THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 239, No. 10, October 1964

Printed in U.S.A.

The Biochemistry of Animal Cells

I. THE EFFECT OF CORTICOSTEROIDS ON LEAKAGE OF ENZYMES FROM DISPERSED RAT LIVER CELLS

YOSHIRO TAKEDA, AKIRA ICHIHARA, HIROAKI TANIOKA, AND HIDEO INOUE

From the Department of Biochemistry, Osaka University Dental School, Osaka, Japan

(Received for publication, November 27, 1962)

In past years there has been great progress in enzyme chemis- try, which has contributed to the clarification of many phys- iological mechanisms in living organisms. Enzymes, however, are not scattered haphazardly in the cell, but are localized in an organized way, often associat’ed with cellular organelles (I). These patterns of enzyme localization control enzyme activities. Typical examples have been shown in mitochondrial respiration coupled with oxidative phosphorylation (2, 3). Results from experiments on subcellular particles should also be true for the intimately related enzymes and cellular structures in whole cells. However, there are few reports on whole cells available. From this point of view, in order to study the behavior of enzymes within the cells, it is desirable to employ dispersed intact cells suspended in a simple, defined medium. Furthermore, the study of the specific functions of organs would also be advanced if investigations were carried out at a cellular level.

There have been a number of reports on procedures suitable for making cell suspensions, with the use of enzymatic digestion (4, 5), filtration through a sieve (6), acid treatment (7), and perfusion of the tissue with chelating agents (8). Cell suspen- sions obtained by the above procedures, however, have two major disadvantages for use in biochemical studies. One is the low rate of respiration of the resulting cells, and the other is the enzyme leakage from the cells. Laws and Stickland (9) and Kalant and Young (10) reported that such cells had remarkably low endogenous respiration and that succinate was the only substrate which could be metabolized.

Henley et al. (11, 12) found that many enzymes, such as lactic dehydrogenase, malic dehydrogenase, and glutamic-pyruvic transaminase, leaked out from rat liver cells when the cells were dispersed. They showed that the leakage of these enzymes was not prevented by addition of various compounds to the suspend- ing medium.

In order to overcome these obstacles and to obtain cells with normal function, experiments were carried out on the addition of ADP to the medium, and the rate of cellular respiration was found to be restored. The preliminary results of this work have been reported (13).

The present paper shows that in suspension, rat liver cells lose many enzymes, including tryptophan pyrrolase, serine and threonine dehydrases, and two-thirds of their glutamic-pyruvic transaminase. The leakage of these enzymes, and particularly of the glutamic-pyruvic transaminase, could be prevented if the rats were treated with various glucocorticoid hormones before the liver cells were dispersed. It is also reported that the anti-

inflammatory actions of these hormones are closely related to their actions in preventing enzyme leakage.

EXPERIMENTAL PROCEDURE

Preparation of Cell Suspension and Homogenate-Of the various methods for the preparation of cell suspensions tested, the meth- ods of Anderson (8) and of Branster and Morton (14) were finally adopted for the present work, because these methods yielded relatively large amounts of cells. The procedure used was as follows. Male albino rats of the Donryu strain, weighing 170 to 200 g, were decapitated. The liver was perfused, via the inferior vena cava and the portal vein, with 30 ml of calcium-free Locke’s solution, containing 0.027 M sodium citrate, at 37”. The liver was then removed, washed with Locke’s solution, weighed, and divided into two portions.

Four grams of the liver were dispersed as a cell suspension. The homogenizer used for preparing the cell suspension was a siliconized glass test tube with a loosely fitting, smooth surfaced pestle, made of acrylate resin. This homogenizer was similar in size and shape to that used by Kamphausen and Morton (15). The liver was suspended in Locke’s solution (5 ml per g of liver) and gently crushed in the homogenizer by hand, with 10 up and down strokes used for the operation. The crude cell suspension thus obtained was filtered through a metallic sieve (200 mesh) and the filtrate was centrifuged at 100 X g for 3 minutes. The resulting supernatant fraction was used, if necessary, for DEAE- cellulose column chromatography as shown in Fig. 4. The precipitate was suspended in 30 ml of Locke’s solution by gentle agitation with a glass rod. The suspension was placed in an ice bath for 10 minutes and then centrifuged. The packed cells thus obtained were dispersed in 10 ml of distilled water. At this stage microscopic examination showed negligible contamination with blood cells and cell debris. This suspension of liver cells was immediately homogenized in a Potter-Elvehjem glass homog- enizer and then sonically disrupted in a IO-kc sonic oscillator for 5 minutes (cell homogenate). The rest of the liver was homoge- nized with 10 ml of distilled water in a Potter-Elvehjem glass homogenizer and sonically treated as described above (original homogenate).

Both homogenates contained about 50 mg of material, dry weight, per ml, and were appropriately diluted before use in experiments.

All of the above procedures, except liver perfusion, were carried out at 4” and pH 7.0.

In order to establish a routine method for the preparation of

3590

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October 1964 Y. Takeda, A. Ichihara, H. Tanioka, and H. In.oue 3591

the cell suspension, the time taken for each step was fixed. Thus 15 minutes were allowed for cell filtration; 10 minutes for the first centrifugation; and 25 minutes for washing, allowing the cells to stand, and the second centrifugation. The cells obtained were immediately homogenized, so that the total time taken from crushing the liver until homogenization was complete was 50 minutes.

Assay IMethods-For assay of glutamic-pyruvic and glutamic- oxaloacetic transaminases, the method of Wada and Snell (16) was slightly modified. The reaction mixture for glutamic- pyruvic transaminase contained potassium phosphate buffer, pH 8.0, 100 pmoles; sodium cr-ketoglutarate, 10 pmoles; n-alanine, 10 hmoles; pyridoxal phosphate, 30 pg; and homogenate, 0.1 ml (about 0.5 mg, dry weight) in a total volume of 2 ml. After 5 minutes of incubation at 37”, the reaction was started by addition of the n-alanine. After further incubation for 10 minutes, the reaction was stopped by the addition of 1 ml of 10% metaphos- phoric acid. The pyruvate formed was measured by the method of Friedemann and Haugen (17). Xylene was used as solvent, and the xylene layer was washed with 4 ml of 0.5 N HCl and then extracted wit,h sodium carbonate solution (16). For assay of glutamic-oxaloacetic transaminase activity, alanine was replaced by n-aspartic acid in the above system, and a more dilute homoge- nate (25 to 50 pg, dry weight) was used. The oxaloacetate formed was converted to pyruvate by the aniline-citrate method (16), and the pyruvate was measured as described above.

For assay of serine and threonine dehydrases, the reaction mixture contained potassium phosphate buffer, pH 8.0, 100 pmoles; n-serine or n-threonine, 10 pmoles; pyridoxal phosphate, 30 pg; and homogenate (15 mg, dry weight). The mixture was incubated for 20 minutes. The pyruvate or oc-ketobutyrate formed was measured by Friedemann and Haugen’s method (17) with the use of ethyl acetate as the extracting solvent for (Y- ketobutyrate.

Glucose &phosphatase, lactic dehydrogenase, and tryptophan pyrrolase were measured by the methods of Cori and Cori (18), Neilands (19), and Knox (20)) respectively.

The activities of the various enzymes are expressed in milli- micromoles per hour per mg of homogenate, dry weight, at 37”. Because the recovery of cells is variable from one preparation to another, comparison of .the total activities of the original homoge- nate and of the cells is impossible. Therefore the retention of each enzyme, i.e. the amount of enzyme remaining in the dis- persed cells, was expressed as a percentage calculated from the ratio of the specific activity of the cell homogenate to that of the original homogenate. This mode of expression may be defended on the basis of findings by Henley et al. that there is not significant general leakage of proteins during preparation of the suspensions w.

Protein was estimated by the modified Biuret reaction of Pardee.’

Treatment of Rats with Corticosteroids-Corticosteroids were injected intramuscularly into rats daily. The dose and number of injections varied, as described in “Results.” Control rats received injections of 0.9% NaCl. During the injection period, food and water were available ad libitum.

When puromycin was injected into the rats together with dexamethasone,Q 15 mg of puromycin were injected into the rats

1 A. B. Pardee, personal communication. 2 1601.Methyl-9a-fluoro-1-dehydrocortisol.

first, and after 1 hour the animals received 1 mg of dexamethasone and 15 mg of puromycin. Subsequently 15 mg of puromycin alone were injected four times at hourly intervals, and 1 hour after the last injection the rats were killed. Thus the total amount of puromycin injected was 90 mg.

Chromatography on Diethylaminoethyl Cellulow Columns- DEAE-cellulose powder (1.5 g) suspended in 0.01 M potassium phosphate buffer, pH 7.0, was introduced into a column, 2 cm in diameter, to a height of 10 cm. The column was washed with the same buffer. The homogenate was dialyzed against the same concentration of buffer for several hours and then centrifuged at 9000 x g for 20 minutes. The resulting supernatant fluid, con- taining 150 mg of protein, was applied to the column and was eluted by successive addition of 40-ml volumes of potassium phosphate buffer (pH 7.0) at 0.01, 0.05, and 0.2 M. The eluate was collected in 5-ml fractions. There was more than 80% recovery of enzyme activity.

Materials-The corticosteroids used were cortisone acetate (Upjohn Company, Kalamazoo, Michigan), dexamethasone acetate (Les Laboratoires Roussel, Paris), and hydrocortisone acetate (UCLAF Company, Paris). Puromycin hydrochloride was a gift from Dr. B. L. Hutchings, American Cyanamid Com- pany, Pearl River, New York. All other compounds used were available commercially.

RESULTS

Enzyme Leakage from Cells in Suspension-Microscopic exam- ination of the dispersed cells showed that they were homogeneous parenchymatous cells and were well dispersed (Fig. 1). They also retained their structural organization without any recogni- zable damage, and there was remarkably little contamination with subcellular constituents or fragments of damaged cells. There are several reports showing that dispersed cells showed no structural damage that was recognizable by electron microscopy (14, 21).

However, when the liver was dispersed as a cell suspension, there was considerable loss of activity of various cellular enzymes (Table I). The enzymes in the soluble fraction especially, such as tryptophan pyrrolase, lactic dehydrogenase, and serine and threonine dehydrases, were almost completely lost from the cells. On the other hand, glucose 6-phosphatase, known to be localized in microsomes (22), stayed entirely in the cells. Our results also

FIG. 1. Phase contrast micrograph of dispersed rat liver cells. Magnification, X100.

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3592 Biochemistry of Animal Cells. I Vol. 239, No. 10

TABLE I Leakage of vam’ous enzymes from normal rat liver cells

Lacticdehydro- genase

Serine dehydrase Threonine de-

hydrase Tryptophan

pyrrolase. Glutamic-pyru-

vie trans- aminase

Glutamic-oxa- loacetic trans- aminase

Glucose G-phos- phatase.

- -

2. mi- I&

- _-

4 5

5

4

10

4 2

4 -

Enzyme activity

Original homogenate

Cell homogenate

mpmoles/hr/mg dry homogenale

2,280 f 9oa 54 f 1.8

42 f 4.9

2.2 f 0.5

1,690 f 287

:4,660 f 1,141

2,590 f 565

02

-

120 f 15a 0

0

0

584 f 91

3,640 f 1,23(

2,626 f 476

T

.-

4

1

1

Retention

%

.3 f 0.5” 0

0

0

35 f 4.6

96 f 3.5

00 f 15

Mean f standard error.

TABLE II

Effect of perfusion on enzyme leakage

Part of the liver was removed before the perfusion and was homogenized. This homogenate was designated “unperfused liver.” The rest of the liver was used as described in “Experi- mental Procedure.”

Enzyme

Enzyme activity

UnK$‘=d 1 Pgzd j cells

m,moles/hr/mg dry homogenate

Glutamic-pyruvic transaminase. 1614 1588 564 Serine dehydrase. 54 54 0 Threonine dehydrase. 47 46 0 Glucose 6-phosphatase. . 2200 2160 2160

showed that the enzymes of the citrate cycle remained in the cells (13). In addition, about 35% of the glutamic-pyruvic transa- minase, and 96% of the glutamic-oxaloacetic transaminase, remained in the cells.

The leakage of enzymes did not occur on perfusion of the liver, but on dispersion of the cells (Table II). It was also found that even when enzymatic methods were used for dispersion of the cells (such as the use of pancreatin, trypsin, or hyaluronidase (4, 5)) leakage of these enzymes was not prevented. Henley, Sorensen, and Pollard reported that the leakage of various enzymes could not be prevented by the addition of various substances during the dispersion of the cells (23). These substances included ATP, hemoglobin, and serum albumin. We also observed that enzyme leakage was not prevented by addition of ATP together with oxidizable substrates such as succinate or fumarate, or of dinitro- phenol, to the medium used for perfusion and dispersion. In addition, even when the cell suspension was ,prepared at 37”, enzyme leakage was not prevented in the presence or absence of oxidizable substrates. These facts suggest that enzyme leakage from dispersed cells may not be energy-dependent. Therefore it

seems possible that the leakage of enzymes from the cells, what- ever the method used for dispersion, is due to a change in cell permeability.

As mentioned above, the enzymes which leaked out were all enzymes of the soluble fraction, and, as seen in Table I, ap- proximately two-thirds of the glutamic-pyruvic transaminase activity of the original homogenate leaked out when the cells were dispersed. Since Katunuma et al. (24) reported that glutamic-pyruvic transaminase is present in both the soluble and mitochondrial fractions, and the enzymes in these two fractions have different properties, it was of interest to see which fraction of the glutamic-pyruvic transaminase leaked out. When the original homogenate was applied to a DEAE-cellulose column, glutamic-pyruvic transaminase activity was eluted in two peaks (Fig. 2). By preliminary chromatography of the supernatant and mitochondrial fractions of rat liver, the pattern of elution of the transaminases was elucidated (Fig. 3). Thus the first peak to be eluted was identified as that derived from the soluble fraction, while the activity of the second peak of the eluate represented mitochondrial activity. The activity ratio ‘,f the first and second peaks was approximately 3 : 1.

On the other hand, chromatography of the cell homogenate yielded two glutamic-pyruvic transaminase fractions in which the ratio of the activities of the soluble and mitochondrial fractions was 1:2.4 (Fig. 4). Since Henley et a2. showed that there is not significant leakage of total protein from cells during their prepara- tion (12), one can compare the activities before and after cell preparation on a dry weight basis. Thus one-fourth of the activity of the original homogenate as shown in Table I (1690 mpmoles) should be attributed to the activity of the mito- chondrial fraction (i.e. 422 mpmoles) while 2.4/3.4 or 70 y0 of the activity of the dispersed cells (584 mpmoles) repr.esents mitochondrial activity (i.e. 412 mpmoles). These calculated values for the mitochondrial activity of the two preparations agree well with the data obtained. This indicates that the majority of glutamic-pyruvic transaminase activity remaining in the dispersed cells is that of the mitochondrial enzyme, w lereas almost all the soluble glutamic-pyruvic transaminase had leaked out.

To prove that the activity which leaked out of the cells was that from the soluble fraction, the supernatant fraction obtained after centrifugation of the filtrate of the cell preparation (see

“Experimental Procedure”) was chromatographed (Fig. 5). It

FIG. 2. DEAE-cellulose column chromatography of glutamic- pyruvic transaminase (GPT) in original homogenate. The activity of the supernatant fraction applied was 1081 X lo3 mpmoles of pyruvate formed per hour, and the protein content was 175 mg. The specific activity was 1789 mpmoles of pyruvate formed per hour per mg of homogenate, dry weight.

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October 1964 Y. Talceda, A. Ichihara, H. Taniolca, and H. Inoue 3593

20 40 60 80 100 120 +O.OlM+O.O5M+- 0.2M’

EFFLUENT I m 1 1

FIG. 3. DEAE-cellulose column chromatography of glutamic- pyruvic transaminase (GPT) in supernatant and mitochondrial fractions of rat liver. Normal rat liver was homogenized and fractionated to subcellular fractions, according to the method of Hogeboom (25). The protein of the supernatant fluid was precipi- tated by addition of solid ammonium sulfate to saturation, and the precipitate thus formed was dissolved in a small volume of 0.01 M potassium phosphate buffer, pH 7.0. The mitochondrial fraction was sonically disrupted. Both fractions were dialyzed before application to the column. The conditions of sonic treat- ment and dialysis are described under “Experimental Procedure.” These fractions were applied to columns separately but are pre- sented in the same figure for comparison. O---O, activity of the supernatant fraction (total activity, 1070 X lo3 mpmoles; protein, 191 mg); and 0- - -0, mitochondrial activity (total activity, 336 X lo3 mMmoles; protein, 71.2 mg).

20 40 60 80 100 +O.OlM+-O.O5Md- 0.2M -

EFFLUEN T (m I )

FIG. 4. DEAE-cellulose column chromatography of glutamic- pyruvic transaminase (GPT) of the cell homogenate. The super- natant fraction of the homogenate contained an activity of 113.4 X lo3 mpmoles of pyruvate formed per hour, and 13G mg of protein.

was found that although there were two active fractions in the

resulting eluate, the fraction representing the soluble part was far more active than that representing mitochondrial enzyme, the ratio of the two activities being 7.7 : 1, whereas this ratio in the

original homogenate was 3: 1, as shown in Fig. 2. Therefore it is evident that the loss of activity on cell dispersion is probably not due to enzyme inactivation, but to leakage of enzymes of the soluble fraction from the cell, and that enzymes of the mito- chondrial fraction remain in the cells even after dispersion. The low activity of mitochondrial enzymes found in the supernatant fraction is likely to be derived from cells ruptured during the

preparation of the cell suspension. EJect of Glucocorticoids on Enzyme Leakage from Cells-It has

been reported that glucocorticoids cause a decrease in the protein content of the carcass and an increase in that of the liver (26,27),

and the increase in liver weight after glucocorticoid administra- tion is due not to an increase in the cell number, but rather to the accumulation of nitrogenous substances in the cell (26). More- over it was also suggested that the accumulation of nitrogenous substances is the result of changes in cell permeability rather than changes in the metabolism (28). It seemed probable, therefore, that glucocorticoids might also prevent the leakage of enzymes from liver cells in suspension.

Experiments were carried out to test this possibility. It was indeed found that when the rats had been treated with cort,isone, a dispersion of their liver cells retained about twice as much glu- tamic-pyruvic transaminase as a dispersion of cells from un- treated animals. Dexamethasone, a synthetic corticosteroid, caused a particularly marked retention of both glutamic-pyruvic transaminase and lactic dehydrogenase, even at less than one- tenth of the dosage of cortisone (Table III). It should be added that there was no sex difference in the rats used for studies on the preventive effect of glucocorticoids on enzyme leakage.

The effect of dexamethasone on glutamic-pyruvic transaminase leakage was followed over a period of time, as shown in Fig. 6. Enzyme retention had already started to increase above the con.- trol value 1 hour after injection of dexamethasone, and it con- tinued to increase to almost 100% of the control value by the 2 day. Thereafter the retention decreased up to the 5 day. The cause of this decrease is still unknown.

The addition of 1 mg of dexamethasone to 50 ml of the perfu- sion fluid did not cause a significant increase in the retention of enzymes.

The leakage of tryptophan pyrrolase and serine and threonine dehydrases could not be prevented by treatment with these hor- mones under the above conditions. It is interesting that the enzymes that are not prevented from leakage by dexamethasone are all readily inducible by this hormone (29-31).

Four days after dexamethasone treatment it was found that the total glut,amic-pyruvic transaminase activity of the original homogenate had increased considerably. It was therefore inter- esting to see which fraction of the glutamic-pyruvic transaminase had increased in activity. Accordingly, the whole liver homoge- nate after dexamethasone treatment was chromatographed on a DEAE-cellulose column. It was found that there was more than 7 times more activity in the soluble fraction than in the mitochon- drial fraction, as shown in Fig. 7, whereas for untreated liver the

20 40 60 80 100 120 +0.01M-1+0.05M ---0.2/v*

EFFLUENT (ml)

FIG. 5. DEAE-cellulose column chromatography of glutamic- pyruvic transaminase (GPT) which leaked out from the cells. The supernatant fraction of the crude filtrate contained an activity of 730 X lo3 mpmoles of pyruvate formed per hour, and 183 mg of protein.

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Biochemistry of Animal Cells. I Vol. 239, No. 10

TABLE III Effect of glucocorticoids on enzyme leakage from rat liver cells

Hormone

Controla

Cortisonec

Dexa- metha- soned

-

1

I

-

2. tni- nals

10

11

5

4

Enzyme activity

Enzyme Retention Original Cell

homogenate homogenate

m~moles/hr/mg dry homogenate %

Glutamic- 1690 f 287b 584 f 91 35 f 4.6 pyruvic transami- nase

Glutamic- 2412 f 312 1470 f 18662 f 7.3 pyruvic transami- nase

Glutamic- 3312 =t 393 2263 f 147 70 & 6.3 pyruvic transami- nase

Lactic dehy- 3150 =t 240 1200 f 60 40 f 15 drogenase

chondrial, fraction. A similar phenomenon was reported on in- duction of phosphoenolpyruvic carboxykinase (32).

Comparison of Efects of Various Corticosteroids on Leakage of Glutamic-Pyruvic Transaminase-Thomson and Mikuta (31) re- ported that the induction rate of tryptophan pyrrolase by corti- sone and cortisol was proportional to the dose of hormone admin- istered. It whould also be mentioned that these glucocorticoids have an anti-inflammatory action (33). Therefore it was inter- esting to see whether the effects of the corticosteroids on trypto- phan pyrrolase induction, anti-inflammatory activity, and prevention of enzyme leakage were interrelated. Table IV shows that when a small dose of the corticosteroids was administered, cortisone and cortisol, which have the same degree of anti-inflam- matory potency, showed no effect on either enzyme leakage or tryptophan pyrrolase induction, whereas the same dose of dexa-

y The values are cited from Table I. 6 Mean f standard error. c Cortisone (10 mg) was injected daily into each rat for 4 days,

and the rats were killed on the 5th day. d Dexamethasone (1 mg) was injected int,o each rat daily for 4

days, and the rats were killed on the 5th day.

;8C

ii if 60

Z 0 2 40

k ii

20

L

0 1 2 3 4 5 DAYS

FIG. 6. Time course of the effect of dexamethasone on glutamic- pyruvic transaminase (GPT) retention in the cells. Dexametha- sone (1 mg) was injected into each rat per day. Figures in paren- theses represent the numbers of rats used.

ratio was 3: 1, as shown in Fig. 2. Since a sample from the treated liver containing an activity of 3720 mpmoles per mg, dry weight, per hour was applied, that of the mitochondrial fraction should be 448 mHmoles per mg of original homogenate, dry weight, because the activity ratio of the soluble and mitochon- drial fractions was 7.3 : 1. As described above, the mitochondrial activity of untreated liver was calculated to be the somewhat similar value of 422 mpmoles per mg of homogenate, dry weight, per hour. This means that the hormone increases glutamic- pyruvic transaminase activity in the soluble, but not in the mito-

FIG. 7. DEAE-cellulose column chromatography of glutamic- pyruvic transaminase (GPT) in the original homogenate after treatment of animals with dexamethasone. Dexamethasone (1 mg) was injected into rats daily for 4 days, and the animals were killed on the 5th day. The original homogenate contained an ac- tivity of 1860 X lo3 mpmoles of pyruvate formed per hour, and 125 mg of protein. The specific activity was 3720 mMmoles of pyruvate formed per hour per mg of homogenate, dry weight.

TABLE IV

Comparison of effect of various glucocorticoids on glutamic-pyruvic transaminase retention and tryptophan pyrrolase induction

Portions (0.5 mg) of the hormone indicated wereadministered to the rats by injection and the animals were killed after 3 hours.

HO~ltlOWS

None

Cortisone

Cortisol

Dexamethasone

Glutamic-pyruvic ransaminase activit

Original Cell 1omoge- homoge-

nate nate I

m/.moles jhr jmg ar3 homogenate

1680 588 1788 624

2100 1038 49 5.9 1884 906 48 4.8

1542 756 49 6.7 2136 9GO 45 3.1

1422 1116 1962 1458

Retention of glutamic-

pyruvic transaminase

Tryptophan pyrrolase

activity of original

homogenate

%

35 34

m,moles jhrjmg dry homogenate

2.0 2.2

78 16.6 74 18.3

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October 1964 Y. Takeda, A. Ichihara, H. Taniolca, and H. Inoue 3595

TABLE V

Effect of puromycin on glutamic-pyruvic transaminase leakage

The treatments were carried out as described in “Experimental Procedure.”

NoneCL. Dexameth-

asone. Puromycin Dexameth-

asone + puromy- tin

Glutamic-pyruvic transaminase NO. activity Retention of of glutamic-

ani- pyruvic IIdS Original Cell transaminas~

homogenate homogenate

m~mmoles~hr/mg dry homogenate ‘35

1690 + 287b 584 f 91” 35 & 4. fb

5 1770 f 400 1552 zt 32485 f 9.2 3 2232It228 912fGG 41fl.O

3 2106 f 340 1584 f 31878 zt 9.8

Tryptophan pyrrolase

activity of original

homogenate

nzpmoles/hr/mg dry homogenate

2.0 f 0.36

t5.5 + 1.3 2.5 f 0.3

2.1 Ik 0.4

a The results of the control experiment are cited from Table I. b Mean f standard error.

methasone, which has a much higher anti-inflammatory potency, caused considerable induction of the enzyme, as well as having a marked effect on enzyme leakage. Deoxycorticosterone acetate, which has no anti-inflammatory action, showed neither trypto- phan pyrrolase induction nor prevention of the transaminase leakage (13). These results suggest that there may be an inter- relation between depression of inflammation, enzyme induction, and prevention of enzyme leakage.

EJect of Puromycin on Leakage of Glutamic-Pyruvic Transami- nase-Puromycin is known to inhibit protein synthesis in animals as well as in bacteria (34, 35). Nemeth and de la Haba (36) showed that puromycin inhibits the induction of tryptophan pyr- rolase by its substrate. In this study of the mechanism of t,he ac- tion of corticosteroids in preventing enzyme leakage, the possible involvement of protein synthesis was tested by means of puromy- tin. It was found that the simultaneous injection of puromycin did not affect the preventive action of dexamethasone on glu- tamic-pyruvic transaminase leakage (Table V), whereas the in- crease of tryptophan pyrrolase activity caused by this corticos- teroid was inhibited completely by the antibiotic. This indicates that protein synthesis is probably not involved in the mechanism of the preventive action of corticosteroid on enzyme leakage.

DISCUSSION

Although there have been many reports on enzyme leakage from ascites tumor cells (37-40), few papers are available on enzyme leakage from intact liver cells in suspension. Henley et al. (11, 12) showed that enzymes such as glutamic-pyruvic and glutamic-oxaloacetic transaminases, and lactic dehydrogenase, leaked out from suspensions of rat liver.

In the present work it was shown that, besides the enzymes which Henley et al. had reported, the cellular activities of trypto- phan pyrrolase and serine and threonine dehydrases were com- pletely lost by leakage. Since these enzymes remained after perfusion of the liver, but had leaked out by the first centrifuga- tion after filtration, it is very likely that leakage occurred at the time of dispersion. This accords well with results report,ed by Henley et al. (11) and Berry (41).

Since the enzymes which leaked out completely are localized in the soluble fraction of the cell, and since glucose 6-phosphatase and enzymes of the citrate cycle, which stayed in the suspended cells (13), are particle-bound or mitochondrial enzymes, it seems reasonable to assume that the enzymes in the hyaloplasm may have a tendency to leak through the cell membrane during the dispersion process. Recently Berry (41) found that glucose 6-phosphatase leaked out to a considerable degree from dispersed cells of mouse liver. This observation is in contrast to the results obtained in the present work and those of Henley et al. (23). Since Berry and Simpson (42) reported subsequently that their preparat.ion of cells showed considerable damage of the cell mem- branes, it is likely that the discrepancy may be due to the degree of cell damage.

It appeared probable that the activity of glutamic-pyruvic transaminase remaining in the cells after dispersion might be that of enzyme localized in mitochondria, since Katunuma et al. (24) showed that the glutamic-pyruvic transaminase activity of rat liver is distributed in both the mitochondria and the soluble part of the cell. Our study indeed showed that the activity of glu- tamic-pyruvic transaminase which leaked out from the cells was that from the soluble fraction, and the activity remaining in the cells was due to mitochondrial enzyme.

Although no extensive morphological investigations were carried out in the present work, there are a number of reports showing that cell suspensions prepared in the way we have de- scribed do not show any microscopic evidence of damage or change (6,14,21), and that the leakage of enzymes is not due to a breakage of the cells, but to changed permeability of the cell membrane (11, 12). Moreover, using ascites tumor cells, Holm- berg (40) showed that leakage of enzymes actually occurs from living cells. Wu (38) reported that there is a difference in the degree of leakage of glycolytic enzymes. Leakage of enzymes from cells occurs whatever method is used for their preparation.

With regard to the effects of corticosteroids on permeability, there are references in the literature showing that glucocorticoids, such as cortisone and cortisol, prevent enzyme leakage from thymocytes (43) and lysosomes (44). Sex hormones and deoxy- corticosterone acetate have the reverse effect.

This prevention of enzyme leakage from cells by glucocorti- coids may be responsible for the increase in liver weight, without a comparable increase in cell number, in rat liver after treatment with these hormones (26,27). There is evidence suggesting that the induced increase of enzyme activity is due to an increase de nooo in the number of enzyme molecules as a result of stimulation of enzyme synthesis (45-47). However, the possibility that enzyme increase may be partly due to accumulation of enzymes within the cell, as a result of a change of cell permeability, is not completely excluded.

The mechanism of the preventive action of glucocorticoids on enzyme leakage is not yet clear, although it is very likely that the hormone may act on the cell membrane, causing a change in per- meability. However, it is unlikely that this change involves pro- tein synthesis, since puromycin does not abolish the action of dexamethasone on enzyme leakage.

The present work indicates some relationship between enzyme leakage and inflammation. Recent clinical observations show that many enzymes appear in the serum of patients with hepatitis and these are very similar to those which leak out from cells in suspension (48). Glucocorticoids also prevent the appearance of these serum enzymes (49, 50). There is ample evidence suggest-

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3596 Biochemistry of Animal Cells. I Vol. 239, No. 10

ing that enzyme increase in the blood can occur in the absence of tissue necrosis when there is an alteration in permeability (51).

SUMMARY

Rat liver cells were dispersed in suspension, and the activities of various enzymes were compared before and after dispersion. It was found that more than 90% of the glucose 6-phosphatase and of the glutamic-oxaloacetic transaminase remained in the cell, whereas tryptophan pyrrolase, and serine and threonine dehydrases, were completely lost when the cells were dispersed. Only 35 y0 of the glutamic-pyruvic transaminase remained in the dispersed cells, and the rest of the activity leaked out. The activity which leaked out represented that from the soluble frac- tion, and the activity which remained in the cells was mitochon- drial activity, as shown by chromatography on diethylamino- ethyl cellulose columns.

Glucocorticoids such as cortisone, cortisol, and dexamethasone (16@-methyl-Sol-fluoro-1-dehydrocortisol) had a preventive action on leakage of glutamic-pyruvic transaminase and lactic dehydro- genase. Their actions on enzyme leakage were parallel with their anti-inflammatory potencies. Thus dexamethasone showed the most effect and deoxycorticosterone acetate had no effect on enzyme leakage.

Glucocorticoids such as dexamethasone increased glutamic- pyruvic transaminase activity in the soluble, but not in the mito- chondrial fraction of rat liver.

Puromycin did not inhibit the preventive actlion of dexametha- sone on enzyme leakage.

1. SIEKEVITZ, P., in J. 111. ALLEN (Editor), The molecular control of cellular activity, McGraw-Hill Book Company, Inc., New York, 19G2, p. 143.

2. CHANCE, B., AND WILLI~IMS, G. R., in F. F. NORD (Editor), Advances in enzymology, Vol. XVII, Interscience Publishers, Inc., New York, 1956, p. 65.

3. HAGIHAR~, B., Biochim. et Biophys. Beta, 46, 134 (19Gl). 4. DULBECCO, R., AND VOGT, M., J. Exptl. Med., 99, 167 (1954). 5. RINALDINI, L. M., Exptl. Cell Reseaxh, 16, 477 (1959). 6. KALTENBACH, J. P., Exptl. Cell Research, 7, 568 (1954). 7. LONGM~IR, I. S., .\ND AP REES, W., Nature, 177, 997 (1956). 8. ANDERSON, N. G., Science, 117, 627 (1953). 9. LAWS, J. O., AND STICKLAND, L. H., Nature, 178, 309 (1956).

10. KAL&T, H.; AND YOUNG, F. G., Nature, 179, 816 (1957). 11. HENI,EY. K. S.. WIGGINS. H. S.. POLLARD, H. M.. AND DUL-

12.

13.

14.

15.

IG. 17.

LAERT~ E., A&. N. Y. ‘Acad. sci., 75, 2iO (195Sj. HENLEY, K. S., WIGGINS, H. S., POLLARD, H. M., AND DUL-

LAERT, E., Gastroenterology, 36, 1 (1959). ICHIHARA, A., TANIOKA, H., INOUE, H., AND TAKEDA, Y.,

Proceedings of the Symposium on Chemical Physiology and Pathology, Vol. 2 (Japanese edition), Medical Culture, Inc., Tokyo, 1962, p. 177.

BRANSTER, M. V., AND MORTON, R. K., Nature, 180, 1283 (1957).

KAMPHAUSEN, H., AND MORTON, R. K., Biochem. J., 63, 647 (1956).

WADA, H., AND SNELI,, E. E., J. Biol. Chem., 237, 127 (1962). FRIEDEMANN, T. E., AND HAUGEN, G. E., J. Biol. Chem., 147,

415 (1943).

REFERENCES

18. 19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

CORI, G. T., AND CORI, C. F., J. Biol. Chem., 199, 661 (1952). NEILANDS, J. B., in S. P. COLOWICK AND N. 0. KAPLAN (Edi-

tors), Methods in enzymology, Vol. I, Academic Press, Inc., New York, 1955, p. 449.

KNOX, W. E., in S. P. COLOWICK AND N. 0. KAPLAN (Editors), Methods in enzymology, Vol. II, Academic Press, Inc., New York, 1955, p. 242.

LEESON. T. S.. AND KAI,ANT. H.. J. Biophys. Biochem. Cytol., 10,95’ (1961 j.

_ ”

SEGAL, H. L., AND WASHKO, M. E., J. Biol. Chem., 234, 1937 (1959).

HENLEY, K. S., SORENSEN, O., AND POLLARD, H. M., Nature, 184, 1400 (1959).

KATUNUMA, N., MIKUMO, K., MATSUDA, M., AND OKADA, M., J. Vitaminok (Osaka), 8, 68 (1962).

HOGEBOOM, G. H., in &. P. COLOWICK AND N. 0. KAPLAN (Editors). Methods in enzumoloau. Vol. I. Academic Press, inc., New York, 1955, p. i6. ““’ ’

WEBER, G., BANERJEE, G., AND BRONSTEIN, S. B., J. Biol. Chem., 236, 3106 (1961).

FEIGELSON, M., GROSS, P. R., AND FEIGELSON, P., Biochim. et Biophys. Acta, 55, 495 (1962).

SILBER, R. H., AND PORTER, C. C., Endocrinology, 52, 518 (1953).

KNOX, W. E., AND AUERBACH, V. H., J. Biol. Chem., 214, 307 (1955).

GOLDSTEIN, L., KNOX, W. E., AND BEHRMAN, E. J., J. Biol. Chem., 237, 2855 (1962).

31. THOMSON, J. F., AND MIKIXA, E. T., Endocrinology, 55, 232 (1954).

32. SHRAGO, E., LARDY, H. A., NORDIJE, R. C., AND FOSTER, D. O., J. Biol. Chem., 238, 3188 (1963).

33. FORSHAM, P. H., in R. H. WILLIAMS (Editor), Textbook of endocrinology, Ed. 3, W. B. Saunders Company, Phila- delphia, 1962, p. 376.

34. YARMOLINSKY, M. B., AND DE LA HABA, G. L., Proc. Natl. Acad. Sci. U. IS’., 45, 1721 (1959).

35. TAKEDA, Y., HAYASHI, S., NAKAGAWA, H., AND SUZ~JKI, F., J. Biochem. (Tokyo), 48, 169 (1960).

36. NEMETH, A. M., AND DE LA HABA, G., J. Biol. Chem., 237,119O (1962).

37. BOSCH, L., Biochim. et Biophys. Actu, 30,444 (1958). 38. Wu, R., Cancer Research, 19, 1217 (1959). 39. MACDONALD, K., Biochim. et Biophys. Actu, 36, 543 (1959). 40. HOLMBERG, B., Cancer Research, 21,1386 (19Gl). 41. BERRY, M. N., J. Cell Biol., 15, 1 (1962). 42. BERRY, M. N., AND SIMPSON, F. O., J. Cell Biol., 15,9 (19G2). 43. MONDER, C., BEN-EZZER, J., AND WHITE, A., Endocrinology,

70, 123 (1962). 44. DE DUVE, C., WATTIAUX, R., AND WIBO, M., Biochem. Pharma-

col., 8, 30 (1961). 45. KENNY, F. T., J. Biol. Chem., 237, 3495 (1962). 46. FEIGET,SON, P., AND GREENG‘IRD, O., J. Biol. Chem., 237, 3714

(1962). 47. SEGAL, H. L., AND KIM, Y. S., Proc. Nutl. Acad. Sci. U. S., 50,

912 (1963). 48. HENLEY, K. S., SCHMIDT, E., AND SCHMIDT, F. W., J. Am. Med.

Assoc., 174, 977 (1960). 49. HARVALD, B., AND MADSEN, S., Acta Med. Stand., 169, 381

(1961). 50. SUMMERSKILL, W. H., CLOWDUS, C. B. F., BOLLMAN, J. L.,

AND FLEISHER, G. A., Am. J. Med. Sci., 241,555 (1961). 51. FISHMAN, W. H., in F. W. PUTNAM (Editor), The plasma pro-

teins, Vol. 11, Academic Press, Inc., New York, 1960, p. 59.

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Yoshiro Takeda, Akira Ichihara, Hiroaki Tanioka and Hideo InoueON LEAKAGE OF ENZYMES FROM DISPERSED RAT LIVER CELLS

The Biochemistry of Animal Cells: I. THE EFFECT OF CORTICOSTEROIDS

1964, 239:3590-3596.J. Biol. Chem. 

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