Intracellular Distribution of Tyrosine-a-ketoglutarate ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 238, No. 1, January1963 Printed

in U.S.A.

Intracellular Distribution of Tyrosine-a-ketoglutarate Transaminase and 4-C14-Hydrocortisone Activities during Induction* GERALD

LITWACK,

MARY

Lou

SEARS,

AND

THOMAS

I.

DIAMoNDsToNEt

From the Biochemical Laboratory, Division of Cardiology, Philadelphia General Hospital, and the Department Graduate School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (Received

for publication,

EXPERIMENTAL

PROCEDURE

Male rats of the Wistar strain were obtained from Huntingdon Farms, Inc. These animals weighed 200 f 20 g at the beginning of each experiment, and they were maintained on a standard chow diet. Tyrosine transaminase activity was determined by the Briggs reaction (8) as modified by Canellakis and Cohen (9). The incubation was carried out for 20 minutes at 37”. Components of the system have been noted previously (4), except that the enzyme preparation occupied 1.0 ml of the system and the total volume of the system was 4.1 ml. A B-minute preincubation period was used before initiation of the reaction with 12 pmoles of n-tyrosine dissolved in phosphate buffer. Data presented are corrected for blanks that contained buffer alone in place of tyrosine. The enzymic preparations were 0.1 ml of a 10% homogenate (in sucrose) plus 0.9 ml of sucrose, or 1.0 ml of the fractions of cytoplasm, mitochondria, microsomes, or nuclei and cellular debris. Enzymic activity is reported as micromoles of p-hydroxyphenylpyruvic acid formed per 20 minutes. Differential centrifugation of 10% homogenates in 0.25 M sucrose was carried out by the modified method of Schneider (10) as described by Hogeboom (11). The nuclei fraction was washed once with 0.25 M and then with 0.14 M KCl. The radioactivity of these washings was counted separately, and the data from the sucrose wash were added to the results from the cyto-

July 26, 1962)

plasmic fractions. The data from the KC1 wash were summarized with the data for the nuclei fraction. These washings permitted a measure of completeness of washing into the supernatant fraction, on the one hand, and ease of desorbing radioactivity, on the other. Hydrocortisone-4-Cl4 in chromatographically pure state and sealed under nitrogen was obtained from the New England Nuclear Corporation or the Nichem Corporation. The specific radioactivity was 25 PC per pmole. Labeled or unlabeled hydrocortisone or both together were administered in the same manner as previously done (5). The inducing level of hydrocortisone was 60 mg per kg. Extraction of fractions containing radioactive hydrocortisone from homogenates or subcellular fractions entailed the use of methylene chloride (12). After extraction was complete, each fraction was taken to dryness at 25” in a vacuum hood and subsequently dissolved in toluene in glass counting vials. To each counting vial was added 1 ml of scintillating fluid. Standard procedures were used to count radioactivity in a Packard Tri-Carb spectrometer (kindly made available to us by Dr. D. J. Prockop). Corrections for quenching were made by counting before and after addition of a sample of standardized C14-labeled benzoic acid. Counting percentage efficiency ranged about a mean value of 28 (22.2 to 33.4). All reported values are corrected to 100% efficiency. Occasionally a nuclear fraction had high quenching, and this was successfully reduced by treatment with H202. RESULTS

* This work was supported by Research Grant A-4187 from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Public Health Service. t Present address, Department of Chemistry, University of Pennsylvania, Philadelphia 4, Pennsylvania.

In Fig. 1 is demonstrated the subcellular distribution of tyrosine transaminase activity as a function of time after administration of hydrocortisone inducer. The general shape of the overall time course curve for liver homogenates has been reported previously (13). It is clear that the activity of whole homogenate is paralleled most closely by the activity of the supernatant. The nuclei contain measurable amounts of enzymic activity, as do the microsomes. The mitochondria appear to be generally devoid of participation in a time course picture. Because of low yields from this fraction, a more intensive extraction was not pursued. In order to ascertain that the induction of enzymic activity was reasonably specific, the transamination of tyrosine, with oxaloacetate in place of cY-ketoglutarate, was followed. The results are shown in Fig. 2. A slight increase in the activity of the nonspecific transaminase is apparent in the course of 4 hours

302

Downloaded from www.jbc.org by guest, on March 18, 2013

We have reported previously the effects of a number of compounds of biological interest upon the nonspecific stimulation of tyrosine-or-ketoglutaarate transaminase activity (l-6). In all cases it was found that stimulation of tyrosine-cu-ketoglutarate transaminase activity required the presence of either the adrenal gland or administered hydrocortisone, although it has been reported that compounds containing the indole nucleus can induce enzymic activity in adrenalectomized rats (7). In an attempt to understand more clearly the mediation by glucocorticoids of the nonspecific stimulation of tyrosine transaminase activity, it was decided to follow the subcellular disposition of enzymic activity and of radioactivity due to administered 4-C14hydrocortisone.

of Medicine,

January

1963

G. Litwack,

M. L. Sears, and T. I. Diamond&me

whole homogenofe

)-

-

specific

fronsaminafion

I-

nonspech%

fransamhotion

0

;

I60 120 240 Minutes Post Inducer FIG. 2. The effects of hydrocortisone inducer on nonspecific transamination of n-tyrosine, with oxaloacetate in place of aketoglutarate, compared to specific ensymic activity. Each point is the average of three to four determinations with whole homogenates. The ratios of specific to nonspecific transamination rates of n-tyrosine are: zero time, 6.5; 120 minutes postinducer, 12.7; 240 minutes postinducer, 16.7. HPP denotes p-hydroxyphenylpyruvate.

O

b /

microsomes

-0

30

60 120 Minutes Post Inducer FIG. 1. Induction curve of tryrosine-or-ketoglutarate transaminase activity in the whole homogenate and in subcellular fractions as a function of time after administration of hydrocortisone inducer. Recovery of eneymic activity reconstituted from the fractions is never as great as the activity of the whole homogenate. This difference is of the order of 20%. Numbers refer to the determinations made for each point. The mean is plotted. HPP denotes p-hydroxyphenylpyruvete.

- 0

30

60 Minutes Post Inducer FIG. 3. Subcellular distribution of 4-C14-hydrocortisone administered with 60 mg of unlabeled hydrocortisone per kg as a function of time. In each experiment, 2 PC were administered. Each point plotted on the graph is the average value of one to four observations. All points are the average of at least two experiments, except for the 30.minute value.


postinducer, the increase over the resting level being 1.65-fold, compared to an increase of the specific transamination of 4.3-fold over the resting level. It should be noted also that the total observed rate of p-hydroxyphenylpyruvate production for the nonspecific reaction was 2.6 pmoles of p-hydroxyphenylpyruvate per 20 minutes per g of fresh liver, whereas the specific reaction had a rate of 40.05 pmoles of p-hydroxyphenylpyruvate per 20 minutes per g of fresh liver at 4 hours postinjection, which is 15 times the rate recorded for the nonspecific transamination. Thus the rate of nonspecific transamination of n-tyrosine at the peak time of induction is considered to be rather small: not more than 7 y0 of the total measured. Fig. 3 depicts the subcellular distribution of 4-C14-hydrocortisone as a function of time after injection. The label concentrates in the various fractions, except for the mitochondrial fraction, in a linear fashion up to 45 minutes postinjection when the steady state is reached. Thereafter, the concentration declines steadily to 4 hours postinjection, when the radioact,ivity is very low in all of the fractions. Kenney and Flora (12) reported the levels of radioactivity in the whole liver 1 to 36 hours postinducer with one animal used per measurement. The rate of decay of radioactivity between 1 and 24 hours postinjection under similar conditions is qualitatively the same. The majority of the label is rapidly concentrated in the supernatant and microsomal fractions, with a measurable amount in the nuclear fraction. The mitochondria do not seem to be highly active in concentrating the label by this extraction technique. The pattern of labeling in the subcellular fractions at 45 minutes postinjection with inducer levels of hydrocortisone is similar to

303

Xubcellular

304

Radioactivity

per g of liver

Inducer* Per fraction

AVerage

Per cent of liver radioactivityt

c.p.m.

Supernatant

Added Omitted

Microsomes

Added Omitted

G561 2791

,-1-1L----1-

65.6 34.4

4048

4.Cr4-hydrocortisone alone; of unlabeled hydrocortisone per g of liver X 100 g of liver

,-,-I

3060

120 Minutes

Post

Vol. 238, No. 1

240

The mediation of glucocorticoids in the induction process of tyrosine transaminase activity has been amply recorded (5, 7, 12-15). The lag of induction of tyrosine transaminase activity of approximately 1 hour appears, from the present work, to relate to the time of maximal concentration of hydrocortisone by the intracellular compartments, especially the microsomal It is of considerable interest to and the cytoplasmic fractions. note that the distribution of enzymic activity at the maximal response level (4 hours postinjection) is similar to the earlier subcellular concentration of hydrocortisone. This could be interpreted to mean that hydrocortisone is exerting a direct effect upon the induction system. This finding is harmonious with the results obtained from liver perfusion experiments by Goldstein, Stella, and Knox (15), who gave support to the idea that hydrocortisone may exert some of its physiological effects directly on liver cells by altering the level of enzyme activities. Kenney and Flora (12) concluded that Celite (a stressor presumably evoking the release of endogenous glucocorticoid sufficient to induce tyrosine transaminase), given with a small dose resulted in whole liver concentrations of hydrocortisone-Cr4, 2 to 3 times greater than those in animals given only the steroid (noninducing dose). Using levels of hydrocortisone presented here, we find that noninducing levels of hydrocortisone-Cl4 are absorbed more efficiently by the liver than is hydrocortisone-04 administered with an excess of carrier. Furthermore, we have noted a shift in concentrations between the microsomal and cytoplasmic fractions (Table I). The major problem remaining is to determine the events after the effective concentration in the cellular fractions (especially the microsomal and the cytoplasmic fractions) that occur up to 45 minutes postinjection and the events preceding the start of enzymic induction at 60 minutes postinjection (Fig. 4).

Inducer

4. The relationship between radioactivity extracted from the whole liver homogenate, derived from 4-Cl*-hydrocortisone administered with unlabeled hydrocortisone inducer, and homogenate tyrosine transaminase activity as a function of time postinducer. The numbers indicate replicate determinations. HPP denotes p-hydroxyphenylpyruvate.

SUMMARY

FIG.

the subcellular distribution of enzymic activity at the peak of induction (4 hours postinducer). Experiments have been carried out in which 0.03 mg of labeled hydrocortisone representing 2 nc was given without carrier hydrocortisone. As judged by enzymic activity measurements of the fractions, this was a noninducing dose. The distribution of radioactivity in two such experiments was similar to results of experiments with inducing levels of carrier, except that a change occurred in the relationship of radioactivity in the microsomal and supernatant fractions. This is summarized in Table I. The relationship of the time course of accumulation of hydrocortisone by the total liver and the induction of enzymic activity is illustrated by Fig. 4. It is obvious that the lag in the development of enzymic activity may be coincident with the time required to saturate the microsomal and supernatant fractions with hydrocortisone. The increase in enzymic activity appears to complement the loss of intracellular hormone concentration until approximately 4 hours postinjection, when the tyrosine transaminase activity is at its peak and the accumulated intracellular hormone concentrations have been depleted.

The increase in tyrosine-a-ketoglutarate transaminase activity during induction is distributed mainly in the cytoplasm and microsomes. Measurable increases also occur in the nuclear fraction, whereas the mitochondria appear not to be involved. Nonspecific tyrosine transamination plays a negligible role in the measurement of induction and also in the subcellular distribution. The distribution of 4-Cr4-hydrocortisone administered with inducing doses of unlabeled hormone is concentrated to the greatest extent within the cells 45 minutes after administration. The concentration of the hormone occurs mainly in the microsomal and cytoplasmic compartments. After the 45-minute period, the intracellular levels of hydrocortisone-Cl4 fall to a minimum The initial lag in at 4 hours, the height of enzymic induction. induction of enzymic activity seems to be related to the time of concentration of hydrocortisone in subcellular fractions. The increment between this phase and the initiation of enzymic activity induction is of approximately 15 minutes’ duration. REFERENCES 1.

LITWACK, 2. LITWACK,

G., Biochim.

G.,

AND

et Biophys. DIAMONDSTONE,

Acta, T.

42,369 (1960). I., Federation

Proc.,

20, 218 (1961). 3. AL-NEJJAR, Z. H., AND LITWACK, Acta, 48, 153 (1961). 4. LITWACK, G., Biochim. et Biophys. 5. LITWACK, G., AND DIAMONDSTONE, 469 (1962).

G.,

Biochim.

et Biophys.

Acta, 66,593 (1962). T. I., J. Biol. Ghem.,

237,


* The noninducing level is 2 RLCof the inducer represents 60 mg per kg added to the labeled hormone. c.p.m. per fraction t Calculated as c.p.m. per

0

Transaminase DISCUSSION

microsomal fractions with 4-F upon induction of tyrosine 45 minutes postinjection

These data are averaged from two experiments. Fraction

of Tyrosine

I

TABLE

Labeling of supernatant and hydrocortisone, depending transaminase activity

Distribution

January 1963

G. Litwack,

M. L. Sears, and T. I. Diamondstone

6. DIAMONDSTONE, T. I., AND LITWACK, G., Biochinz. et Biophys. Acta, 67, 583 (1962). 7. ROSEN, F., AND MILHOLLAND, R. J., Federation Proc., 21, 237 (1962). 8. BRIGGS, A. P., J. Biol. Chem., 61, 453 (1922). 9. CANELLAEIS, 2. N., AND COHEN, P. P., J. Biol. Chem., 222, 53 (1956). 10. SCHNEIDER, W. C., J. Biol. Chem., 176, 259 (1948). 11. HOGEBOOM, G. H., in S. P. COLOWICK AND N. 0. KAPLAN

12. 13. 14. 15.

305

(Editors), Methods in enzymology, VoZ. I, Academic Press, Inc., New York, 1955, p. 16. KENNEY, F. T., AND FLORA, R. M., J. Biol. Chem., 236, 2699 (1961). LIN, E. C. C., AND KNOX, W. E., J. Biol. Chem., 233, 1186 (1958). KENNEY, F. T., J. Biol. Chem., 237, 1610 (1962). GOLDSTEIN, L., STELLA, E. J., AND KNOX, W. E., J. Biol. Chem., 237, 1723 (1962).
