Effect of treatment with phenobarbital on rates of synthesis of rat liver nuclear and cytoplasmic proteins

(;on. Pharmac. I.,1. 9. pp 315 to 320 ~'. Pcrya.loJr Press l.td 1978 Printed 01 Great Brttam

0306-3623,'78/1001-0315502.00/0

E F F E C T O F T R E A T M E N T WITH P H E N O B A R B I T A L O N RATES OF SYNTHESIS O F RAT LIVER NUCLEAR AND CYTOPLASMIC PROTEINS MAHMOUD DEWAIR and HEINRICII MATTHAEI Department of Molecular Genetics, Max-Planck-lnstitute for Experimental Medicine, Hermann-Rein-Str. 3, D-3400 Gfttingen, W. Germany (Receired 13 February 1978)

Abstract--I. Double label technique and polyacrylamide gel electrophoresis have been used to study the effect of one and two injections of phenylbarbiturate (barbiturate) on rates of synthesis of cytoplasmic and nuclear proteins in rat liver. 2. Within 2 hr following one injection with barbiturate, increases in rates of synthesis of cytoplasmic proteins of molecular weights about 68, 50-55 and 35,000, respectively, were observed. Stimulation of synthesis of low molecular weight nonhistone chromatin proteins is also observed within this period. 3. The overall rates of synthesis of cytoplasmic and nuclear proteins seems to return to normal level 24 hr after one injection of barbiturate. A stimulation in rates of synthesis of some chromatin proteins was, however, first observed after 24 hr following the first barbiturate injection. 4. After 24 hr following a second barbiturate injection made 24 hr after the first one, syntheses of proteins of similar molecular weights were found to have been stimulated again.

INTRODUCTION

More than 200 chemical c o m p o u n d s are known to induce the in eiro synthesis of the monooxygcnases as well as other components of rat liver microsomes. These c o m p o u n d s are classified into two types (Conney, 1967). Phenobarbital and 3-methyl-cholanthrene are most oftenly studied as representatives of type I and type II, respectively (Conney, 1967). Treatment with phenobarbital stimulates the synthesis of tyrochrome P-450 in rat liver (Orrenius et al., 1969; Ryan et al., 1975; Haugen et al., 1976) and in primary cultures of hepatocytes (Michalop et al., 1976), N A D P H generating enzymes in rat liver (Kaufmann et al., 1976), UDP-glucuronyl transferase in oro ~Wishart & Dutton, 1975), and egasin in mouse liver (Owerbach & Lusis, 1976). Studies on the mechanism of enzyme inductions by barbiturate and by other inducers suggest that the induction process involves the stimulation of protein synthesis (Conney, 1967; Rudden & Rainey, 1970; Shenoy & Peraino, 1976) as well as RNA synthesis and modification (Smith et al., 1976). These studies and others suggest that the induction process takes place as a result of gene activation. O n the other hand, strong evidence has been accumulated, indicating the important role of chromatin proteins in the regulation of gene activity (Stein & Kleinsmith, 1975) changes in rates of synthesis of nuclear and chromatin proteins have been reported to occur after induction in animal cells (Rudden & Rainey, 1970; Augenlicht et tll., 1975). Studies on changes in rates of synthesis of nuclear and cytoplasmic proteins following the administration of phenobarbital may help to understand the relationAbbreviations: SDS, sodium dodecyl sulfate. PMSF, Phenylmethansulfonylfluoride. EDTA-Na2, The disodium salt of ethylene diamine tetra acetic acid. 315

ship between the turnover rates of different cellular proteins and the process of enzyme induction. METHODS Two of four brother Sprague-- Dawley rats of about 180 g body weight were used in each of the five experiments described here (Table I). 1.5 ml containing the radioactive amino acid or sodium phenylbarbiturate in 140 mM NaC1 or just this salt solution were injected. In experiment No. 1 (Exp. 1, Table I), one of two brother rats received 3.5 mCi [3H]L-leucine and the other one received 500pCi [~4C]L-leucine. In experiment No. 2 (Exp. 2, Table 1) one of two brother rats received 5 mCi [3H]L-leucine and the other rat received 500#Ci [~'*C]L-leucine. In experiment No. 3 (Exp. 3, Table 1) each of two of four brother rats received 100 mg,'kg body weight of sodium phenylbarbiturate and 30 min later 5 mCi of [SH]L-leucine, whereas each of the other two rats was injected with 140 mM NaCI solution and 30min later with 500/zCi [t4C]L-leucine. In experiment No. 4 (Exp. 4, Table 1), injections with the radioactive amino acid followed 24 hr after the barbiturate and saline injections. For experiment No. 5 (Exp. 5, Table 1), each of the four brother rats received a second injection with barbiturate (50mg/kg body weight) or saline 24hr after the first injection, and 24 hr later (48 hr following the first barbiturate injection) rats were injected with the radioactive amino acid solutions. In each of the five experiments rats were killed 90 min after injection with the radioactive amino acids, livers were washed separately, combined and homogenized together. Injections were all done intraperitonally, and at the same time of the day. The radioactive leucine had specific activities of about 60Ci/mMole for 3H and about 300 mCi/mMole for 1'*C. Isolation of nuclei All procedures were done at 4"C. Combined livers were cut into pieces, suspended in 50ml/liver of medium-I (250mM sucrose~ 50raM Tris-HCl, 25mM KCI, 5 m M MgCI2, 0.5% Triton X-100, final pH = 7.4). After homogenization with a glass teflon homogenizer, the homogenate was filtered through cheese cloth and centrifuged

316

M A I t M O t t) DI;WAIR and t|l{IXRI< It MAI l i l k l l

Table I. ~t1 >~(" ratios in different protein f r i l c t i t m s Protein fract ion (" NS IA ItA

15xp. 1

Exp. 2

3.4 3.23 3,35 3.31

5111 4+95 5.00 4.9~

at 8(10 g for l I ) m m The supernatant ,aas kept as the postnuclear homogenate. The nuclear pellet v, as suspended once more m medium-I and centrifuged as above. Washed nuclei were purified further by homogenization m medium-1, addition of 2 ,.ohimes of medium-2 (2..3 sucrose. 5 0 m M Tris ttCI. 2 5 m M K('I. 5 r a m Mg('l,. 0.5",, Triton X-IO0, pH = 7.4). stirring for Itlmin+ and centrifugation at 2().()CX)g for 3 0 m m Nuclei ~ere linally washed once more in medium-l. Examination of the so prepared nuclei using phase contrast microscopy re\ealed round intact nuclei free of cytoplasmic debris.

Fractiomttion o/ mwh'ur pr~lei#i,s Nuclei ,acre washed once by homogenization in 75 m M Na('l, 211 m M EDTA-N:I,. stirring and centrifugation. The supernatant v, as discarded, l'he washed nuclei were extracted t~ice with 140mM NaCI. II/mM Tris I1('1 pll 7.4 and once with 10raM l'ris HCI pH 7.4. The soluble material extracted with these t ~ o solutions \vas combined as the nuclear sup (NSI fraction: this was dialyzed against water, lyophilized, dissohed in 2". SDS. 65 m M Tris It('1 pH 6.8. dialyzed against this solution and kept at - 20 ('. The insoluble material remaining after extraction of the NS proteins is the c h r o m a t m fraction. Chromatin proteins were fractionated into t,ao fractions by a dissociation of chromatin, followed by a one step-recon.stitution, mainl) as described by Patel & T h o m a s 119731. The chromatin was homogenized briett.,, in IOrnM Tris ItCI pH 7.4 at a concentration of about 1.5 mg DNA per ml: thereupon 2',ol containing 3 M NaCI. 7.5M Urea. 1 0 m M l'ris HCI pH 7.4 v, ere added under stirring at 4 C. The mixture was stirred further for 6 hr. The resulting clear ,,iscous solution was dialyzed twice, for 12 and 8 hr. each time against 2 0 v o l o f l 4 0 m M NaCI. 10raM Tris It('1 pll 7.4 and then centrifuged at 20.000 0 for 30 mm. ] h e supernatant contained what v+e call lo\,. affinity ILA) proteins, and the pellet contained a complex of ,ahat v.c call high altinity {HA) proteins and DNA. or t t A P - D N A complex. The I,A proteins were dialyzed against '~.atcr, lyophilizcd, dissolved in 2". SDS. 65 m M Tris H(+I pH 6.8, dialyzed against the same solution and kept at 20 ('. The pellet containing the H A P - I ) N A complex was homogenized in 2"., SDS. 1 0 m M Tris HCI ptt 8 at a concentraticm of about I m g DNA per ml. stirred at 30 (" for lShr. centrifuged tit 200,000g for 24 hr at 20 C to pellet the I)NA. The supernatant contained wh,it ~c call the high affinity iliA) proteins: these were precipitated ~ith 9vol of acetone at - 1 5 C, 24 hr. collected by centrifugation, dried, dissol',ed in 2".. SDS. 65 m M Tris HCI pH 6.8. dialyzed against the same solution and kept at • 20 C. The cytoplasmic soluble protein fraction, i.e. the post nuclear supernatant, was centrifuged otlce at 20,000 g for 2 0 m m . and the supcrnatant was centrifuged once more at 36,(RR) 0 for I h r : the resulting supernatant was used to prepare the cytoplasmic proiem fraction studied here. This fraction ~,as extracted tv, ice with chloroform-isoamyl alcohol 13:11 to remove lipids, wtlereby the denatured proteins were precipitated at and collected from the interface between the orgamc and aquous phases; they were washed with 96% ethanol ether 11:1 k ether, dried, dissolved in 2". SDS. 65 m M Tris tlCI pil 6.8. dialy:,'cd against the same

311 i+(- ratio Exp. 3 ~.17

5.s 5.3 6.11

Exp. 4 5.~7 5.56 5.4 5.6

I-xp. 5 77

7.13 s2 "7.7

solution and kept at - 2 0 ('. All solutions used contained I m M PMSF. For pol)acryhtmide gel electrophoresis the method of Laemmli 'aas used (l.aemrnli. 197(1}. f o r radioactivity counting the pol)acrylamide gels gerc cut into 1.7 m m slices. For drying the slices were left in glass counting ,,ials for about 10hr: thereafter I).Sml ol 30",, hydrogen peroxide was added to each slice, the +lals were tightly closed, incubated at 60 (" for 12 hr and cooled to room temperature: 15ml of Dimilume-30 were than added. Counting ~sas done in a "Iri-Carb 3375 spectrometer {Packard Instrument lnternationall. The external standard ratio method v, as used to correct for quenching when necessar',. Onl.~ gel slices v, hich ease at least 2.5 limes :is many counts as the background ,,',ere used for calculation of ~1I ia(. cpm ratios. Determination o! .~pecilw radiouclil itt

Aliquots of the protein solutions m the electrophorcsis buffer ,acre made 10",, with ~flchloroacetic acid. boiled for 10rain and left at 15 (" m e r n i g h t : the precipitated prteins were collected b~. centrifugation, dried, dissoked in I N N a O t l at 37 (" and di',ided into two paris. O n e part was used for protein estimation according to Lowry ILowry et al.. 1951). the other part for radioactivity counting. .%latermls

l h e radioactive amino acids from Amersham-lSuchler. Braunschweig. (iermany. Sodiumphenylbarbiturate and PMSF from E. Merck, Darmstadt. Germany. Acrylamide. N. N'-methylenbisacrylamide and S D S from BiochemicaService-lhesheim. German',. and the l)imuhime-30 IYom Packard Instrument International. R E ~ t I .'1",%

F r a c t i o n a t i o n of n u c l e a r p r o t e i n s : T h e n u c l e a r s a p INS) p r o t e i n s are t h o s e p r o t e i n s o b t a i n e d by extracting the nuclei with 1 4 0 m M NaCI. 1 0 m M T r i s HCI p H 7.4. R e m a i n i n g after this e x t r a c t i o n , t h e c h r o m a t i n fraction c o n t a i n e d p r o t e i n s a n d D N A in a ratio of a b o u t 2: I. T h e c h r o m a t i n p r o t e i n s were f u r t h e r fract i o n a t e d by d i s s o c i a t i o n a n d o n e step r e c o n s l i t u t i o n , w h e r e b y a b o u t 80°,, of the c h r o m a t i n n o n h i s t o n e proteins were irreversibly d i s s o c i a t e d from t h e c h r o m a t i n a n d collected in a fraction to w h i c h we refer here as t h e low affinity (I.AI proteins. T h e h i s t o n e s plus a b o u t 200<, of t h e n o n h i s t o n e p r o t e i n s r e a s s o c i a t e d with the D N A u n d e r these c o n d i t i o n s were later diss o c i a t e d from the D N A with S D S s o l u t i o n a n d referred to as t h e high affinity (IIA) proteins. F i g u r e 1 s h o w s the S D S - p o l y a c r y l a m i d e gel electrop h e r o g r a m s of c y t o p l a s m i c (C). n u c l e a r s a p {NS). c h r o m a t i n low affinity (LA) a n d c h r o m a t i n high att]nity (HA) p r o t e i n fractions. A p r o t e i n (s) of m o l e c u l a r weight a b o u t 45.000 s e e m s to be p r e s e n t in all t h r e e n u c l e a r p r o t e i n fractions. T h e c y t o p h i s m i c p r o t e i n s s h o w n in Fig. 1. gel "'C'" r e p r e s e n t p r o t e i n s

Effect of treatment with phenobarbital

317

1800

3CO

c

~

400-

T

U

6860.

o

r5o

0 5"if200" 2'.5 I.

% --

'~-

~-"~~

~ . . . . '1',~

/1

il

45. 40.

55

¢ o

21.

E

5 f 390 .4 $

12. NS

LA

HA

I-ig. I. Polyacrylarnidc gel electropherograms of cytoplasmic ((') nuclear sap INS), low affinity (I,A) and high affinity proteins IliA). The lincs to the left mdicatc the molecular ~ei~hts x 10 " obtained from the soluble postribosomal supernatant of the rat li~,er homogenate. Since 0.5". Triton X-100 w,as used during all homogenization steps, it is to bc expected that this fraction contains also proteins solubilized from thc mitochondrial, plasma and microsomal membranes. Incorporation of radioactive leucine into different protein fractions without treatment with barbiturate: In order to be sure that differences in 3H:"4C ratios observed in diffcrcnt proteins after treatment with barbiturate are not artifactual, two control experiments wcrc carried out {Exp. 1 and 2, Table 1). in each of these two experiments one of two brother rats was injected with [3H]L-leucine and the other one with [t4C]t,-leucine. The tw.o livers were combined and proccsscd under the same conditions used later on to study the cffccts of barbiturate treatment. Table 1 shows the ~'lt ~4(, ratios in the different protein fractions from the two control experiments (Exp. 1 and Exp. 21. It can bc noticed that the ratios are T;ible 2. Relatixc specitic radioactivities in the different protein fractions

Protein (? NS LA IIA

.-%o

4

C

I'ract i o n

-LA

Rclativc sp. radioactivity ",," 37 13 26 24

*The relatixc sp. radioactivity is the amount of cpm per mg of the correspondintz protein fraction dixided by the sum of amounts of cpms m 4mg: I mg of each of the four fractions, multiplied by I(X).

£cL 2OO

I00

¢

g 112 ~

(D

112

°o E Ut~ 175

~

E

~

1-

6O

0 u ~r

m

o

l 20

1 40

0

Sltee number Fig. 2. Radioactivity distribution and "~H.'4C ratios in polyacrylamide gels of the cytoplasmic (C), nuclear sap (NS), low affinity (LA), and high affinity proteins from the control experiment I (Exp. I, Table lj. One of two brother rats was injected with [3H]t,-leucine and the other rat with [J'~C]L-leucine, 1.5 hr later rats were killed, livers were combined, homogenized and used for isolation of thc different protein fractions. similar for all four fractions. Figure 2 shows the radioactivity distribution and the 3H:'14C ratios along the polyacry, lamide gels of the four protein fractions studied. The results shown in figure I are those of experiment I, the results of experiment 2 werc very similar, except that the average 3H. 14C seems to be higher for all fractions [about 5 compared to about 3.3 in experiment 1), this is due to the use of a larger amount of [3H]t.-leucine in experiment 2. Relative specific radioactivities of different protein fractions: The relative amounts of ~4C as well as 3H cpm per mg proteins in each of the four protein fractions were calculated from the two control experiments (Exp. I and Exp. 2, Table I). The total radioactivity incorporated into 4 mg proteins. 1 mg of each of thc four fractions was considered as 100, and the amount of cpm:mg of each of the four fractions was

31g

~.'l,klh",l()l'l)

[)1 ~,'.,',,IR and IIt:INRI('II M.x] [ H x l l

calculated relative to this total m percents. The results arc shown in Table 2. It can be sccn that the highest relative specific radioactivity is in the cytoplasmic fraction, whereas the least one is m the nuclear sap (NS) fractions. LA and ttA fractions had almost equal relative specific radioacti~itics, that of the LA being slightly higher. As a matter of fact, the radioactivity incorporetcd into the NS fraction was too little to allow for a reliable estimation of the 311..)'~C ratios in the polyacrylamidc gels, except for a fe~ slices, corrcspondingly in each gel. Effect of treatment v. ith phcn',lbarbiturate on the rate of incorporation of leucine into different protein fractions: Table 2 IExp. 3, Exp. 4 and Exp. 5) shows the 3|i ~4(, ratios for each of the four protein fractions studied. Within the tirst 2 hr after the lirst phcnylbarbituratc injection (l(X)mg kg body v, cight: Exp. 3, l a b l c 1 ) a significant increase in the 3 H ~ 4 C ratio was observed for the cytoplasmic protein fraction as well as for all the three nuclear protein fractions. The 3H. ~'tC ratios after 24 hr following the first barbiturate injection (Exp. 4, Table I1 were generally lower.

Mot.

Wt

x I0 - ~

6,8 60

45 40

I -(o)

t

2~

I

,2

t

,,1_ L 400

tx

iif 5°° :~ . . . . .

~..E~'~a~..~~_~ 6

,~

20O

1250 O. T

'!_

m

i

9

~

9000 ( b ' ,

-%

~. ~ .z~,~ . . ~ , ¢ ~ ¢ ~

%0

. . . . . . , ~ _ ~ . /..

Tii

4 E u

450

O0

? 15

1250

~3

l~l •

.

.

~ I,-

.0200

.

.

.

.

.

.

.

.

.

o

,IOO

0

0

20 Sl~ce

4() number

Fig. 3. Radioactivity distribution and ~H. t4C ratios m polyacrylamide gels of the cytoplasmic protein fractions. a, b and c from experiments 3. 4 and 5, corresponding to treatments with barbiturate for 0.5 hr, 24 hr and 48 hr. respectively.

M o t Wl" x 10-3 (~8 60 45 40

I

ii ~

:o)

1

P,

I I

~2

~

.1

60

i

::f E Q

I:

'

I

15C

3C

o x T

E

E ~

o_ u

T

Y

1

°'

E 125'

~

~

-

i

,i,

'-

;

O

~0

30

0

T 112

t)

r,- 0 t

C '8C

,!i::i

.

.

.

.

.

.

.

20 SI~ce

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

40 nJmber

Fig. 4. Radloacti'..lt'. distribution and "~H L'~C" ratio~, m polyacr)lamidc gels of the nuclear ,,,ap (NS) protein fractions a. b and c from experiments 3. 4 and 5, correspondmg to treatments v. ilh barbituratc for 0.5 hr, 24 hr and 48 hr. rcspecti~ el.'. After 24 hr follo~sing a second barbiturate injection ( 5 0 m g k g body weight: Exp. 5, Table 1) increases in 3H.: ~'~C rat los for all fractions st udied were observed : this increase is signiticant for the LA fraction at least. The patterns of radioactivity distribution and 3H t'sC ratios for the cytoplasmic fractions from Exps 3 . 4 and 5 arc shown in Fig. 3a, b and c respectively. Within the first 2 hr, follnwing the lirst barbiturate in.jection (Exp. 3), an increase in 3H ~'~(," ratio was obscrvcd for proteins o[ molecular s~.eights about 68-70. 50. 55 and 35.(XX). Twcnty-four hr after the tirst barbiturate injection IExp. 4) only a slight increase in 3H 14(, was seen for some proteins of low molecular weights labour Ig,lX)O). Twenty-four hr after a second barbiturate injection ( 5 0 m g kg body weight) and 48 hr after the tirsl injection (Exp. 5), an increase in 3H ~'~C ratios for protein bands of molccular weights 68, 50 55. 35 and Ig.(XX) could bc found. The radioactivity distribution and the 3l-t 14C ratios in polyacrylamide gels of the NS fractions from Exps 3, 4 and 5 arc shown in Fig. 4a, b and c, respectively. The radioactivity incorporated in proteins of this fraction was. howe~,er, tot) low to alloy, for a reliable estimation of 3H. 14C ratios, except in few gel

319

Effect of treatment with phenobarbital Mot..Wt.

x IO - 3

68~o

45 4 o

2~

~2

20 0

7.5 f 800' 6.5 5.5 E

o.

tO 400

T

tO0

-

E

bands of molecular weights about 38, 30 and 16,000 within the first 2 hr after the first barbiturate injection (Fig. 6a). After 24 hr following the first barbiturate injection, the 3H/14C ratios were almost the same for all protein bands in this fraction, within __+ls from the average value (Fig. 6b). After 24 hr following the second barbiturate injection, there were increases in 3H..q'*C ratios for almost all proteins in this fraction. especially for proteins of molecular weights about 45 50, 40, 30 and 12-20,000.

o "T m

I) I S C I : S S I O N (b)

l;o

~'

G

4 300

'

~

u

g !

~2

t ° 750

"(c)

a

a

A set of preliminary experiments similar to the experiments described here was previously performed (unpublished). In those experiments smaller amounts of radioactive amino acids were used (2 mCi [3H]Lleucine or 200,uCi [~'*C]L-leucine per rat). The radioactivity incorporated was too little, therefore, to allow reliable estimations of the 3H/14C ratios in most of the protein bands in the polyacrylamide gels. Those bands, however, which contained the largest amounts of radioactivity showed 3H 14C ratios similar to those

].°0 i

,o~

375

Mol. Wt.

xlO -3

68 60

45 40

"(o)

r

~ , , ~,~,~

5687f 700' _

_

_~,__- ' ~ , ~ _ , ~ , ~ %

,2

21 a

180

,

........

T 9O

E .300 20 Shce

40 number

0

u

E u t.)

Fig. 5. Radioactivity distribution and 3H. t4C ratios in polyacrylamide gels of the chromatin low allinity [LA) protein fractions a. b and c from experiments 3, 4 and 5, corresponding to treatments with barbiturates for 0.5 hr, 24 hr and 48 hr, respectively. slices. Increases in 3H. 14C ratios could be. however, observed in a protein band of molecular weight of about 100,000 within the first 2 hr after the first barbiturate injection (Fig. 4a) and for a band of molecular weight 45,000 after 24 hr following the first barbiturate injection (Fig. 4b). As to the LA protein fractions, the radioactivity distribution and the 3H,,'t4C ratios from Exps 3, 4 and 5 are shown in Fig. 5a, b and c, respectively. Increases in 3tl:t'~C ratios for low molecular weight proteins of molecular weights 30,000 and 14,000 were observed within the first 2 hr after the tirst barbiturate injection (Fig. 5a). A significant increase in the 3H,14C ratio was to be observed for a protein band of molecular weight 45,000 after 24 hr following the first barbiturate injection (Fig. 5b). After 24 hr following the second barbiturate injection an overall increase in the 31-1."14C ratio was observed for the whole LA fraction and particularly for proteins of molecular weights about 30,(XX)' and 50,000. For HA fractions, the radioactivity and 3 H : ~ C ratios from Exps 3, 4 and 5 are shown in Fig. 6a, b and c, respectively. The HA protein fraction showed significant increases in 3H,,'~'*C ratios for protein

,°20 .~,

E Q. 6O

250

E t.) ,:r

I

C)

z~

-

~o

l0 9

8F 65

400

ge

0

E

E

o. t)

112 '~ 0

20 Shoe

40

O

number

Fig. 6. Radioactivity distribution and 3H,"~4C ratios in polyacrylamide gels of the chromatin high affinity (HA) proteins fractions a, b and c from experiments 3, 4 and 5, corresponding to treatments with barbiturate for 0.5 hr, 24 hr and 48 hr. respectively.

MAIIM(iUI) [)I!WMR and HI.I\RI( II MAI l l l J l l

3211

of the corresponding bands in the experiments reported here. l v , o independent control experiments v, crc performed {Exp. I and Exp. 2, Tablc 1 and Fig. 2) in order to determine lhe limits of accuracy of the double htbcl technique as uscd hcrc and in similar studies. The 3H laC ratios were practically' equal in all of the four total protein fractions studied: they depended only on the amounts of radioactive amino acid used ITablc 11. Furthermore, all prolein bands in one gel had similar 3H ~'~(" ratios, within +_ Is from the axeragc ~alue. In the barbiturate experiments. therefl/re. 311 ~aC ratios at least 2 +_s-~.alues higher or Im~er than the a,,erage ratio in the gel could be considered to indicate significant changes in the rate o f a m i n o acid incorporation. Positive changes in rates of amino acid incorporation occurring within 2 hi aftcr a first barbiturate rejection may be particularly interesting, in the cytoplasmic fraction, significanl stimulation of amino acid incorporation is seen in proteins of molecular weight around 68JX)0. this may be rat liver albumin, as well as m proteins of molecular weights 50--55,(XXL which may be cytochromes P-450 (Haugen et al., 1976). In tim c h r o m a t m fraction, increased 3H i'tC ratios were obscr'~cd for Imv molecuhir weight proteins of both lhc low and high attinity proteins. Stimulation of syntheses of chromatin proteins has been observed in rat liver after treatment with barbiturate [Rudden & Raincy. 1970). and in mouse salivary glands after injection with isoproterenol (Stein & Baserga, 19701. Another observation about changes in rates of amino acid incorporation concerns a protein(sl of molecuhir weight about 45,000, whose rate of s y n thesis seems to be stimulated only 24 hr after one barbiturate injection. The overall rates of synthesis of rat liver proteins studied here are significantly lower 24 hr after one barbiturate injection compared to those observed within the first 2hr. Twenty-four hours following a second barbiturate injection, an overall and more general stimuhttion of radioactive a m i n o acid incorporation seems to take place. In general the rates of protein synthesis seem to return to a normal level after 24 hr lollowing one, barbiturate injection, except for few c h r o m a t i n proteins, whose rates of s,,nthcsis are still significantly higher at this time. ()no of these proteins has a molecular weight of about 45,000: it seems to bc found in all nuclear protein fracticms studied, especially, however, in the low altinit3 protein fraction. The solubility' of this nonhistonc protein in 0.14 M NaCI makes it suitable for isohltion and further characterization. We wish to thank Mr J. Bormann for his ad,,ice on tile exaluation of data. .lchnouh'dllt'menl

R I.'FER E'N('ES

At'~iFXtl{lll L. II.. BII:SMaN H. & RAJtiWSKY J. 11975) ("hromosomal proteins of rat brain: Increased synthesis and aflhlit,, t~ar I)NA foiler,rag a pulse of tile carcinogen cthylnitrosourca in vii'<,. J. Cell. Physiol. 86, 431 438. ('OXXlV A. tl. (1967) Pharmacological implication of microsomal enzyme induction. Pharmac. Rec. 19, 317 366

H.xt'(;FX l). A.. Coo', M. J. & Ni:uFi~i I). ~'. 11976i Indu<.lion of multiple fornls of mour, e li,,er cytochronlc P-450. E\idence for genetical]', controlled de no,.o protein ~,',uthesis in response to treatment 'a.lth fl-naphtholta,.one or phenobarbital. ,l. hlol. ('hem. 251, II',;17 IS27 KAI'FMANN | . (i.. [-),ANS R. K. & "]llt RM.\N R. (i i19761 Induction of hepatic H A l ) P i t - g e n e r a t i n g ellZ',.nlcs b} phcllOhitrhllal IFcatmClll. /'l'lhl /Iro(. I-edn t01..'3"ol 5 i'\p. BioL 35, 1709 1709. K A t e R , JoNl~:iR! W. R , l.ol.B L A.. I'll', ] . & (.il.l.B~il'-, II. V. t l966l Studic~ on the ineehanisn/ of drug induced microsonla] ¢n2")111c activities. \.'. Phenobarbital snmu]ation of cndogenotis illessellger R NA and pol) urid)]lc acid-directed I-[ I'l('lphen)lalanine incorporation. Moh't. I)hormal. 2, 171 Ig6. LAF,MMII I I K. (19?(it (.'lCalag¢ of struclura[ proteins during the assen'lbl) of the head of bacteriophage T4. .~,cllllrt'. 227. 680 685.

LoWR', O. t|.. Rt)SI!\BROt'.(;II N. J.. I"~RR A. L. & R-~,NI).~.II R. J. 119511 Protein measurement ,~ilh the folm phcnoI reagerlt..I, hi(ft. ('hem. 193, 265-275. MI( IIAl.(li > (i.. S a r t i i R ('. A . S A I I I I R (i. 1.. & Plll)l II. 11976) { ' y l o d m ~ m e P-4511 induction h) phenobarbital lind 3-melhvlcholanthrenc m primar', cullure of hepalocytes..~,'~ il'm l' 193, 907 9l)g. [MiIIA.E 'f.. Lt.;ASA(II %,'.. I)ARR.t(Q S. t~ Rill SSl:..xU f1975) Opposite tit"cots of phcnoharhilal prelrealrllcnl till allatoxin I~Jl-induccd inhibition of transcription m ral add rl-iOtlSe Ii~cr. tlio< hem. Phur/llll(. 24, 1851 IX54. ORRI;NII!S ,~.. F.RI(NSON J. L. I-. & ERNSII.R I,. (19691 Phenobarbital induced synthesis of the mlcrosomal drug-metabolizing e n l ) n l o s)stonl alld its relationship to

the prolil;eration of endopl:ismic membranes. A morphological arid bio¢l'lcmical stud.,,. J. ('ell BiM. 25, 627 639. O,ai!Rlt..xctl I). & l.l:SlS A. J 11976) Phenobarbital induelion of egas)n: A~ailabilit~ of cgasyn in tit,~ determines giucuronidasc binding to nlembraries. Biocheol hiophl.g. Rex. ('ommun. 69, 62X 634.

PATH G. I.. & lltoXIAS T. 1.. il973i Some binding parametcrs of ¢hromatm acidic proteins v, ith high alt}nltv fi>r deoxyribonucleic acid. Proc h a t h . . l e a , / Sci ['.5..-t. 70. 2524 252g. RI:I)I)IN R W. & RMNt.', ( ' H. 1197Oi S'tlmulation of nuclear proimn synthesis m r a l liver after phenobarbital administration, lTiochcm, hinphy~. Res. (',,mmun. 40, 152 16(i. R'f,,,N I).. l.i A. Y. II.. W i s l S. & l.i',i~ W. 119751 Multiple

forms of cytochrome P-450 m phenobarbital and 3-meth)lbohmthrene-treated rats. Separation and spectral properties. J. biol. ('hem. 25tl, 2157 2163. Stnixo,, S. I . & I"lRAi'.o C. ll976i Ilepatic nucleic acid and protein changes in phenobarbital reduced hepatomegal,,. I"edn Pro,. I'edn 4m. S,,(.s e\p. tli,,l. 35, 1710 1710. SMIIII S. J. I.ix l). K.. I.IIiNARII T. B. I)A(LMAN R. W. VI!SAIi [ . S. 119761 Phenobarbital-induced increases m mcth)laiion of ribosomal prectirsor ribonucleic acid J.lfo/(,l', Phtlrtlltl(. 12, ~20 b;3].

SqI'IN (i. & BASliR(i-% R. 1197t)l l h e synthesis el acidic nuclear proteins m the prercphcati\c phase of the isoprotcrenol-siimulatcd sali\ary gland. J. biol. Chem. 245, 6097 61115. SII;IN (i..~. & Kl I:INSMIIII I.. J. 119751 ('hro,l~soola/ Proleinx and Their Rob' in Rl'quhition ~!I Gem' Expression. Academic Press. New York. "I's'tRI(iW I. B.. Z,XKII.,%R()\A N. [{,, (iRONOVA (). A. & L.','AKti()\I( II V. L 119761 Possible mechanism of indue-

lion of li\er microsomal rnonooxygenascs by phenobarbital. Biochem. hiophv.v Acta 421, 44 56. Wlslbxi,tl (..i.J. & Dt;ilox G. J. 11975) Release by phenobarbital of the repression of UL)F'-glycuronyhransferase acli\ity in ozo. Biochem. t'hormm. 24. 451 454.