THE DISTRIBUTION OF CYTOCHROME OXIDASE AND SUCCINOXIDASE IN THE CYTOPLASM OF THE

MAMMALIAN LIVER CELL

BY GEORGE H. HOGEBOOM, ALBERT CLAUDE, AND ROLLIN D. HOTCHKISS

(From the Laboratories of The Rockefeller Institute for Medical Research, New York)

(Received for publication, July 22, 1946)

The relation between enzymatic functions and cell structure remains one of the most interesting and least explored problems confronting the cytolo- gist and biochemist. Investigations by various methods have yielded a certain amount of information on the subject, but the validity of the re- sults, especially those secured by microscopical stain reactions, has been questioned. The present day knowledge regarding the distribution of enzymes in the cell is mostly derived from histochemical studies such as those of Gierke, Graeff, and others (l-4), from investigation by the in- genious microtechniques of Linderstdm-Lang and Holter (5), and from the more recent studies on the analysis of isolated parts of cells (6-11).

The experiments described in the present report offer additional data on the distribution of two enzyme systems, cytochrome oxidase and succinoxi- dase, in the cytoplasm of the liver cell of the rat. In these experiments, use was made of the observation of Claude (12,13) that an extract suitable for fractionation by differential centrifugation can be prepared from animal tissues in such a way that it consists almost entirely of material from the cytoplasm, the nuclei being removed practically uninjured, together with residual intact cells, by preliminary centrifugation. This “cytoplasmic extract” can be fractionated upon further centrifugation into the following three distinct portions: (1) large granules, 0.5 to 2 p in diameter, separated by 25 minutes centrifugation at 2000 X g, and representing mitochondria for the most part; (2) small particles or microsomes, 60 to 150 mpin diameter, separated by 90 minutes centrifugation at 18,000 X g; and (3) the super- natant fluid remaining after the two sedimentable fractions have been removed.

The three fractions just described were examined for their cytochrome oxidase and succinoxidase content. In the present experiments, as in additional ones of the same type that involve other enzymes and will be reported in subsequent papers, attention was paid not only to the specific activity of the respective preparations in terms of their Q values but also to another quantitative aspect of the results by determining the total amount of activity recovered with each fraction, and expressing it in per- centage of the total activity possessed by the original extract.

615

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616 ENZYME SYSTEMS OF LIVER CELL

Experiments a,re also under way in which a comparison is being made of the activity of the various cell fractions derived from both normal liver and liver of animals treated with the carcinogen, p-dimethylaminoazobenzene, in order to follow, if possible, the changes brought about by the chemical in the course of the malignant transformation of the hepatic cells. The pre- liminary results obtained in the study are included in this report.

EXPERIMENTAL

Materials and Methods

Animals and Diet-The animals used in most of the experiments were young adult albino rats of the Wistar strain, fed a diet of bread and milk, and weighing 125 to 150 gm. In four experiments, the rats were of the Sherman strain and were fed either a basic diet (control group) consisting of unpolished rice (Texas brand), washed casein (Merck), olive oil, and carrot (14), or the same basic diet, to which p-dimethylaminoazobenzene was added to a final concentration of 0.06 per cent (test group).

Preparation and Fractionation of Liver Extract-The procedure by which the liver extract was prepared and subsequently fractionated in the cen- trifuge has been described in detail in preceding papers (12, 13). In brief, the method was as follows:

From ten to fifteen rats were used in a single experiment. Each animal was killed by a blow on the head and allowed to bleed profusely through a neck incision; its liver was removed without delay and placed on ice. The pooled livers were forced in small fragments through a masher fitted with a 1 mm. mesh screen that retained the connective tissue framework and al- lowed the parenchymatous part of the organs to be collected as a pulp. The liver pulp was ground alone in a mortar for about 3 minutes; the sol- vent, 0.85 per cent NaCl solution, was added very slowly at first, then more rapidly until a final volume equivalent to 5 times the weight of the pulp had been used. The resulting suspension was submitted to three successive centrifugations of 3 minutes at 1500 X g, and the sediments, composed of tissue debris, unbroken cells, free nuclei, and red corpuscles, were discarded. The supernatant solution from the last centrifugation will be referred to as the liver extract.

The liver extract, which contained for the most part elements derived from the cytoplasm of the hepatic cells (12, 13), was subjected to fractiona- tion by means of differential centrifugation, its constituents being segre- gated into the three main portions already mentioned, according to the scheme illustrated diagrammatically in Fig. 1. In this fractionation a single centrifuge type was used, namely the SB, size 1 model, manufactured by the International Equipment Company, Boston. The preliminary cen- trifugation at 1500 X g was accomplished by means of the horizontal yoke

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HOGEBOOM, CLAUDE, AND HOTCHKISS 617

No. 242 with Pyrex cups of 250 ml. capacity; separation and washing of the large granules were carried out under a force of 2000 X g in the conical head No. 823, with narrow neck Pyrex tubes of 50 ml. capacity; the microsomes were separated by means of the multispeed attachment and No. 295 head.

The initial separation, or concentration, of the large granules was accom- plished by submitting the liver extract to 25 minutes centrifugation at

LiverE;xtpact

I 5eparation of mic0osomes

90 minutes pt 18,000 x g

M.,c. s2

FIG. 1. Fractionation of liver extract by differential centrifugation: diagram of procedure. The fractions especially investigated were L.G.c., L.G.w., Sz, and M.w. (see the text).

2000 X g. The supernatant (S) was retained for further fractionation. The main portion of the sediment was resuspended in a known volume of 0.85 per cent NaCl solution and will be referred to as the concentrated large granule preparation (L.G.c.). There remained a small bottom layer in the pellet that contained tissue dhbris. This fraction (Sdl) was reserved separately.

In order to effect purification and to free the preparation from the soluble substances of the original liver extract, the large granules were washed by

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618 ENZYME SYSTEMS OF LIVER CELL

submitting them to three successive cycles of centrifugation at 2000 X g for 25 minutes with intervening resuspensions. The opaque, yellowish sediment from the last centrifugation was resuspended in saline and will be referred to as the washed large granule preparation (L.G.w.). At each suc- cessive centrifugation, a diminishing fraction at the bottom of the pellet, which consisted of a few residual red cells, nuclei, and agglutinated large granules, was removed and added to fraction Sdl.

The supernatant (S), set aside when the mass of the large granules was re- moved, still contained large granules in small amount. In order to insure greater homogeneity for the microsome fraction the remaining large gran- ules were discarded, together with a portion of the microsome substance itself, by a centrifugation of 5 minutes at 18,000 X g. The supernatant & was saved, while the resulting mixed sediment (Sdz) was resuspended in saline and used for determination of solids and activity tests.

Separation of the microsome material was then brought about by sub- mitting S1 to centrifugation at 18,000 X g for 90 minutes. The supernatant Sz was saved and constituted the third liver fraction. The sediment from this high speed centrifugation appeared as a pellet of jelly-like material, completely transparent and dark amber in color. The entire deposit was resuspended in saline to give an opalescent suspension which will be re- ferred to as the concentrated microsome fraction (M.c.).

Washing of the microsome material was accomplished by submitting it to two cycles of centrifugation at 18,000 X g for 90 minutes and resuspen- sion in saline. The deposit from the last centrifugation will be referred to as the washed microsome preparation (M.w.).

Determination of Cgtochrome Oxidase Activity-The determination of cytochrome oxidase was made aerobically at 38” according to the method of Schneider and Potter (15). The Warburg manometric vessel contained 0.35 ml. of enzyme preparation, 0.1 ml. of 0.5 M phosphate buffer, pH 7.4, 1.0 ml. of 1.3 X UF4 M cytochrome c in 0.85 per cent NaCl solution, and 0.15 ml. of 0.005 M AlCb. The side arm contained 0.15 ml. of 0.114 M

sodium ascorbate, the center well 0.2 ml. of 5 N KOH. A control vessel, containing all the reagents except the enzyme preparation, was run in parallel. The ascorbate solution was tipped into the vessel after equilibra- tion for 15 minutes. 2 to 3 minutes were then allowed for reestablishment of temperature equilibrium, and the manometer readings were made there- after every 5 or* 10 minutes. The average volume of oxygen absorbed during the first 20 minute period was used in calculating the &02 values for each preparation (microliters of oxygen taken up per hour per mg. of dry weight). In addition, the total activity of each fraction was calculated from the aliquot used in the flask and expressed in per cent of the total activity exhibited by the unfractionated extract (recovery values).

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HOGEBOOM, CLAUDE, AND HOTCHKISS 619

It was noted, as pointed out elsewhere (16), that the autoxidation of ascorbic acid varied somewhat from one experiment to the next and was usually depressed in the presence of active cytochrome oxidase prepara- tions. For this reason, it was necessary to make the determination at three levels of enzyme activity for each fraction and to calculate the ascorbic acid a&oxidation by extrapolation to zero enzyme concentration. This procedure was accepted as valid, since it yielded a linear relation between oxygen uptake and amount of enzyme preparation. Use was made of a 20 minute period for calculation of Qo, and recovery values, since the oxy- gen uptake was regularly constant during that interval but tended, in some instances, to decrease slowly at later periods. The endogenous oxygen uptake, without substrate, of all preparations at the dilutions used for the cytochrome oxidase determination was found in preliminary experiments to be negligible. A second control flask to correct for endogenous oxygen uptake was therefore not used.

Determination of Succinoxidase Activity-The determination of aerobic succinoxidase activity was carried out manometrically at 38” (15). The vessel contained 0.80 ml. of enzyme preparation; 0.1 ml. of 0.5 M phosphate buffer, pH 7.4; 0.4 ml. of cytochrome c solution (1.3 X 10V4 M); 0.15 ml. of 0.005 M CaC12; and 0.15 ml. of 0.005 M AlCL. The side arm contained 0.15 ml. of 0.5 M sodium succinate, and 0.2 ml. of 5 N KOH was placed in the center well. A control without substrate for determination of endogenous oxygen uptake was necessary only in the case of the extract, the endogenous uptake of this preparation being approximately 10 per cent of that occurring in the presence of succinate. The assay of each preparation was made at two or more levels of enzyme activity, and the procedure followed in equili- brating the vessels, reading the manometers, and calculating the Qo2 and per cent recovery of activity from the extract was similar to that described for the cytochrome oxidase determination. The oxygen uptake observed with succinate as substrate was linearly proportional to the amount of enzyme preparation at the dilutions used in the present determinations.

Determination of Anaerobic Succinic Dehydrogenase Activity-The demonstration that ferricyanide is reduced in the presence of tissue slices and succinate with the formation of acid and that the rate of acid formation can be measured anaerobically in bicarbonate medium (17) has provided a convenient method for the determination of succinic dehydrogenase. The results of preliminary experiments indicated not only that the ferricyanide method was applicable to cell-free tissue suspensions as well as to tissue slices, but also that the rate of CO2 production approached fairly closely the theoretical value predicted from the rate of oxygen uptake in aerobic determinations (i.e., 4 microliters of CO2 evolved anaerobically per 1 micro- liter of 02 absorbed aerobically).

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620 ENZYME SYSTEMS OF LIVER CELL

The determination of succinic dehydrogenase by this method was made as follows: 1.5 ml. of 0.05 M NaHC03, 0.2 ml. of 0.5 M sodium succinate, 0.8 ml. of 0.85 per cent NaCl, and 0.5 ml. of the enzyme preparation were mixed in a Warburg vessel, and 0.4 ml. of an 8 per cent solution of NasFe(CN)a in 0.025 M NaHC03 was placed in the side arm. The vessel was quickly gassed with a 5 per cent Cot-95 per cent Nz mixture and equilibratea at 38” for 15 minutes. The contents of the side arm were then added to the vessel, 2 minutes were allowed for reestablishment of temperature equilibrium, and readings were taken thereafter at 5 minute intervals. A control vessel without succinate was run in parallel. Qo$j, values were calculated from the average CO2 output observed during the initial 10 minute period. The evolution of CO:! was constant over this period but at later intervals declined at a fairly rapid rate (approximately 10 per cent per 5 minutes).

Results

Distribution of Cytochrome Oxidase and Xuccinoxidase in Cytoplasmic Extract of Normal Rat Liver-In three consecutive experiments the distri- bution of cytochrome oxidase and succinoxidase among the three main fractions of normal rat liver was determined. The results are shown in Table I. It can be seen that the removal from the extract of all particulate components sedimentable at 18,000 X g for 90 minutes (E --+ S,) resulted in complete removal of demonstrable cytochrome oxidase and succinoxidase activity. An average of 70 per cent of the former and 74 per cent of the latter enzyme system was recovered in the unwashed mitochondrial fraction (L.G.c.). Only a small proportion of the activity (less than 4 per cent of the cytochrome oxidase and approximately 7 per cent of the succinoxidase) was found in the microsome fraction (M.w.).

Several interesting points are brought to light when the Qo, and recovery values for the unwashed mitochondrial fraction (L.G.c.) are compared with corresponding figures observed after the granules had been washed three times with saline (L.G.w.). The cytochrome oxidase Q value rose sharply on washing and almost all of the cytochrome oxidase originally present in the unwashed granules remained associated with these elements. The succinoxidase Q value, however, rose only slightly upon washing, and the per cent recovery values indicated that an appreciable proportion of the total succinoxidase activity had been lost. It was evident, therefore, that the washing procedure resulted in definite purification of cytochrome oxi- dase with very little loss of enzyme activity, whereas purification of succin- oxidase was only slight and accompanied by a considerable loss of the total enzyme activity.

Xuccinic Dehydrogenase Activity of Particulate Fractions-Since the uptake of oxygen by the succinoxidase system is effected through the mediation of

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HOGEBOOM, CLAUDE, AND HOTCHKISS 621

cytochrome oxidase, it is conceivable that the latter enzyme may, under certain conditions, become the limiting factor in the aerobic oxidation of succinate. This possibility was remote in the case of the mitochondrial fraction which showed high cytochrome oxidase activity but could not be eliminated, on the basis of the data in Table I, in the case of the microsomes which contained very little cytochrome oxidase.

In order to obtain a more direct measure of activity against succinate, both sedimentable components of the liver extract were tested anaerobically

TABLE I Distribution of Cytochrome Oxidase and Succinoxidase in Cytoplasmic Extract of

Rat Liver

Liver fraction

E (liver extract)

L.G.c. (large granules, unwashed)

L.G.w. (large granules, washed 3 times)

M.w. (microsomes, washed 2 times)

S2 (supernatant after re- moval of large granules and microsomes)

-

1

.-

-

Experi- ment

NO.

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

- I

-

Dry Total weight volume of prep- aration

of prep- aration

mg. er P tn.

26.0 29.8 24.7 32.0 44.0 38.8 20.2 28.2 25.2 27.5 38.6 23.5 16.6 17.9 16.6

-

ml.

250

250 315 20.0 20.0 25.0 20.0 20.0 25.0 22.5 22.3 33.8

-

Q% I I -

27 (100) 8.4 32 (100) 9.0 36 (100) 10.0

202 74 66 185 68 58 195 67 55 318 73 63 272 64 70 295 66 62

<lO <4 7 <lO <4 4

0 0

-

0 0

0 0

(100) (100) OocJ)

78 76 69 47 59 50 8 5

0 0

for succinic dehydrogenase content by the ferricyanide method. The results of these determinations, which are summarized in Table II, demon- strate that the ratio, activity of large granules to activity of microsomes, in the oxidation of succinate, was roughly the same whether measured aerobi- cally or anaerobically. The results thus indicate that cytochrome oxidase was not a limiting factor in the det,erminations of the aerobic succinoxidase activity of the microsomes.

Additional Studies of Succinoxidase System of Mitochondrial Fraction-As shown by the recovery values in Table I, repeated washing of the large granule preparations in isotonic saline solution resulted in a considerable loss in their total succinoxidase content. Although this effect is not entirely

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622 ENZYME SYSTEMS OF LIVER CELL

understood, it is probably related to the physical properties of the granules. The following observations and experiments clarify the finding to some extent.

In the three experiments summarized in Table I, a marked decrease in the dry weight of the mitochrondrial fractions occurred when the granules were washed in saline. Additional observations have shown that the loss in dry weight is considerably greater and more rapid if the washing is conducted in hypotonic media, such as water or dilute buffers. Further- more, when simply suspended in water, the granules can be observed to swell enormously and eventually to disintegrate. If the water suspension is allowed to stand for several days at 4”, only a small fraction of the original

TABLE II

Comparison of Aerobic Succinoxidase and Anaerobic Sucrinic Dehydrogenase Activities of Large Granules and Microsomes

Preparation Succinic

dehydrogenase ON2 . cot

Succinoxidase Q%

Ratio, N2

Qcor : 0%

Large granules 1 205 63 3.3 (washed 3 times) 2 251 70 3.6

3 229 62 3.7 Microsomes (washed 1 29 7 4.1

2 times) 2 30 4 7.5 3 36

-

large granules can be recovered by low speed centrifugation, the remaining material consisting of soluble substances, including protein, and a particu- late component which is of considerably smaller size than the original mitochondria (12, 13).

The results given in Table III illustrate the rate of decline in the succin- oxidase activity of large granules kept in isotonic and hypotonic media, and demonstrate that the stability of the enzyme system is considerably greater in the former than in the latter type of medium. One is led to assume that either the dehydrogenase or an unknown component of the system, inter- mediate between the dehydrogenase and cytochrome c, was lost through lability or dilution.

In another experiment, a preparat.ion of large granules was washed twice with saline, suspended in hypotonic NaHC03 (0.025 M), and tested both aerobically and ana.erobically for activity against succinate. When retested after standing for 72 hours at 4’, the preparation had lost over 90 per cent of its aerobic succinoxidase activity but only 28 per cent of its original anaerobic succinic dehydrogenase activity. The particulate

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HOGEBOOM, CLAUDE, AND HOTCHKISS 623

material of the aged preparation was then isolated by centrifugation at 18,000 X g for 1 hour, washed twice in 0.025 M NaHC03, and finally resus-

TABLE III E$ect of Hypotonic Media on Succinoxidase Activity of Large Granules

Exp~i?=t Tonicity

1 Isotonic Hypotonic

2 Isotonic

Hypotonic 3 Isotonic

Hypotonic

Composition of medium Per cent of original succinoxi-

dase activity after standing

24 hrs. 48 hrs.

0.85% N&l Hz0 0.85% NaCl containing

0.01 M phosphate, pH 7.4

100 50 77

66 44 68

0.01 M phosphate pH 7.4 46 44 0.85% NaCl containing 96 37

0.025 M NaHCOa 0.025 M NaHCOs 34 7

TABLE IV E$ect of Hypotonic Medium on Aerobic Succinoxidase and Anaerobic Succinic Dehy-

drogenase Activity of Large Granules

Preparation

L.G.0 (original preparation in 0.025 M NaHC03)

L.G.1 (L.G.oafter standing 72 hrs. at 4”) L.G.2 (L.G.1 recovered by centrifuga-

tion and resuspended) S (supernatant after centrifugation of

L.G.1) L.G.8 (L.G.z washed twice and resus-

pended) L-G.8 + S

-

Aerobic determination T Per ml.

xeparation per hr.

cm??& 0,

420

17 <IO

@O*

70

3 <2*

Per ml. preparation

per hr.

cmm. co,

2035

1470 875

38

58

518f

N¶ Qcoz

340

245 142

10

30

267t

* The cytochrome oxidase Qo, for preparation L.G.2 was 360 c.mm. of 02 per mg. per hour. The low succinoxidase activity of the preparation was therefore not a result of loss of cytochrome oxidase content.

t These values are calculated on the basis of the aliquot of L.G.3 tested. The aliquots of both L.Gsa and S were the same as those employed when the two prepara- tions were tested separately.

pended in the same medium. The activity of the various preparations is shown in Table IV.

The following points of interest can be derived from the data presented in Table IV. (1) The results of the anaerobic measurements indicate that

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624 ENZYME SYSTEMS OF LIVER CELL

the succinic dehydrogenase system is considerably more stable in hypotonic media than would appear from assays of the complete succinoxidase system. (2) The anaerobic succinic dehydrogenase activity of the sedimentable material remaining after treatment of large granules with a hypotonic bicarbonate solution can, however, be reduced to low levels by repeated washing in the same medium (L.G.l --+ L.G.2 -+ L.GJ. (3) A soluble fraction (S), obtained after lysis of large granules in hypotonic bicarbonate

TABLE V

Effect of p-Dimethylaminoazobenzene on Distribution of Cytochrome Oxidase and Xuccinoxidase in Cytoplasmic Extract of Rat Liver

Experi merit NO.

-7

Diet

p-Dimethyl- aminoazoben- zene, 36 days

p-Dimethyl- aminoazoben- eerie, 50 days

Control diet, 44 days

Control diet, 56 days

Liver fraction

Extract Large granules

(washed 3 times) Microsomes (washed

2 times) Extract Large granules

(washed 3 times) Microsomes (washed

2 times) Extract Large granules

(washed 3 times) Microsomes (washed

2 times) Extract Large granules

(washed 3 times) Microsomes (washed

2 times)

- I( Cytochrome oxidase

Q%

32 (100)

QOS

7.5 46

33 223

(1~) 48

<2

7.8 29

<5 <l 2

24 (100) 6.7 243 54 60

<lO <2

37 (1~) 234 63

<5 <1

4

9 55

3

Per cent ~~CO”.Ty

(100) 35

<2

(100)

25

2

(100) 47

3

WI 50

2

solution, contains a substance which greatly enhances the succinic dehydrogenase activity of the lysed and washed particulate material (L.G.3).

E$ecl of Ingestion of p-Dimethylaminoaxobenxene-Table V presents the results of four experiments designed to determine the effect of p-dimethyl- aminoazobenzene on the cytochrome oxidase and succinoxidase activity of the cytoplasmic extract and its two particulate components. The condi- tions in the four experiments listed in Table V are comparable except that the animals in Experiments 1 and 2 received p-dimethylaminoazobenzene added to the basic diet.

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HOGEBOOM, CLAUDE, AND HOTCHKISS 625

The livers of the animals that received the carcinogenic compound showed no gross lesions. The only change noted microscopically in representative stained sections was a wide variation in the size of the parenchymal cells and their nuclei, but in no instance was cirrhosis or tumor formation detected. The livers of the animals that received the control diet appeared grossly and microscopically normal.

The data of Table V, in the light of those obtained with the liver of rats fed a normal diet (Table I), show that neither the butt,er yellow nor the control diet appreciably affected the cytochrome oxidase and succinoxidase activity of the liver extract. The activity (&on) and per cent recovery of both enzyme systems in the mitochondrial fractions derived from the animals fed the control diet (Experiments 3 and 4, Table V) were, however, slightly less than comparable values recorded in Table I. In Experiment 2, Table V, in which the large granules obtained from animals fed butter yellow were assayed for cytochrome oxidase, neither the Qo, nor the per cent recovery of this enzyme was greatly different from comparable values obtained in Experiments 3 and 4 with the control animals. The succinoxi- dase Qo, and per cent recovery values obtained in one butter yellow experi- ment (Experiment 1, Table V) were somewhat lower than the control values and in the next butter yellow experiment (Experiment 2, Table V) were considerably lower than the control values.

DISCUSSION

Intracellular Distribution of Enzymes-The observations reported in the present paper lead to the conclusion that most, if not all, of the cyto- chrome oxidase and succinoxidase activity of the cytoplasmic extract is associated with relatively large granules, approximately 0.5 to 2 /* in diameter. The findings supporting this conclusion can be summarized as follows: (1) When both the large granule and microsome fractions were removed from the extract, the supernatant Sz showed no demonstrable cytochrome oxidase or succinoxidase activity. (2) An average of 70 per cent of the former and 74 per cent of the latter enzyme system was recovered in the mitochondrial fraction. (3) Only a small proportion of the activity of the extract (less than 4 per cent of the cytochrome oxidase and approxi- mately 7 per cent of the succinoxidase) was found in the microsomes. Although the activity of the microsomes was appreciable, it was probably accounted for by the presence of large granules or of large granule fragments in the microsome fractions. In this respect, direct observations of the latter preparations in the dark-field microscope indicated that the number of large granules present was probably sufficiently high to account for the enzymatic activity of the microsomes. (4) When the over-all recovery of enzyme originally present in the extract is considered, it should be noted

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626 ENZYDIE SYSTEMS OF LlVER CELL

that two inhomogeneous fractions, Sdl and Sdz, were discarded. Sdl consisted essentially of agglutinated large granules and a small number of nuclei and red blood cells, and Sdz of a mixture of large granules and micro- somes. On the basis of dry weight measurements and microscopical obser- vations, it has been estimated that the number of large granules present in the discarded fractions is usually 10 to 20 per cent of those originally present in the extract. Furthermore, occasional determinations have shown that the discarded large granules possess roughly the same enzyme activity as that of the main large granule fraction. The amount of activity, in terms of recovery, which can be accounted for as present in large granules thus approaches 90 per cent for both enzyme systems.

Taken together, these observations suggest that the cytochrome oxidase and succinoxidase systems, when isolated by centrifugation from the cyto- plasm of the liver cells of the rat, are entirely localized in the so called large granules. This point is of considerable importance since it indicates that, in the living cell itself, these cytochrome-linked enzyme systems are situated in corresponding cytoplasmic elements.

The fact that the two enzyme systems under study are associated with insoluble components of the cell has been well known for many years and has been frequently alluded to in the literature both as a point of fundamen- tal biological significance and as the primary reason that neither system is well understood. As early as 1913, Warburg (6) noted that the large granules were responsible for most of the oxygen uptake exhibited by cell- free extracts of guinea pig liver. Bensley and Hoerr (18) were able to separate the large granule fraction by means of centrifugation, and con- centrates thus obtained were shown by Lazarow and Barron (8,9) to possess succinoxidase activity. The latter investigators, however, were unable to find a.ny significant difference in the activity of two particulate fractions separated by differential centrifugation. Stern (7), working with heart muscle, demonstrated that particles 50 to 200 rnp in diameter were associ- ated with both cytochrome oxidase and succinoxidase activity. In the present experiments the distribution of the cytochrome oxidase and succin-

oxidase systems has been placed on a quantitative basis, and both systems have been shown to be associated, probably exclusively, with granules of a definite range of size.

Problem of Cytological Identification of Particulate Fractions-Although the exact nature of the large granules is still unsettled, microscopical observations indicate that in the present experiments the large granule fraction consisted for the most part of those cytoplasmic inclusions known under the term mitochondria. Evidence for this view, which has been reviewed by Claude (12, 13), can be summarized as follows: Of the particu- late components present in the cytoplasm of the normal liver cell that can

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HOGEBOOM, CLAUDE, AND HOTCHKISS 627

be seen in either fixed or unfixed preparations, only mitochondria and secre- tory granules possess a range in size comparable to that of isolated large granules. Particulate glycogen and microsomes, on the other hand, are of sufficiently smaller size to permit effective separation from the large gran- ules by differential centrifugation. The abundance of secretory granules in the intact liver cell and, presumably, in the cytoplasmic extract can be varied considerably by simple alteration of the food intake of the animal; viz., when the animals are fasted, secretory granules accumulate in great numbers in the liver cells, whereas in animals fed normally to the time of death the liver cells contain very few secretory granules. In the present experiments with normal rats (Table I) the animals were given an ample supply of food, and the proportion of secretory granules present in the large granule fraction isolated by centrifugation was probably small.

A possible objection to the belief that the large granule fraction consists for the most part of mitochondria is based on the observation that isolated large granules are uniformly spherical, whereas the mitochondria of liver cells are frequently elongated. It is well known, however, that injuries of various sorts to the cell will cause mitochondria to become spherical. This can be demonstrated, for example, by observing cultured cells under dark-field illumination as they rupture and their mitochondrial content disperses in an isotonic medium.

It is, of course, tempt)ing to believe that the mitochondria of the living cell contain all of the cytochrome oxidase and succinoxidase present in the cytoplasm and therefore, that the mechanism by which molecular oxygen is utilized in the cytoplasm of the cell can be ascribed in large measure, if not entirely, to a definite morphological entity. Before this view can be definitely accepted, however, the relation between the large granules of the extract and mitochondria must be further clarified.

Work from this laboratory has indicated that the microsome substance represents the chromophilic component of the ground substance of cyto- plasm (19). Centrifugation experiments (19) and electron microscope studies of cultured cells (20) and of sections of guinea pig liver (21) support the view that the microsome material exists in the cell in t#he form of particulate, submicroscopic units. The part played in the economy of the cell by the microsomes is at present unknown.

Components of Succinoxidase System-A number of observations, several of them already mentioned, have shown that the large granules can be affected by changes in the tonicity of the medium, probably through the existence of a limiting membrane, and that their disintegration in hypotonic media is accompanied by a rapid and pronounced loss in aerobic succinoxi- dase activity. By the use of the ferricyanide method, it has been possible to follow more directly the changes affecting this cytochrome-linked enzyme

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628 ENZYME SYSTEMS OF LIVER CELL

during disintegration of the granules. Thus, it was found that the anaero- bic succinic dehydrogenase activity was largely retained in a hypotonic medium even though the aerobic activity had almost disappeared (Table IV) and that the anaerobic activity could be reduced to low levels only by repeated washing of the granules in hypotonic medium. This apparent difference in stability is difficult to explain on the basis of the present data. It is suggested, however, that the sharp decline in aerobic activity with time may be largely a dilution phenomenon, whereby substances inter- mediate in the chain of hydrogen transfer between succinate and cyto- chrome c are released from the granules to such an extent that their con- centration relative to the cytochrome oxidase system is reduced below a critical level. Such a dilution effect might be expected to occur more readily in a reaction dependent on cytochrome oxidase than in a reaction involving ferricyanide, since cytochrome oxidase is firmly bound to parti- culate material, whereas ferricyanide is present in solution in great excess.

The data in Table IV also demonstrate the existence of a soluble com- ponent of the succinic dehydrogenase system, a finding which confirms experimental results reported previously (7, 22). More recently (23), it has been found that the intact succinic dehydrogenase system can be obtained in a soluble form through treatment of the large granules with acetone and subsequent extraction of the residue with 0.01 M NaHC03. Further studies dealing with the properties of the dehydrogenase will be reported at a later date.

Ejj’ect of p-Dimethylaminoaxobenxene on Xuccinoxidase Activity of Liver Fractions-The preliminary data presented in Table V indicate that the ingestion of p-dimethylaminoazobenzene over a period of less than 2 mont.hs produces a considerable reduction in the succinoxidase content of the washed la.rge granules but has little, if any, effect on the succinoxidase content of the liver extract. Although an explanation for this finding must await further experimentation, it seems possible that the large granules obtained from rats fed the carcinogenic substance were abnormally fragile and therefore allowed the escape during the washing procedure of an unusually large amount of a soluble component of the succinoxidase system.

SUMMARY

1. A study of the distribution of two enzyme systems, cytochrome oxi- dase and succinoxidase, in the cytoplasm of the liver cell of the rat has been presented. A liver extract, containing only cytoplasmic material and suitable for fractionation by differential centrifugation, was utilized as an experimental tool.

2. Both enzyme systems were shown to be associated, probably exclu- sively, with sedimentable granules of a relatively large size (0.5 to 2 p in diameter). A smaller sedimentable component (60 to 150 rnp in diameter),

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HOGEBOOM, CLAUDE, AND HOTCHKISS 629

which was present in large amount in the liver extract, showed little, if any, enzyme activity and the remaining soluble material of the extract showed no activity. A number of observations suggested that the enzymatically active large granule fraction consisted chiefly of those cytoplasmic inclusions known as mitochondria.

3. The large granules were markedly affected by changes in tonicity of the surrounding medium, their disintegration in hypotonic media being accompanied by a rapid and pronounced loss in aerobic succinoxidase activity. This decline in succinoxidase could not be explained either by loss of cytochrome oxidase activity, which remained at a high level, or, from the results of anaerobic determinations, by loss of succinic dehydro- genase itself. By repeated washing of the large granules in a hypotonic medium, it was possible, however, to reduce their anaerobic succinic dehydrogenase activity to a low level, apparently through the release of a soluble component of the enzyme system.

4. The ingestion over a period of less than 2 months of the carcinogenic substance, p-dimethylaminoazobenzene, produced a reduction in the succino.xidase content of the large granules of rat liver.

BIBLIOGRAPHY

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Harbor, 7, 312 (1939). 8. Lazarow, A., in Biological symposia, Lancaster, 10,9 (1943). 9. Barron, E. S. G., in Biological symposia, Lancaster, 10, 27 (1943).

10. Kabat, E. A., Science, 93.43 (1941). 11. Dounce, A. L., J. Biol. Chem., 147, 685 (1943). 12. Claude, A., Science, 97, 451 (1943). 13. Claude, A., J. Exp. Med., 84, 51,61 (1946). 14. Sugiura, K., and Rhoads, C. P., Cancer Res., 1, 3 (1941). 15. Schneider, W. C., and Potter, V. R., J. Biol. Chem., 149,217 (1943). 16. Umbreit, W. W., Burris, R. H., and Stauffer, J. F., Manometric techniques and

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Rollin D. HotchkissGeorge H. Hogeboom, Albert Claude and

OF THE MAMMALIAN LIVER CELLSUCCINOXIDASE IN THE CYTOPLASM

CYTOCHROME OXIDASE AND THE DISTRIBUTION OF

1946, 165:615-629.J. Biol. Chem. 

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