Preparations P-l and P-2. However, since the mechanism of the coupling process and the fraction of the total phosphorylating potential being utilized are unknown, such assays are, of course, subject to consider- able uncertainty.

From the foregoing it is evident that Preparations P-l and P-2 are con- siderably more active than intact mitochondria on a weight basis. The QO, (microliters of oxygen uptake per mg. dry weight per hour at 37”) for the oxidation of BOH by intact mitochondria is about 10 to 15; for Preparations P-l and P-2 &o, is about 50 to 70.

Other Properties of Enzyme Complex-The isolated multienzyme Prepa- rations P-l and P-2 contained considerable digitonin, about 40 per cent, as revealed by measurement of the nitrogen content of dried samples, assum- ing that the protein moiety contained 16 per cent N. On the basis of digitonin-free dry weight, the total lipide content was found to be about 23 per cent, almost all of which was phospholipide (calculated as lecithin). The distribution of phosphorus in pooled Preparations P-l and P-2 was determined (Table VIII). It is seen that some 84 per cent of the total phosphorus is extractable with alcohol-ether mixtures and is probably phospholipide. A significant amount of nucleic acid phosphorus was also found. Spectrophotometric examination of this fraction revealed that the phosphorus was satisfactorily accounted for by approximately equivalent amounts of materials absorbing at 260 to 270 rnp, assuming a “normal” distribution of purine and pyrimidine nucleotides. The preparations con- tained considerable phosphorus with properties corresponding to those of the phosphorus of phosphoprotein; existence of such a fraction in mito- chondria is already known (25).

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502 OXIDATIVE PHOSPHORYLATION. I

The enzyme preparations were subjected to ultracentrifugal analysis. When suspended in distilled Hz0 or 0.15 M KCI, Preparation P-l showed an average sedimentation constant of szo = 220 X 1013, corresponding to a particle weight of about 50,000,OOO if the usual assumptions of spherical shape, specific gravity, etc., are made. The preparations were polydis- perse, as judged by spreading of the boundary with time. Particles of this magnitude represent about l/3000 of the particle weight of an intact rat liver mitochondrion. However, because of the high content of digitonin of the preparations and other factors, these values can only be regarded as gross approximations.

Although the preceding calculations were predicted on spherical shape, preliminary measurements of light scattering indicate asymmetry.4 Elec-

TABLE VIII

Phosphorus Content of Enzyme Preparation

Determined on pooled Preparations P-l and P-2.

Fraction y phosphorus er mg. Fraction of total total i-f phosphorus

Acid-soluble................................ 1.0 “ inorganic phosphate. 0.67

Phospholipide . . . 82.4 Nucleic acid................................ 12.5 “Phosphoprotein”. . 2.2

fier cent

1.0 0.6

84.0 12.8

2.2

tron micrographs of shadowed preparations indicate the presence of small, uniform particles arranged in various degrees of agglutination.

DISCUSSION

The enzyme preparations described in this paper have provided great advantages over intact mitochondria in studies of the mechanisms of elec- tron transport and oxidative phosphorylation being carried out in this laboratory. Although the preparations are still highly organized in both the functional and the structural sense, they are not merely fragmented mitochondria which retain a full complement of mitochondrial enzymes. The digitonin treatment has evidently preserved the electron transport chain and associated phosphorylation mechanisms but has caused com- plete loss of the organized Krebs cycle and fatty acid cycle activity.

It is striking that only digitonin treatment yielded extracts retaining some phosphorylating activity. Extracts of mitochondria obtained by vibration, exposure to butanol-water mixtures (26), drying with acetone,

4 Unpublished observations, Dr. J. I,. Gamble, Jr.

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C. COOPER AND A. L. LEHNINGER 503

exposure to hypotonic media, grinding, and treatment with cholate or deoxycholate (27) were found to be totally inactive. In this survey the criterion of “solubility” was retention of activity in the supernatant layer after centrifugation at 25,000 X g for 25 minutes. Digitonin has already been employed with considerable success in dissociating lipoprotein struc- tures, as in the case of retinal enzymes (28) and cell nuclei (29), and is be- lieved to exert its dispersing effect through its ability to react with certain sterols to form digitonides. It appears significant that mitochondria con- tain considerable lipide (30), some of which is apparently located in the membrane or cristae (31). Considerable lipide is present in particulate succinoxidase preparations (32); it has been found that enzymatic activity is to some extent dependent on the integrity of lipide components in such preparations (33).

The properties of the isolated enzyme system suggest very strongly that oxidative phosphorylation, as it occurs in intact mitochondria, is subject to some measure of control exerted by the properties of the highly differen- tiated ultrastructure of the mitochondrion, including the semipermeable membrane. For instance, the P: 0 ratio in intact mitochondria is maximal at certain levels of osmolarity of the reaction medium (34, 35). How- ever, such dependence on osmolarity was not observed with Preparation P-l; in fact the P:O ratio was depressed on addition of even low con- centrations of NaCl, KCl, or sucrose. Furthermore, intact mitochondria require addition of Mg++ for maximal P:O ratios; the isolated enzyme re- quires no addition of Mg+. Ca * is a very potent uncoupling agent in intact mitochondria (15, 21). However, Catt- does not uncouple phosphor- ylation even in quite high concentrations in the isolated enzyme. These findings indicate that the uncoupling action of Ca* in intact mitochon- dria is an indirect one, mediated through some aspect of the structural organization of the mitochondrion; in support of this view is the finding that Ca+ causes swelling of mitochondria under certain conditions.5 Also pertinent is the finding that the isolated enzyme system is not adversely affected by incubation in orthophosphate, a treatment which inactivates phosphorylation in intact mitochondria (16). The restorative action of Mn* (21, 36) on mitochondria inactivated by Ca* or orthophosphate may also be a matter of an effect on mitochondrial structure rather than on the phosphorylating enzymes per se. These findings suggest that other uncoupling factors may also act indirectly via effects on structural features; the action of certain hormones, particularly thyroid hormones, is now being examined.

Although a number of multienzyme preparations apparently derived from mitochondria have been reported in the past to oxidize succinate or

6 Unpublished observations, Dr. D. I?. Tapley.

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504 OXIDATIVE PHOSPHORYLATION. I

DPNH, little or no coupled phosphorylation has been observed with such preparations. Green et al. reported slight phosphorylation accompanying oxidation of succinate by a particulate system in 1951 (37); however, no further work on these preparations has been reported. The “DPNH oxi- dase” preparations more recently described by Huennekens et al. (38) and by Green et al. (39) have not been reported to have any properties suggest- ing existence of coupled phosphorylation. It is probable that these prepa- rations represent complexes of the electron transport factors from which the normally associated phosphorylating enzymes have been lost through the conditions of the isolation procedure. In a brief note Martius (40) has described a “vitamin K1 reductase” system which requires both phos- phate and ADP for activity; it is suggested that this is reoxidized by cyto- chrome b. Raw (41) has recently reported occurrence of phosphate trans- fers in a complex system containing, in addition to a number of other enzyme preparations, a digitonin extract of mitochondria. The dependence of the reaction described by Raw on inosine triphosphate indicates that the reaction may not be the same as that reported in this paper, in view of our finding that ADP is the specific phosphate acceptor.

It is still not clear whether the DNP-insensitive phosphorylative changes observed by Pinchot (42) in Alcaligenes faecalis extracts associated with oxidation of ethanol represent phosphorylation linked to the respiratory chain. Recently, however, Brodie and Gray (43) have observed DNP- sensitive phosphorylation in extracts of another microorganism, Mycobac- terium phlei.

The authors are greatly indebted to Nancy Buseman for skillful and efficient technical assistance, to Thomas M. Devlin for measuring the dif- ference spectra, to Albert Mildvan for the measurements with the oxygen electrode, to Herbert J. Rapp for assistance in determination of sedimen- tation rates, and to Dr. Gerhardt Hutz for electron micrographs.

SUMMARY

From digitonin extracts of rat liver mitochondria a multienzyme com- plex of relatively low particle weight has been separated. Such prepara- tions catalyze the oxidation of D( -)-/3-hydroxybutyrate to acetoacetate via the cytochrome system. The oxidation is accompanied by coupled phosphorylation of adenosine diphosphate to yield adenosine triphosphate. P:O ratios as high as 2.8 have been observed; most values lay between 1.5 and 2.4. The 5’-diphosphates of inosine, uridine, cytidine, thymidine, and guanosine are essentially inactive as phosphate acceptors. The phos- phorylation is uncoupled by 2,4-dinitrophenol, gramicidin, methylene blue, Dicumarol, arsenate, and pentachlorophenol, but is not affected by Ca++.

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C. COOPER AND A. L. LEHNINGER 505

The preparations oxidize only P-hydroxybutyrate and succinate at signifi- cant rates, among a number of metabolic intermediates examined. When chemically reduced diphosphopyridine nucleotide was added as substrate, oxidation ensued, but the P:O ratios were very low compared to those ob- served with P-hydroxybutyrate as substrate. The enzyme complex con- tains flavin nucleotides and cytochromes c, a, aa; it is also rich in phospho- lipide. These multienzyme preparations provide a much more direct approach to the study of the enzymatic mechanisms of oxidative phosphor- ylation than is possible with intact mitochondria.

BIBLIOGRAPHY

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Cecil Cooper and Albert L. LehningerOXYGEN

-HYDROXYBUTYRATE TOβTHE SPAN EXTRACTS OF MITOCHONDRIA: I.

AN ENZYME COMPLEX FROM OXIDATIVE PHOSPHORYLATION BY

1956, 219:489-506.J. Biol. Chem. 

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