Effects of Phenobarbital on the Synthesis and Degradation ...Effects of Phenobarbital on the Synthesis and Degradation of the Protein Components of Rat Liver Microsomal Membranes*

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 244, No. 8, Issue of April 25, pp. 2017-2026, 1969

Printed in U.S.A.

Effects of Phenobarbital on the Synthesis and Degradation of the Protein Components of Rat Liver Microsomal Membranes*

(Received for publication, October 17, 1968)

Y. KURIYAMA$ AND TS~NEO OMURA

From the Institute for Protein Research, Osaka University, Osaka, Japan

P. SIEKEVITZ AND G. E. PALADE

From The Rockefeller University, New York, New York 1002i

SUMMARY The synthesis and turnover of the total proteins of endo-

plasmic reticulum membranes and of two enzymes, i.e. NADPH-cytochrome c reductase and cytochrome bs, isolated and purified from these membranes, have been studied during treatment with phenobarbital, one of the drugs known to induce an increase in the activity of hydroxylating enzymes and in the amount of smooth endoplasmic reticulum membranes in rat hepatocytes.

A single phenobarbital dose induces a prompt increase in the rate of NADPH-cytochrome c reductase synthesis without affecting the production of cytochrome bg. The induced reductase is chromatographically and immuno- chemically identical with the enzyme normally produced by the liver.

Repeated phenobarbital doses cause a large increase in NADPH-cytochrome c reductase amount concomitantly with a moderate rise in-cytochrome bs content. Both effects appear to result from a drastic reduction in rates of enzyme degradation.

Cessation of phenobarbital treatment is promptly followed by a progressive reduction in NADPH-cytochrome c reductase amount caused by a large increase in the rate of degradation of the enzyme in the face of continued synthesis.

The findings indicate that the rates of synthesis and degradation of the NADPH-cytochrome c reductase vary conversely in the induction process. The relevance of these tIndings to the control of the turnover of membrane enzymes is discussed.

As a continuation of our earlier studies (l-3) on the biogenesis of endoplasmic reticulum membranes, we have examined the

*Part of this work was supported by United States Public Health Service Grant ROl HD-61689 (to P. S.).

$ On leave of absence from Central Research Laboratory, Sankyo Company, Ltd., Shinagawa-Ku, Tokyo, Japan.

special case of the phenobarbital-induced increase of smooth endoplasmic reticulum membranes in rat hepatocytes. Ever since the early work of Conney et al. (4-6), of Remmer et al. (7- 9), and of others (10, ll), it has been repeatedly (12-19) shown that soon after phenobarbital injection there is a proliferation of smooth endoplasmic reticulum membranes with an increase of some, but not all, of the enzymes usually associated with these membranes in normal rat hepatocytes. This increase in enzyme activity is probably due, as work with inhibitors of protein and RNA synthesis has indicated (6, 13, 20, 21), to an increase in enzyme amount. Despite the lack of knowledge concerning the immediate phenobarbital action, it is clear that the end result is a differential proliferation of particular membranes in the liver cell. These smooth endoplasmic reticulum membranes appear morphologically similar to those existing before induction (7-9, 12, 13, 15, 18, 19); however, enzyme concentrations in the total smooth membrane fraction isolated from the liver of normal phenobarbital-injected rats are quite dissimilar in that after phenobarbital treatment the specific activities of certain enzymes (namely those of the NADPH-oxidase chain) are increased several fold, while those of others either are not changed or actually are decreased. These circumstances provide us with a system of membrane biogenesis of which the following questions can be asked: (a) is the increase in enzyme activity due to increased enzyme synthesis or decreased enzyme breakdown, (b) and, concomitantly, are the turnover rates of individual proteins any different from those of the enzymes of normal liver membranes; (c) are new membranes synthesized pari passu with new enzymes, that is, is there a one-step organization of new smooth membranes, or (d) are new membranes formed independently of new enzyme synthesis, the new enzymes being added on at random to both new and previously existing smooth endoplasmic reticulum membranes?

In this paper, we have extended to the phenobarbital-treated rat earlier work (3) carried out on normal animals on the turnover rates of two purified membrane proteins, the NADPH-cytochrome c reductase and the cytochrome bg. One of these en-

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2018 Phenobarbital and Microsomal Protein Turnover Vol. 244, No. 8

zymes, the NADPH-cytochrome c reductase, goes up in amount much faster than does total smooth membrane protein, and hence its specific activity increases several fold, while the increase in the amount of the other, cytochrome by, is much less than the increase in over-all smooth membrane proteins so that its specific activity goes up only slightly (cj. Reference 22).

Our findings on the synthetic and turnover rates of these enzymes are discussed in relation specifically to questions a and b above and generally to current concepts of membrane biosyn- thesis.

METHODS

!i?eatrnent of Animals and Preparation of Microsomes-In all experiments Sprague-Dawley male rats weighing 160 to 180 g were used. Except where noted, they were kept on a 15% casein diet for about 2 weeks before the initiation of the experiments.

In short term experiments on the effect of a single phenobarbital injection on the rates of synthesis of microsomal enzymes, the drug was injected intravenously as a 10% solution in isotonic 0.9% NaCl solution through a caudal vein. In turnover experiments, which required repeated daily injections, the phenobarbital solution was injected intraperitoneally. The dose of the drug was always 10 mg/lOO g, body weight, of the animal. Ex- cept where noted, radioactive ammo acids (on-leucine-1-14C, 25 mCi per mu, New England Nuclear) and arginine (guanidino- r4C-L-arginine, 30 mCi per mM, Radiochemical Centre, Amer- sham, England) were injected intravenously through a caudal vein, as a solution of 50 E.cCi per ml in isotonic NaCI; the exact amounts are given below for each experiment.

Before killing, the rats, both normal and phenobarbital- treated, were fasted for 24 hours. Except when noted, livers were perfused. Total microsomes were obtained and washed as described (3) from control and phenobarbital-treated animals except that 10 mM EDTA in 0.15 M KaCl was used for washing instead of 0.15 M KCl. The washed microsome preparation represents mostly membranes, as most of the proteins of the contents and most of the ribosomal RNA have been extracted (3).

Preparation and PuriJication of Enzymes-The purifications of NADP-cytochrome c reductase and cytochrome bs were carried out as described previously (3) except that larger columns (1.5 cm x 50 cm) of Sephadex G-100 were initially used. In experiments in which cytochrome bs was not extracted, a lower concentration of trypsin (O.OOl’% instead of 0.03% (3)) was used to solubilize the reductase from microsomal membranes.

Preparation of Antireduetase y-Globulin-The reductase was purified from liver microsomes of untreated rats essentially as previously described (3) with several small modifications to make the procedure suitable for large scale operation. The purified enzyme was found to be homogeneous by sedimentation analysis and by disc gel electrophoresis.

Male white rabbits were immunized with the purified reductase as follows. Each animal received subcutaneously two injections of 5 mg each of purified enzyme mixed with Freund complete adjuvant (Difco); the interval between the injections was 3 weeks. Two weeks after the second subcutaneous injection, 2 mg of purified enzyme were injected intravenously as a solution in isotonic NaCl. Two days later, another intravenous injection of 2 mg of purified enzyme was given. The serum was obtained from the blood of the immunized animals, and a y-globulin frac-

tion was prepared by fractionation with ammonium sulfate. The antireductase rabbit y-globulin thus obtained strongly inhibited the reduction of cytochrome c catalyzed by purified rat reductase; it also inhibited the NADPH-cytochrome c reductase activity of rat liver microsomes, but did not inhibit at all their NADH-cytochrome c reductase activity. The preparation and properties of the antireductase -y-globulin will be reported in detail elsewhere.1

The purity of the antireductase y-globulin was tested by two different procedures: the Ouchterlony agar diffusion technique and the adsorption of y-globulin by untreated microsomes and by microsomal trypsin digestion residue (i.e. microsomes from which the reductase has been extracted). The details of the agar diffusion experiment are given in the legend of Fig. 3, while the details of the antibody adsorption experiments will be reported in detail elsewhere.’ Here it is sufficient to mention that the residue of microsomal trypsin digestion, which does not contain NADPH-cytochrome c reductase (3), did not adsorb the antibody at all, while untreated microsomes effectively ad- sorbed it. The results suggested that the antibody-enzyme complex obtained with untreated microsomes was specific.

Enzyme Assay-The microsomal and purified NADPH-cytochrome c reductases were assayed in a reaction mixture which contained 2 X 10e5 M yeast cytochrome c, 1 X 10m4 M NADPH (Sigma), and enzyme in 2 ml of 0.1 M potassium phosphate buffer of pH 7.5. Crystalline cytochrome c was obtained from Sankyo Company, Tokyo, and was further purified by preparative chro- matography on a CG-50 ion exchange column. With this purified preparation, a value of -35.0 pmoles of cytochrome c reduced per min per mg of protein at 25” was obtained. This represents a 100% increase in specific activity over that previously recorded (3) with the horse heart cytochrome c obtained from Sigma. The latter was used only in the experiments whose results are given in Figs. 11 to 13. Cytochrome bs in microsomes, and in purified preparations, was determined as previously described (3).

RESULTS

Identity of NADPH-cytochrome c Reductases of Normal and Phenobarbital-treated Animals-The enzyme was extracted from hepatic microsomes of normal and phenobarbital-treated rats and the corresponding extracts were filtered through separate Sephadex G-100 columns in exactly the same way. Each of the two reductase-rich fractions was then placed on a DEAE- cellulose column and eluted as shown in Fig. 1. Since phenobarbital treatment caused a 3-fold increase in enzyme activity, most of the reductase activity in the eluate obtained from treated animals was due to new enzyme. It is clear (Fig. 1) that the two peaks of reductase activity had identical positions on the column. It was also found that the specific activities of the purified enzymes were also the same in both cases. Likewise, the antibody to normal NADPH-cytochrome c reductase inac- tivated to the same extent its antigen, as it did the enzyme from phenobarbital-treated animals (Fig. 2). Moreover, the agar double diffusion technique gave a single, continuous precipita- tion band (Fig. 3) of the antibody with microsomal extracts from normal and phenobarbital-treated rats, thus showing that the antibody fraction contained a single reacting component with the same protein in both extracts. All of these results indicate that the activities found in the microsomes of normal and of

IT. Omura, manuscript in preparation.


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Control

I I * z

1.5 -

1.0 -

I.5 -

50 100 I50

Volume of eluote (ml)

FIG. 1. Chromatographic separation of normal and induced NADPH-cytochrome c reductase. Liver microsome fractions were obtained from normal and phenobarbital-treated animals (five daily injections) and the reductase from each was solubilized and initially fractionated on a Sephadex G-100 column. The reductase-rich fractions from these columns were combined, placed on DEAE-cellulose columns (1 X 10 cm), and eluted with a linear concentration gradient of KC1 from 0 to 0.25 M in 0.05 M phosphate buffer, pH 7.5. Enzyme activity was assayed on 4-ml fractions of the eluant. For technical details see “Methods” and Reference 3.

Anti-reductase added (mg/ml)

FIQ. 2. Inhibition of normal and induced NADPH-cytochrome c reductases by antibody prepared against normal reductase. The antibodywas incubated for 10 min at 25’ in 0.1 M phosphate buffer, pH 7.5, with microsomes from either normal or phenobarbital- treated animals. The reduction of cytochrome c by NADPH in the microsomes was then measured at 25” as described under “Methods.”

phenobarbital-treated animals are due to the same enzyme protein.

Pulse Labeling of Miwosomd Membrane Protein Components- Since some of the experiments that we contemplated depended on rates of protein synthesis, we investigated the time needed

for an adequate pulse labeling (Fig. 4). As the results indicated that 40 min was adequate, this time interval was used in later experiments. The different shapes of the curves in Fig. 4 indicate that the proteins of the washed microsomes were labeled more rapidly (cf. Fig. 6) and the cytochrome ba more slowly than the reductase. Experiments to be published separately indicate that the microsomes contain apocytochrome bg and suggest that the delayed appearance of radioactivity in cytochrome bb can be explained by the additional time required to attach the heme to its apoprotein.

Effect of Single Injection of Phenobarbital on Rates of Synthesis of Cytochrome 66 and of NADPH-cytochrome c Recluctase-It has been known for quite some time that a single phenobarbital injection can cause an increase in the activities, or amounts, of the enzymes involved (cf. Reference 22). Fig. 5 indicates that intravenously given phenobarbital caused a rapid increase of reductase activity, a slower increase of P-450 amount, and no increase at all in cytochrome bg amount over 18 hours; over this interval there was no significant increase in liver weight or in microsome recovery per g of liver.

Combining the information in Fig. 4, which indicated that 40 min were sufficient for pulse labeling, with that in Fig. 5, which suggested that a single phenobarbital injection might cause an increase in the amount of reductase, we decided to examine the phenobarbital effect on the rates of synthesis of the total protein of the microsomes and of the purified reductase and cytochrome bg. Since there was observed an increase in reductase activity

FIG. 3. The immunological identity of NADPH-cytochrome c reductase isolated from hepatic microsomes of normal and phenobarbital-treated rats. NADPH-cytochrome c reductase from normal and phenobarbital-treated animals and the antibody to the reductase from normal animals were prepared and purified as described in the text. The double diffusion test was run in 1.2% agar (special agar Noble, Difco) in 0.05 M phosphate buffer, pH 7.5, at 4” for 72 hours. The well diameters were 7 mm and the center to center spacing between wells was 18 mm. Well A contained 20 mg per ml of purified antireductase r-globulin; Well B contained the trypsin digestion extract of microsomes obtained from phenobarbital-treated animals (25 mg of protein per ml, equivalent to about 2 mg of pure reductase per ml) ; Well C contained the trypsin digestion extract from microsomes of normal animals (60 mg of protein per ml, equivalent to about 2 mg of pure reductase per ml).


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700

600

200

r- ,/p..’

y’ I I I I I I I I

IO 20 30 40 ,

+-ik- 0

Minutes after the. injection of C’4-leucine

FIG. 4. Incorporation of I%-leucine into cytochrome bs, reductase, and total proteins of washed microsomes. Normal rats were fasted for 24 hours before an intravenous injection of DL-W- leucine (5 &i/100 g, body weight). Two experiments were performed, with very similar results, with three animals for each time point in each experiment; the data represent therefore the pooled microsomal fraction from the livers of six rats. The animals were killed at the times given after the phenobarbital injection and microsomes and purified enzymes were obtained as described under ‘(Methods.”

"0 II 2 11 4 II 6 11 8 11 IO ’ “1 12 14 1 ’ 16 I ’ 18 11

Hours after injection of PB

FIQ. 5. Changes in NADPH-cytochrome c reductase activity and in amounts of cytochromes P-450 and bs after a single phenobarbital (PB) injection. The data are averages of two experiments which gave nearly identical results; three animals were used in each experiment for each time point. The rats were fasted for 24 hours before an intravenous phenobarbital injection (10 mg/lOO g, body weight) and also for the duration of the experiment. Hepatic microsomes were obtained and microsomal enzymes were assayed as described under “Methods.” The results are expressed as per cent of reductase activity or cytochrome content of the controls. Control values (100%) were 0.34 mpmole of cytochrome ba, 0.162 unit of reductase, and 0.85 mpmole of cytochrome P-450, all per mg of microsomal protein.

(or amount) during the experiment (Fig. 5), the raw specific radioactivity values for each reductase time point were multiplied by the factor: (amount of microsomal enzyme in phenobarbital-treated rats)/(amount of microsomal enzyme in normal

animals). This correction was not necessary for cytochrome bs or for total microsomal proteins, since these did not change in amount during the experiment (Fig. 5). Fig. 6 thus shows the uncorrected and corrected curves for only the reductase. Two separate experiments were performed, with almost identical results. It can be seen from Fig. 6 that the phenobarbital effect on the rate of synthesis of NADPH-cytochrome c reductase was rapid, no lag being discernible so that even after 1 hour a significant increase was observed. By contrast, the increase in the rate of synthesis of cytochrome bs was negligible, hence the indication that the phenobarbital effect was specific on enzyme synthesis and was not connected with a change in the size of the pool of free amino acids available for the synthesis of the two proteins. Comparing Fig. 5 with Fig. 6, we found that while the rate of reductase synthesis reached a peak at 5 to 7 hours after phenobarbital injection (nearly 4-fold the normal value) and then declined to twice the normal value by 18 hours, enzyme concentration continued to increase slowly past -7 hours after phenobarbital injection. This finding is commented upon under “Discussion,” as is the observation (Fig. 6) that the rate of synthesis of the total protein of washed microsomes reached a peak before that of the reductase.

Turnover of NADPH-cytochrome c Reductase and Cytochrome bs during Continued Daily Administration of Phenobarbital-The

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FIG. 6. Change in the rates of synthesis of cytochrome 65, reductase, and total proteins of washed microsomes. The animals were prepared as indicated for Fig. 5. In addition, at 1,3,5,7,12, and 18 hours after the intravenous phenobarbital (PB) injection, nn-leucine-l-r% (5 ,.&X/100 g, body weight) was injected intravenously, and the animals were killed after 40 min. At each time point, the livers of three rats were pooled for the preparation of washed microsomes and for the separation and purification of microsomal enzymes. The raw data for the reductase were corrected for concurrent changes in microsomal enzyme content (cf. Fig. 5) by multiplying the corresponding specific radioactivities by the ratio: (enzyme content in the microsomes of phenobarbital- treated rats)/(enzyme content in the microsomes of control rats).


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FIG. 7. Changes in liver weight, microsome recovery per g of liver, and cytochrome 66 and reductase contents per mg of microsomal protein during phenobarbital (PB) treatment. The animals were started on a 15% casein diet 2 weeks before the initiation of the experiments and received intraperitoneally a daily dose of phenobarbital (100 mg per kg, body weight). Three animals were used for each time point. Animals not injected but kept on the same diet were used as controls. The results are expressed in per cent of control values which were: 4.5 g, wet weight, of liver per 100 g, body weight, 13.2 mg of microsomal protein per g of liver, and 0.314 mpmoles of cytochrome bg and 0.188 unit of reductase, both per mg of microsomal protein.

next experiments were designed to investigate what happens to enzyme synthesis and enzyme degradation over long periods of continuous increase in enzyme (particularly reductase) concentration. Such a situation can be obtained by daily phenobarbital injections. Fig. 7 shows the increase in liver weight, in re- coverable microsomes per liver,2 and in cytochrome bg and reductase contents per microsomes over an 8-day period of phenobarbital treatment.

The experimental animals received a single injection of 14C- leucine and 4 hours later the first phenobarbital injection, which was then followed by daily phenobarbital treatment. Control rats were carried through the same protocol, except for the phenobarbital treatment. Fig. 8 gives the decrease with time in specific radioactivity of the total protein of liver microsomes from normal and phenobarbital-treated rats. The half-lives for control animals graphically determined from the initial slopes were similar to those obtained earlier (3) .3 However, if the data

2 Consonant with the increase of the endoplasmic reticulum volume in hepatocytes (U-19), the amount of protein recovered in the microsomal fraction per g of tissue equivalent increases in phenobarbital-treated animals (cf. Fig. 7).

3The half-lives obtained in the present case were: 70 hours for total micrcsomal protein, 70 hours for the reductase, and 100 hours for cytochrome bs. These values are on the lower end of the range previously reported (3).

for the phenobarbital-treated animals were corrected for the change in liver weight and microsome recovery (body weight increased by only 10% during the time and was disregarded) by the following two factors-(u) liver weight of phenobarbital- treated rats/liver weight of control rats and (b) microsome recovery in phenobarbital-treated rats/microsome recovery in control rats-the corrected data for the phenobarbital-treated rats were obtained. They indicate (Fig. 8) that during the period that phenobarbital was acting to increase the amount of certain enzymes and of smooth endoplasmic reticulum membranes the turnover rate of microsomal membrane protein was slowed down, so that the half-life of the microsomal (mostly membrane) protein was lengthened to twice the value that it had in normal animals (cf. Reference 3). We also measured the specific radioactivities of the proteins of the trypsin digestion residue (cf. Reference 3), and the values of this fraction (membrane protein) were almost the same as that of the washed microsomal proteins in all cases.

The two enzymes, the reductase and cytochrome bg, were isolated and purified and their specific radioactivities were determined (Table I). The raw data for the enzymes prepared from phenobarbital-treated rats were corrected as noted in Table I for changes in liver weight and amounts of microsomes and enzymes recovered, all as compared to the corresponding data for normal rats (Fig. 7). It is clear that, despite the scatter of

0 2 4 6 8

Days of PB-treatment

FIG. 8. Decay of specific radioactivity of total microsomal proteins (washed microsomes) from normal and phenobarbital (PB)-treated rats. The animals were treated as given for Fig. 7. In addition, all animals received an intravenous injection of W-l- leucine (10 &X/100 g, body weight) 4 hours before zero time. Treated animals received in addition a daily intraperitoneal injection of phenobarbital (100 mg per kg, body weight) beginning at zero time. For each time point three control and three treated rats (fasted for the preceding 24 hours) were killed and microsomes were prepared from their pooled livers. For the day 0 point, the animals were killed 4 hours after the W,-1-leucine injection without any phenobarbital treatment. The specific radioactivity values for phenobarbital-treated animals were corrected by multiplying each of them with the following two factors: (a) liver weight of phenobarbital-treated rats/liver weight of normal rats and (b) microsome recovery in phenobarbital-treated rats/microsome recovery in normal rats (cf. Fig. 7). The arrows indicate half-lives graphically determined from the initial slopes.


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TABLE I

Specific radioactivities of hepatic microsomal NADPH-cytochrome c reductase and cytochrome 66 in normal and in phenobarbital-

treated animals after single injection of 14C-1-leucine The experimental details are given under “‘Methods” and in the

legends to Figs. 7 and 8.

Days of phenobarbital

treatment

1

-

Specific radioactivities

YADPH-cytochrome G reductase cytochrome 6s

Phenobarbital-treated Phenobarbital-treated

Control Control corye;;ed Correcteda cor;e$-&d Corrected=

920 632 366 204 153

492 291 169 133

cpm/mg #r&in 513

1170b 476 13206 296 865 197 768 165

363 660b 250 720b 161 547 134 544

(1 Corrected cpm/mg = uncorrected cpm/mg

X liver weight phenobarbital rats

liver weight-control rats

X recovery microsomes in phenobarbital rats

recovery microsomes in control rats

X content enzyme/mg microsomes of phenobarbital rats

content enzyme/mg microsomes of control rats

b We assume that the comparable increases in reductase and cytochrome bg radioactivities from day 2 to day 4 are due to shifts in microsomal recovery, which are inadequately corrected by the above procedure.

points, the turnover rates of the reductase and of cytochrome bs in phenobarbital-treated rats are quite different from those in normal animals. First, the difference between the half-lives of the reductase and cytochrome bg in normal animals (Table I) agrees with an earlier finding of ours (3) that the cytochrome bg has a significantly longer half-life than the reductase.a In addition, it appears that in phenobarbital-treated animals both of these enzymes are stabilized, so that there is very little degradation of the enzyme occurring. Put in another way, practically all of the decrease in the uncorrected specific radioactivity of the enzymes (Table I) is due to an increase in the amount of enzyme being synthesized from nonradioactive precursors; there is practically no concomitant degradation of radioactive enzyme to be replaced by nonradioactive enzyme. This conservation of total radioactivity was also noticed in the case of the reductase by Shuster and Jick (23,24) and is commented upon under “Dis- cussion.” It is of some significance that, in phenobarbital- treated rats, the total protein of washed microsomes seems to be degraded more rapidly (Fig. 8) than were the two isolated and purified proteins, the reductase and the cytochrome bs (Table I).

The marked differences in enzyme turnover between normal and phenobarbital-treated animals may be more apparent than real, since it is possible that radioactive amino acids resulting from the degradation of labeled proteins are more efficiently reutilized (cf. Loftfield and Harris (25) and Gan and Jeffay (26)) in phenobarbital-treated animals than in controls. This possibility was investigated by repeating exactly the above experiment

except that l4C-guanidino-Larginine was used as radioactivity source, since it has been shown (27, 28) that the guanidino group is not reutiliied in the liver because of its rapid loss during the operation of the urea cycle (cf. Schimke (29) and

200

100

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Total microsomal protein

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2 4 6 8 Days of PB-treatment

FIG. 9. Decay of specific radioactivities of the total proteins of washed microsomes and of the microsomal trypsin digestion residue from normal and phenobarbital (PB)-treated rats. The animals were treated as in Figs. 7 and 8 except that W-guanidino+ arginine monohydrochloride (30 mCi per nq Radiochemical Centre, Amersham, England) was used instead of W-leucine. The raw radioactivity values were corrected as given for Fig. 8.

1 Cyt. b,

.c 0 I I u I I 5 k I Reductase -

ho 0

FlOO

PB-treated 0

0

Days of PB treatment

FIG. 10. Decay of specific radioactivities of cytochrome bb and NADPH-cytochrome c reductase in normal and phenobarbital (PB)-treated rats. Details of the experiments are the same as for Fig. 9; the raw specific activity values were corrected as given in Table I.


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Stephen and Waterlow (30)). Because the specific radioactivities of the various fractions were much less than in the case of 14C-leucine-injected animals, the samples were counted until an error of less than 5% was reached. The results (Figs. 9 and 10) indicate the following. (a) I n normal rats the half-lives of total microsomal protein, total trypsin digestion residue proteins, and the two purified proteins were 20% to 30% shorter than those found when 14C-leucine was used (the difference shows that 14C-guanidino-labeled arginine was indeed less reutilized than was leucine). (b) Despite the shortening of all half-lives in normal animals, the half-life of cytochrome bb was still more than 50% longer than the half-life of the reductase. (c) In phenobarbital-treated animals, degradation of total proteins and of trypsin digestion residue proteins was slowed and, in the case of the two purified enzymes, apparently completely stopped. It must be emphasized that, as in the 14C-leucine experiments already described, the raw specific activity data obtained from

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FIQ. 11. Changes in liver weight, microsomal recovery per g of liver, and cytochrome bs and NADPH-cytochrome c reductase in liver microsomes after cessation of phenobarbital (PB) treatment. Phenobarbital was injected daily as described under “Methods.” In this experiment, the animals were fed a standard laboratory diet (35% protein) beginning with 2 weeks before the initiation of the experiment. All rats were fasted for 24 hours before being killed. The results are given as per cent of the control (non- treated animal) values which were: 3.0 g, wet weight, of liver per 100 g, body weight, 17.7 mg of microsomal protein per g of liver, 0.61 mpmole of cytochrome bs per mg of microsomal protein, and 0.091 unit of reductase per mg of microsomal protein. The difference between the 100% values in this experiment and the one outlined in the legend to Fig. 7 could be explained by a higher protein diet (35’% as compared to 15%) which seemed to give a 2- fold increase in cytochrome bg content of liver microsomes; the difference in the specific activity of the reductase can be accounted for by the use of higher purity cytochrome c in the later experiments (cf. “Methods”). In this experiment also, the livers were not perfused, which could account for the lower liver wet weight and the higher microsomal recovery recorded in this experiment as opposed to that given in the legend to Fig. 7.

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Days after the last injection of PB

FIG. 12. Decay of specific radioactivity of cytochrome bl, NADPH-cytochrome c reductase, and total microsomal protein (washed microsomes) after cessation of phenobarbital (PB) treatment. Twelve 170- to 190-g Sprague-Dawley rats were fed ad lib&m a standard diet (35% protein) (Lab Chow, Ralston Purina Company) and received daily for 5 days 10 mg of phenobarbital per 100 g, body weight, intraperitoneally. On the last day of the phenobarbital treatment, nn-leucine-l-K! (50 &i/100 g, body weight) was injected with phenobarbital. On 1, 3, 5, and 8 days after the last injection, groups of three animals were killed after having been fasted for 24 hours. The livers were removed and pooled and microsomes and purified enzymes were obtained as described.

the phenobarbital-treated animals were corrected for increases in liver weight, microsomal recovery, and microsomal enzyme content. Thus, the data obtained with 14C-guanidino-labeled arginine agree with those obtained with 14C-leucine in showing the apparent lack of degradation of cytochrome bs and the NADPH-cytochrome c reductase in phenobarbital-treated animals.

Turnover of NADPH-ytochrome c Recluctase and @tochrome bs after Cessation of Phenobarbital Treatment-We next looked at the other end of the process of phenobarbital induction, namely at the rapid decrease in enzyme activity toward normal levels after cessation of the treatment. Fig. 11 shows the decrease in liver weight/body weight and of microsomes recovered per g of liver, and also indicates the slight decrease in cytochrome bs per mg of microsome protein and the large decrease in the reductase per mg of microsomal protein, all detected by 24 hours after the last phenobarbital injection and continuing thereafter for the 8 days of the experiment. The decrease in the excess amount of the reductase can be further expressed as a half-time of random degradation, and this value was graphically determined as -45 hours.

On the last day of the phenobarbital treatment, the animals were injected with 14C-leucine, and groups of animals were killed at various time points thereafter. Fig. 12 shows the decay in the specific radioactivity of the protein of washed microsomes and of the purified reductase and cytochrome b6. When these values were corrected for losses in liver weight and microsome recovery (Fig. ll), as detailed above and in the figure legends, the corrected values plotted in Fig. 13 were obtained. It is evident that the loss in radioactivity from reductase is much


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5,000 -

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z zooo- F \ E 0”

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FIG. 13. Decay of corrected specific radioactivity of cytochrome bg, NADPH-cytochrome c reductase, and total microsomal protein (washed microsomes) after cessation of phenobarbital (PB) treatment. For total microsomal proteins, the specific radioactivity values in Fig. 12 were multiplied by the following factors: (a) liver weight of phenobarbital-treated rats/liver weight of normal rats and (b) microsome recovery in phenobarbital- treated rats/microsome recovery in normal rats. For cytochrome bs and NADPH-cytochrome c reductase, the specific radioactivity values in Fig. 12 were corrected as indicated in Table I.

faster than from cytochrome bs or from the mixed proteins of the microsomes. Indeed, the half-life of reductase radioactivity as calculated from the initial points of the curve is -30 hours. When this value is compared to the loss in enzyme activity (equated in this case to enzyme amount), it is clear that although the enzyme is being continuously degraded there is still equally continuous synthesis of new enzyme molecules; degradation is proceeding, however, much more rapidly. Indeed, the fact that the specific radioactivity falls at all is an indication that during this period of net enzyme loss enzyme synthesis is still continuing. It is to be noted again (cf. Figs. 4, 6, and 8, and Table I) that there is a difference in the rates of labeling or of turnover between the reductase and the mixed proteins of washed microsomes.

DISCUSSION

It is clear, from many reported studies, that phenobarbital affects a regulatory mechanism by which the cell controls its content of certain membranes and membrane-bound enzymes. The net effect is a proliferation of smooth surfaced endoplasmic reticulum membranes, and concomitantly a large increase of some, but not all, of the enzymes associated with this type of membrane in the normal cell. Phenobarbital is metabolized by the enzyme system whose increase in amount it induces, and repeated injections of the drug are needed to obtain maximal effects which have been extensively studied (cf. Reference 31). Early effects occurring within the first hours after phenobarbital injection have received less attention. In this regard, electron

microscope studies have yielded little information, for the mag- nitude of membrane proliferation is still too low to allow any quantitative evaluation. Ernster and Orrenius (22) have investigated some of these early changes, such as a binding of phenobarbital to microsomes, and an increase in enzyme activity (oxidative demethylation) in the rough surfaced endoplasmic reticulum. Later on, the same activity rises in smooth surfaced endoplasmic reticulum and, then the phospholipid content and finally the protein content of the microsome fraction begin to increase.

Our data extend some of theirs (22) by showing that an actual increase in enzyme amount accounts for the rapid rise in reductase activity recorded over the first 6 hours after a single phenobarbital injection. Moreover, our chromatographic and im- munochemical studies indicate that the induced activity can be ascribed to an increased production of the same protein species as in controls. Contrary to the results of Ernster and Orrenius (22), however, we found that the increase in cytochrome P-450 content lags noticeably behind the production of reductase (Fig. 5). The situation is reminiscent of that encountered in normal hepatocyte differentiation (1, 2).

Our findings also show that an even earlier event than the increase in rate of reductase synthesis is an increase in the rate of incorporation of amino acid into total microsomal membrane protein, which becomes maximal at 3 to 4 hours after a phenobarbital injection (Fig. 6). These data refer to a KCl-EDTA- washed microsomal fraction which has lost during preparatory washings ~50%, -5O%, -2O%, of its original protein, RNA, and phospholipid, respectively, and appears to consist primarily of microsomal membranes with little content and about half of their ribosomes. Similar results have been obtained, moreover, with the trypsin digestion residue of washed microsomes which is known to retain practically no ribosomal RNA (3). Hence, the increase in the rate of protein synthesis recorded in the microsomal fraction may reflect the laying down of a “primary membrane” (possibly not specific), which precedes the synthesis and deposition thereon of phenobarbital-induced enzymes.

The results obtained in studying protein synthesis and turnover during induction by repeated phenobarbital injections indicate that the increase in reductase amount is achieved initially by a stepping up of the rate of synthesis (Figs. 4 to 6) and later on in the process by a marked reduction in the rate of reductase degradation. An increase in the rate of reductase synthesis has also been recently reported by Schimke et al. (32). As far as degradation is concerned, our results confirm those already published by Jick and Schuster (24) ; in addition, they show clearly an increase in the rate of reductase synthesis in early induction, and extend the over-all findings to the mixed proteins of the microsomal membranes; they also indicate that the increase in microsomal cytochrome 55 content is due primarily to a reduction of its rate of degradation. The phenobarbital effect on reductase synthesis is in general agreement with earlier findings which im- plicated protein synthesis in drug induction. It was shown, for instance, that puromycin administration inhibits the phenobarbital-induced increase in enzyme activity (6, 13), and that amino acid incorporation into microsomal protein in o&o is increased by prior administration in viva of phenobarbital to rats (33). The necessity for increased mRNA production is indicated by the finding that phenobarbital injections caused a stimulation of RNA polymerase activity of rat liver nuclei (34), and that simultaneous injection of actinomycin D and phenobarbital


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prevents the induced increase of enzyme activity (13). How- ever, the initial effect of phenobarbital treatment remains still unknown.

During the second phase (involution) of the induction process, the progressive reduction in enzyme amount is again achieved by altering concomitantly the rates of synthesis and degradation of the enzyme; the first is drastically reduced, but not completely curtailed, while the latter is markedly stepped up. Comparable regulatory situations have already been encountered in a num- ber of cases. For instance, evidence of concomitant increase in synthesis and decrease in degradation of hepatic microsomal phospholipids during phenobarbital induction was recently published by Orrenius (35) and a reciprocal adjustment, i.e. decrease in synthesis and increase in degradation, was observed by Hirsch and Hiatt (36) for ribosomal RNAs and proteins during fasting. Regulation of soluble enzymes has been studied by Schimke (29) for liver arginase, by Schimke, Sweeney, and Berlin (37) for tryptophan pyrrolase, and by Segal and Kim (38) for glutamic- alanine transaminase. An over-all review of this topic has been published by Schimke (39).

In the steady state condition of the fully differentiated hepatocyte of the adult animal, the rates of synthesis and degradation of a protein must be strictly related to one another, such that the cell responds to increased synthesis by increasing degradation and vice versa; a constant concentration of enzyme is thus maintained in the cell. In the case of the reductase, this relationship is such that an enzyme molecule taken at random has a half-life of from 3 to 3.5 days (3) or 2.5 days when leucine or guanidino- labeled arginine, respectively, is the radioactive precursor. The corresponding values for cytochrome bs are 5 days (3) and 4 to 4.5 days, respectively. Phenobarbital disturbs this relationship, initially by increasing the rate of synthesis of the reductase and secondarily by decreasing the rate of degradation of the reductase and of cytochrome bg. While a sizable amount of information on protein synthesis is available, little is known concerning its regulation in eukaryotic cells; moreover, information regarding its regulated corollary, protein degradation, is practically nil. The close relationship between synthesis and degradation has been amply confirmed by the finding that inhibited states of one affect the other in mammalian systems (37, 40, 41) and in bacteria (40, 42, 43).

In interpreting our results, we have assumed that there is no extensive reutilization of labeled amino acids made available by the degradation of the radioactive proteins whose turnover rates we have been studying. It can be argued, however, that phenobarbital causes an increase in reutilization of these amino acids (on account of increased rate of protein synthesis), and that our results could be explained by an extremely efficient reutilization of label, rather than by a reduced rate of degradation of radioactive proteins.

We favor the last alternative because (a) the phenobarbital effect has been studied over long intervals during which the livers of the treated animals were subjected to a constant infusion of dietary amino acids; (b) radioactivity was equally conserved in the reductase and in cytochrome bb even though their rates of synthesis, hence possibility of reutilization of the label, were greatly different; (c) the amount of reductase remained at the same high level (Fig. 5) when the rate of synthesis of the enzyme began to decrease (Fig. 6), a situation best explained by assuming that the enzyme amount is maintained by decreasing the rate of degradation in the face of a decreasing rate of synthesis; and (d)

the results of the experiments (Figs. 9 and 10) with the less re- utilizable guanidino-labeled arginine fully supported those obtained with leucine. Although the half-lives of the two enzymes in control animals were 20% to 30% shorter with arginine than with leucine, it still appeared as if in phenobarbital-treated animals there was no degradation of the enzymes during that period in which their actual amounts increased in the cell. Our results are in apparent disagreement with those of Schimke et al. (32) who, by using guanidino-labeled arginine, found no apparent difference in the degradation rate of total microsomal proteins between phenobarbital-treated and normal animals. The total microsomal protein fraction is, however, a complex mixture of proteins, only a small percentage of which is increased in amount by phenobarbital induction (cj. Reference 22); hence, in drug induction, the turnover rate of the total microsomal proteins is expected to reflect to a much greater extent the condition of noninduced (Figs. 8 and 9) rather than that of induced proteins, such as NADPH-cytochrome c reductase, and cytochrome P-450, or even cytochrome bs (Table I and Fig. 1O).4 The small increase in the amount of the latter (Fig. 7) seems to be due solely to an apparent cessation of its degradation, while the large increase in reductase amount (Fig. 7) results from an increase in synthetic rate (Fig. 6) as well as an apparent cessation of degradation (Table I and Fig. 10). We admit, however, that even with guanidino-labeled arginine limited reutilization is not ex- cluded; hence we consider that our findings strongly support a reduction in the rate of degradation of enzymes during the induction process, for which our data give maximal, but not neces- sarily actual, values.

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4 In this respect, it may be of interest to point out that Schimke et al. (32) obtained results similar to ours when they studied the nutritional regulation of a single protein, arginase, isolated and purified from liver cell sap. A low protein (casein) diet raised the concentration of the enzyme by concomitant increase in its rate of synthesis and decrease in its rate of degradation, whereas a high protein diet decreased the enzyme content by the reciprocal adjustment of these rates.


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Y. Kuriyama, Tsuneo Omura, P. Siekevitz and G. E. PaladeComponents of Rat Liver Microsomal Membranes

Effects of Phenobarbital on the Synthesis and Degradation of the Protein

1969, 244:2017-2026.J. Biol. Chem.

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