265
°Biochimica et Biophysica Acta, 399 (1975) 265--276 © Elsevier Scientific Publishing Company, A m s t e r d a m -- Printed in The Netherlands
BBA 27699
ONTOGENY OF CYCLIC AMP-DEPENDENT PROTEIN PHOSPHOKINASE DURING HEPATIC DEVELOPMENT OF THE RAT
P.C. LEE and RICHARD A. J U N G M A N N
Department of Biochemistry, Northwestern University Medical School, Chicago, Ill. 60611
(U.S.A.)
(Received February 18th, 1975)
Summary The ontogeny of protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) and cyclic AMP-binding activity in subcellular fractions of liver was examined during prenatal and postnatal development of the male rat. 1. Protein kinase activity and cyclic AMP-binding activity were found in the nuclear, microsomal, lysosomal-mitochondrial, and soluble liver fractions. 2. The protein kinase activity of the soluble (105 000 X g SUlSernatant) fraction measured with histone F1 as substrate was stimulated by cyclic AMP. Cyclic AMP did not stimulate the protein kinase activity of the particulate fractions. 3. The protein kinase activity of all subcellular fractions increased rapidly from the activity observed in prenatal liver (3--4 days before birth) to reach maximal activity in 2-day-old rats. Thereafter, the protein kinase activity declined more slowly and regained the prenatal levels at 10 days after birth. 4. Considerable latent protein kinase activity was associated with liver microsomal fractions which could be activated by treatment of microsomes with Triton X-100. The latent microsomal protein kinase activity was highest in prenatal liver, at the time of birth, and 2 days after birth. During the subsequent postnatal development the latent microsomal protein kinase activity gradually declined to insignificantly low levels. " 5. During the developmental period examined (4 days before birth to age 60--90 days) marked alterations of the cyclic AMP-binding activity were determined in all subcellular fractions of rat liver. In general, cytosol, microsomal, and lysosomal-mitochondrial cyclic AMP-binding activity was highest in 10--11 day-old rats. Nuclear cyclic AMP-binding activity was highest 3--4 days before birth and declined at birth and during the postnatal period. There was no correlation between the developmental alteration of cyclic AMP-binding activity and cyclic AMP dependency of the protein kinase activity in any of the subceUular fractions. This suggests that the measured cyclic AMP-binding activi-
266 ty does not reflect developmental alterations of the cyclic AMP-binding regulatory subunit of cyclic AMP-dependent protein kinase.
Introduction Alterations of the activities of certain enzymes in vertebrate livers are known to form an integral part of the developmental process [1,2]. Cyclic AMP-dependent protein phosphokinase has been recognized in recent years to constitute a key hepatic enzyme mediating the phosphorylative and functional modification of a number of liver proteins [3]. Cyclic AMP (adenosine 3',5'monophosphate) is known to regulate the activity of the protein kinase in response to hormonal stimulation. A recent report by Christoffersen et al. [4] has demonstrated that hepatic enzymes involved in the metabolism of cyclic AMP undergo ontogenetic changes during the prenatal and early postnatal age of the rat. In addition, developmental changes of the activity of cyclic AMPdependent protein kinases coincide and correlate with changes of the hepatic glycogen metabolism in the rat [ 5]. In view of the importance of the cyclic AMP-dependent protein kinase system in mediating the hormonal control of hepatic function, we have examined the subcellular distribution and certain properties of hepatic protein kinase and cyclic AMP-binding activities at distinct stages of prenatal and postnatal development of the rat. Materials and Methods
Chemicals and animals. All biochemicals reagents were obtained from Sigma Chemical Co., St. Louis, Mo. [7 -32p] Adenosine 5'-triphosphate, ammonium salt (77.7 Ci/mmol), and [8-3H]adenosine 3',5'-monophosphate (27.5 Ci/mmol) were purchased from I.C.N. Isotope and Nuclear Division, Irvine, Calif., and Amersham Searle Corp., Arlington Heights, Ill. Male rats and pregnant rats at various times of gestation were obtained from Holtzman Co., Madison, Wisc. Preparation o f liver homogenate. Fetuses of both sexes were delivered by Caesarean section. Newborn and adult rats were sacrified by decapitation. The liver of rats older than 10 days was profused with a solution of 0.14 M NaC1/10 mM sodium citrate to rid of excess blood. Excised and profused livers were placed in 2vol. of ice-cold 0.01 M Tris buffer, pH 7.4, containing 0.25 M sucrose, 4 mM CaCl2, 5 mM MgC12 and homogenized in a Dounce homogenizer. The homogenate was filtered through gauze and again homogenized in a glass-to-glass Dual tissue grinder (Kontes, Vineland, N.J.). The resulting homogenate was used for the isolation of the subcellular fractions which were prepared using procedures described by Fleischer and Kervina [6]. The purity of the subcellular fractions and the degree of cross-contamination were judged according to the criteria given by Fleischer and Kervina [6]. Applying these criteria only a slight degree of cross-contamination was observed. Preparation of nuclei. Nuclei were prepared by the method of Widnell and Tata [7]. Accordingly, the homogenate was centrifuged at 800 × g for 15 min. The crude nuclear pellet was resuspended in 0.01 M Tris buffer, pH 7.4, con-
267 taining 2.3 M sucrose/5 mM MgC12 and sedimented by centrifugation at 105 000 X g for 60 min. The nuclei were again suspended in 0.01 M Tris buffer, pH 7.4, containing 2.3 M sucrose/5 mM MgC12 and sedimented by centrifugation at 105 000 X g for 60 min. The resulting purified nuclear pellet was suspended by homogenization with a glass-to-glass Potter-Elvehjem tissue grinder in 0.01 M potassium phosphate buffer (pH 7.0) to yield the nuclear fraction. Microscopic examination of these purified nuclei stained with hematoxylin and eosin showed no or relatively little contamination with cytoplasmic particles. The protein to DNA ratio of purified nuclei was 2.5 and did not change markedly with age of the rat. Comparison of the DNA content of the homogenate with that present in isolated nuclei revealed a routine recovery of 45--50%. The recovery of nuclei from the homogenate did not change with the age of the rat. Preparation of lysosomes-mitochondria. The 800 X g supernatant fraction was centrifuged at 13 000 X g for 15 min to yield the 13 000 X g pellet which was resuspended in 0.01 M Tris, pH 7.4, containing 0.25 M sucrose and sedimented by centrifugation at 13 000 X g for 15 min. The resulting 13 000 X g pellet was suspended by homogenization with a glass-to-glass Potter-Elvehjem tissue grinder in 0.01 M potassium phosphate buffer (pH 7.0), and designated lysosomal-mitochondrial fraction. The recovery of this fraction from the homogenate was routinely about 60% based on the recovery of succinate-cytochrome c reductase from the homogenate [6]. The percent recovery did not change with the developmental stage of the rat. Preparation of microsomes and cytosol. The 13 000 X g supernatant fraction was centrifuged at 105 000 X g for 60 min. The resulting supernatant, designated cytosol, was used for further experimentation. To achieve further purification the 105 000 X g pellet was suspended in 0.25 M sucrose and layered onto a 6.0 ml cushion of 1 M sucrose/0.01 M potassium phosphate buffer, pH 7.0. Centrifugation was carried out at 105 000 X g for 60 min. The sedimented pellet was homogenized in 0.01 M potassium phosphate buffer and designated microsomal fraction. The microsomal fraction isolated in this way contained both the rough and smooth microsomes. The recovery of this fraction was about 40% based on the recovery of glucose-6-phosphatase activity from the homogenate [6]. The percent recovery did not change with changing age of the rat. Determination of protein phosphokinase activity. The protein kinase assay was carried out as described previously [8] in a total incubation volume of 0.2 ml containing: 8--10 #g of protein to be assayed for protein kinase activity; 100 pg of histone F1; 10 #mol of sodium glycerol phosphate buffer, pH 7.0; 4 nmol of [7 -32p] ATP (0.15 pCi); 2 pmol of magnesium acetate; 2 pmol of NaF; 0.4 pmol of theophylline; 0.2 pmol of dithiothreitol; with or without cyclic AMP as indicated in the text. Incubation was started by the addition of [7_32p] ATP and carried out for 15 min at 37°C. The reaction was terminated by the addition of 2 ml of 20% trichloroacetic acid containing 1% sodium dodecyl sulfate. The samples were filtered using Millipore filters (0.3 pM). The filters were washed four times with 3 ml of 20% trichloroacetic acid/l% sodium dodecyl sulfate, dried, dissolved in 10 ml PCS (Amersham/Searle), and analyzed for radioactivity. Under the experimental conditions incorporation of
268 32 p into substrate protein proceed linearly with respect to incubation time and with increasing concentrations of protein exhibiting kinase activity. Assay o f cyclic AMP-binding activity. Binding of cyclic [ 3H] AMP to protein was determined by membrane filter assay as described previously [8] in a total incubation volume of 0.25 ml containing: 25 pmol of cyclic [ 3H] AMP; 16.5 pmol of Tris buffer, pH 7.4; 1.7 pmol of theophylline; 2.5 pmol of MgCI~ ; and 20--25 pg of protein to be assayed for cyclic AMP-binding activity. Incubation was carried out for 60 min at 2° C. The samples were filtered using Millipore filters (0.3 pm) which had been presoaked in ice-cold 0.25 mM Tris buffer, pH 7.4, containing 10 mM MgC12. Filters were washed five times each with 5 ml of 0.25 mM Tris/10 mM MgCI2, dissolved in 10 ml of PCS, and analyzed for radioactivity. Under the experimental conditions binding of cyclic [3H]AMP to protein was linear with protein concentrations up to 0.1 mg. Under the conditions no marked change of cyclic AMP-binding activity occurred after 60 min incubation. Protein concentration. Protein concentration was determined by the method of Lowry et al. [9] with bovine serum albun~i'n, fraction V, as reference standard. DNA concentration. DNA concentration was determined by the diphenylamine method of Burton [10] with calf thymus DNA as reference standard. Determination of radioactivity. Samples collected on Millipore filters were dissolved in 10 ml of PCS (Amersham/Searle) which was used as the scintillation fluid. All samples were counted in a model 3375 Packard Tri-Carb liquid scintillation spectrometer as previously reported [8]. Results It appears that protein phosphokinases are highly compartmentalized and found in close proximity to their phosphoprotein substrates. The present study not only indicates the degree of rat liver protein kinase compartmentalization but demonstrates alterations of the specific activities and relative subcellular distribution as a function of the age of the rat.
Cyclic [ 3H] AMP-binding activity in rat liver subcellular fractions A comparison of the affinity of rat liver subcellular fractions to bind cyclic. [ 3H] AMP reveals that throughout the age periods investigated cytosol exhibits the highest cyclic [ 3H] AMP-binding activity of the subcellular fractions assayed (Fig. 1). The specific cyclic [3H]AMP-binding activity ranges from 3 to 7.5 pmol of cyclic [ 3H] AMP bound per mg of protein in the cytosol fractions, about 1--2 pmol of cyclic [ 3H] AMP bound per mg of protein in the microsomal and lysosomal-mitochondrial fractions, to relatively low binding activities in the nuclear fractions which bind 0.08--0.22 pmol of cyclic [ 3H]AMP per mg of nuclear protein. A correlation of the cyclic [ 3H] AMP-binding activity of individual liver subcellular fractions as a function of age reveals a complex changing pattern which differs depending on the subcellular fraction investigated. The specific cyclic [ 3H] AMP-binding activity of cytosol appears to be similar in prenatal rats (3--4 days before birth) and in rats 25 days old, and older {Fig. 1). There is a distinct decline of the cytosol specific cyclic
269 SO CYTOSOL
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55-~
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AGE ( D a y s )
Fig. 1. D e v e l o p m e r i t a l a l t e r a t i o n s of c y c l i c [ 3 H] A M P - b i n d i n g a c t i v i t y in s u b c e U u l a r f r a c t i o n s of r a t liver. F o r p r e p a r a t i o n of s u b e e l l u l a r f r a c t i o n s a n d d e t e r m i n a t i o n o f cyclic [3 H] A M P - b i n d i n g a c t i v i t y see Materials a n d M e t h o d s . E a c h v a l u e r e p r e s e n t s t h e m e a n +- S.E. of t w o d e t e r m i n a t i o n s on five s e p a r a t e groupS of a n i m a l s . Significant c h a n g e s f r o m t h e v a l u e s o b t a i n e d w i t h p r e n a t a l rats ( 3 - - 4 d a y s b e f o r e b i r t h ) are d e n o t e d b y s y m b o l s (* = p ,~ 0 . 0 1 ; ** = p < 0 . 0 0 5 ; ~ = p ~ 0 . 0 0 1 ) .
[ 3H] AMP-binding activity during the prenatal period and at the time of birth reaching its lowest activity 2 days after birth. At this age liver cytosol cyclic [ 3H] AMP-binding activity is approx. 60% lower than the average activity observed 3--4 days before birth or in rats 4 days old, and older. No change of the cyclic [ 3H] AMP-binding activity of the lysosomal-mitochondrial fraction is apparent during the early prenatal and postnatal period {Fig. 1) but the activity increases in 10--11- and 25--26-day-old rats. At 19 days of gestation (3--4 days before birth} nuclear cyclic [ 3H] AMPbinding activity is significantly higher than during the postnatal period. The lowest nuclear cyclic [ 3H] AMP-binding activity is measured at birth. It increases slightly during the postnatal period but remains lower than the nuclear cyclic [ s HI AMP-binding activity observed in fetal liver. Marked fluctuations of the microsomal cyclic [ 3H] AMP-binding activity are observed as a function of age culminating in an approx. 2-fold increase of cyclic [ 3H] AMP-binding activity in 10--11- and 25--26-day-old rats as compared to the binding activity at the other age group examined (Fig. 1). From the specific cyclic [ 3H] AMP-binding activities and the protein concentration in each of the purified subcellular fractions the amount of cyclic AMP-binding activity of the subcellular fractions and the relative subcellular distributions can be calculated. The relative subcellular distribution of the cyclic [ 3H] AMP-binding activity in rat liver during hepatic development is shown in Table I. About 90--95% of the total liver cyclic [ 3H] AMP-binding activity is present in the cytosol, with lesser proportional activity in the particulate fractions. Although there are marked fluctations of the cyclic AMP-binding activities in the particulate subcellular fractions as a function of the age of the rat,
270 TABLE I S U B C E L L U L A R D I S T R I B U T I O N O F C Y C L I C A M P - B I N D I N G A C T I V I T I E S IN R A T L I V E R D U R I N G PRENATAL AND POSTNATAL DEVELOPMENT T o t a l a c t i v i t y r e p r e s e n t s t h e s u m of t h e activities o f all p u r i f i e d s u b c e l l u l a r f r a c t i o n s isolated f r o m t h e s a m e h o m o g e n a t e . F o r p r e p a r a t i o n and r e c o v e r y o f p a r t i c u l a t e f r a c t i o n s see Materials a n d M e t h o d s . No c o r r e c t i o n s h a v e b e e n m a d e for r e c o v e r y o f p a r t i c u l a t e f r a c t i o n s . Since t h e r e c o v e r y of t h e p a r t i c u l a t e f r a c t i o n s is n o t q u a n t i t a t i v e , t h e c a l c u l a t e d values for c y t o s o l are c o r r e s p o n d i n g l y high.
Age ( d a y s )
-3 Birth 2 4-- 5 10--11 25--26 35--36 60--90
Cyclic [ 3 H ] A M P - b i n d i n g a c t i v i t y (% of t o t a l ) Cytosol
Lysosomes-mitochondria
Microsomes
Nuclei
92.2 93.0 90.3 94.2 94.8 95.7 93.0 94.7
3.9 4.0 5.2 2.8 2.6 2.1 2.6 2.2
2.3 2.3 4.1 2.8 2.5 1.9 4.2 2.8
1.55 0.71 0.30 0.21 0.14 0.17 0.25 0.20
cyclic AMP-binding activity in the cytosol remains relatively constant throughout the prenatal and postnatal developmental period. The relative proportion of cyclic AMP-binding activity decreases markedly in the lysosomal-mitochondrial, and in the nuclear fractions during the developmental period investigated. A temporary increase of the relative amount of microsomal cyclic AMP-binding activity is seen in 2--3-day-old rats and again in 35--36-day-old rats. Protein phosphokinase activity in rat liver subcellular fractions Fig. 2 graphically summarizes a comparative study of the protein kinase activity in liver subcellular fractions during prenatal and postnatal hepatic development. Several important features distinguishing the soluble protein kinase activity in the cytosol from the particulate protein kinase activities in the nuclear, microsomal, and lysosomal-mitochondrial fractions can be noted: (1) Among the subcellular fractions examined the range of the specific activities of cytosol protein kinase is generally higher (150--350 pmol of 32 p incorporated/ mg protein per min) than in the particulate fractions. (2) Under the conditions of the assay protein kinase activity in intact nuclei is unresponsive to stimulation by cyclic AMP tested at various concentrations of cyclic AMP (10 - 8 10 -s M). Microsomal and lysosomal-mitochondrial protein kinase activity is only marginally stimulated by cyclic AMP (10 -6 M). Responsiveness of the particulate protein kinase activities to cyclic AMP does not change markedly during prenatal and postnatal development. (3) Soluble cytosol protein kinase activity possesses considerable cyclic AMP dependency. Addition of cyclic AMP at a concentration of 10 -6 M to the assays results in a maximal 1.5--2-fold stimulation of protein kinase activity in the various cytosol fractions. The cyclic AMP dependency is reduced in liver cytosol obtained from rats at the time of birth and in 2-day-old rats which is expressed by a decline of the activity ratio (protein kinase specific activity measured in the presence of cyclic
271 t5 1 7 . * U)
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Fig. 2. D e v e l o p m e n t a l a l t e r a t i o n s of p r o t e i n kinase a c t i v i t y in subceUulax f r a c t i o n s of r a t liver. F o r p r e p a x a t i o n of s u b c e l l u l a r f r a c t i o n s a n d p r o t e i n kinase assay see Materials a n d M e t h o d s . P r o t e i n kinase a c t i v i t y was a s s a y e d w i t h h i s t o n e F1 as s u b s t r a t e w i t h o u t a n d w i t h 1 " 10 -6 M cyclic AMP. O p e n bars, p r o t e i n kinase a c t i v i t y a s s a y e d w i t h o u t cyclic AMP; h a t c h e d bars, p r o t e i n kinase a c t i v i t y a s s a y e d in t h e p r e s e n c e o f 1 • 1 0 - 6 M cyclic AMP. N u m b e r at t h e t o p of e a c h b a r r e p r e s e n t s t h e p r o t e i n kinase a c t i v i t y r a t i o ( p r o t e i n kinase a c t i v i t y a s s a y e d in t h e p r e s e n c e of cyclic AMP versus p r o t e i n kinase a c t i v i t y a s s a y e d w i t h o u t cyclic AMP). E a c h v a l u e r e p r e s e n t s t h e m e a n -+ S.E. of t w o d e t e r m i n a t i o n s o n five s e p a r a t e g r o u p s o f animals. Significant c h a n g e s f r o m t h e values o b t a i n e d w i t h p r e n a t a l rats ( 3 - - 4 d a y s b e f o r e b i r t h ) are d e n o t e d b y s y m b o l s (see l e g e n d of Fig. 1).
AMP versus protein kinase specific activity measured in the absence of cyclic AMP) from a value of about 2.0 (in prenatal rats and in rats of the various age groups from age day 4--90) to 1.5. The specific activities of the soluble and of all particulate protein kinase activities increase markedly in 2-day-old rats from the levels found in prenatal animals but return to the prenatal levels in liver from rats 10 days old, and older. Cytosol and lysosomal-mitochondrial protein kinase exhibit additionally increased specific activities at the time of birth and in 4--5-day-old rats. Table II lists the calculated relative distribution of the total protein kinase activity in each subcellular fraction at distinct times during hepatic development. Under the experimental conditions cytosol protein kinase activity represents about 71--83% of the total activity present in the various liver homogenates. The relative level of cytosol protein kinase activity increases to the highest levels measured in liver from newly born, 4--5-, and 10--11-day-old rats. Thereafter, the relative amount of cytosol protein kinase declines again to about 71% of the total liver activity. The levels of lysosomal-mitochondrial protein kinase activity increase gradually from 8.5% of the total liver activity in prenatal rats to 17.5% of the total activity in 35--36-day-old rats and drop slightly to 13% in 60--90-day-old rats. The relative amount of protein kinase activity in the microsomal and nuclear fractions exhibits the most pronounced changes. Whereas the levels of microsomal protein kinase activity increase
272 T A B L E II SUBCELLULAR DISTRIBUTION OF PROTEIN PRENATAL AND POSTNATAL DEVELOPMENT
K I N A S E A C T I V I T I E S IN R A T L I V E R D U R I N G
T o t a l a c t i v i t y r e p r e s e n t s t h e s u m o f t h e activities of all p u r i f i e d s u b c e l l u l a r f r a c t i o n s i s o l a t e d f r o m the s a m e h o m o g e n a t e . P r o t e i n kinase a c t i v i t y w a s a s s a y e d w i t h o u t a d d i t i o n o f cyclic AMP using h i s t o n e F1 as s u b s t r a t e . F o r p r e p a r a t i o n a n d r e c o v e r y of p a r t i c u l a t e f r a c t i o n s see Materials a n d M e t h o d s . N o c o r r e c t i o n s h a v e b e e n m a d e for r e c o v e r y o f p a r t i c u l a t e f r a c t i o n s . Since t h e r e c o v e r y o f p a r t i c u l a t e f r a c t i o n s is n o t q u a n t i t a t i v e , t h e c a l c u l a t e d values for c y t o s o l are c o r r e s p o n d i n g l y high. Age ( d a y s )
-3 Birth 2 4-- 5 10--11 25--26 35--36 60-90
P r o t e i n kinase activity (% of t o t a l ) Cytosol
Lysosomes-mltochondria
M~erosomes
Nuclei
72.2 83.0 73.0 78.0 77.9 75.3 71.1 71.6
8.5 7.2 10.2 7.6 13.0 11.1 17.5 13.0
4.3 4.8 9.2 4.2 6.6 10.0 9.6 12.9
15.0 5.1 7.6 10.2 3.1 3.6 3.5 2.5
gradually from 4.3% of the total liver activity in prenatal rats to 12.9% in 60--90-day-old rats, with an intermittent decrease in 4--5-day-old rats, the relative levels of nuclear protein kinase activity decline drastically from 15% of the total activity in prenatal rats to only 5.1% at the time of birth, recover to 10.2% in 4--5-day-old rats, and finally decline to only 2--3% of the total activity in 10--1l-day-old rats, and older animals.
Effect of Triton X-I O0 treatment on microsomal cyclic AMP-binding activity and protein kinase activity A special feature of the microsomal protein kinase activity during the developmental period examined was its characteristic response to Triton X-100 treatment. It is well established that microsomes contain considerable latent protein kinase activity when assayed in vitro [11,12]. Treatment of microsomes with detergents, particularly the non-ionic detergent Triton X-100, unmasks and activates latent endogenous microsomal protein kinase activity. We have examined the effect of Triton X-100 on the protein kinase activity of the various liver subcellular fractions obtained from rats at distinct prenatal and early postnatal age periods. When protein kinase activity is assayed in vitro, Triton X-100 at concentrations of 0.5% and higher inhibits both protein kinase and cyclic [ 3H] AMP-binding activity in all subcellular fractions. Lower concentrations of Triton X-100 (0.1%) have no effect on cytosol, nuclear, and lysosomal-mitochondrial protein kinase activities b u t microsomal protein kinase activity is significantly increased. The degree of increase of microsomal protein kinase activity after Triton X-100 treatment appears to depend upon the age of the animal from which the liver microsomal fraction is obtained. Table III summarizes the results of several experiments in which the effect of Triton X-100 treatment on liver microsomal protein kinase activity was investigated during prenatal and early postnatal hepatic development. In agree-
273 TABLE III EFFECT OF TRITON X-100 ON RAT LIVER MICROSOMAL PROTEIN KINASE ACTIVITIES DURING PRENATAL AND POSTNATAL DEVELOPMENT T r i t o n X - 1 0 0 w a s a d d e d to m i c r o s o m a l f r a c t i o n s at a f i n a l c o n c e n t r a t i o n o f 0.1%. The f r a c t i o n s w e r e inc u b a t e d f o r 10 m i n a t 2°C a n d a l i q u o t s w e r e u s e d f o r p r o t e i n k i n a s e assay w h i c h w a s c a r r i e d w i t h h i s t o n e F1 b u t w i t h o u t c y c l i c A M P . T h e final c o n c e n t r a t i o n o f T r i t o n XolO0 in t h e a s s a y m i x t u r e w a s 0 . 0 0 5 % . F o r e x p e r i m e n t a l d e t a i l s see M a t e r i a l s a n d M e t h o d s . E a c h v a l u e r e p r e s e n t s t h e m e a n ± S.E. o f f o u r d e t e r minations on two separate preparations. Significance of changes from the values obtained without Triton X - 1 0 0 a r e d e n o t e d b y s y m b o l s (* P ~ 0 . 0 1 ; ** P ~ 0 . 0 0 5 ; * * * P ~ 0 . 0 0 1 ) .
Age (days)
P r o t e i n k i n a s e a c t i v i t i e s ( p m o l 32p i n c o r p o r a t e d / m i n p e r m g ) - Triton X-100
+ Triton X-100
+ Triton X-100 Ratio
-3 Birth 2 4-- 5 10--11 25--26 35--36 60---90
114+- 6 . 0 1 1 9 +- 3.7 152± 3.2 1 0 8 ± 10.9 1 2 9 -+ 1 0 . 0 101 ± 4.2 75-+ 5.2 99 -+ 1 1 . 5
189 + 1 4 . 4 " * 201 + 4 . 9 * * 271+ 12.7"** 151 -+ 8.9* 153 • 13.2 1 2 5 -+ 1 6 . 0 83 + 6 . 2 106 + 10.6
- Triton Xol00
1.7 1.7 1.8 1.4 1.2 1.2 1.1 1.1
ment with the data of Fig. 2 microsomal protein kinase activity reaches its highest specific activity in liver from 2-day-old rats. With the exception of microsomes from 35--36- and 60--90-day-old rats, treatment of microsomes with 0.1% of Triton X-100 prior to the protein kinase assay results in varying degrees of activation of latent microsomal protein kinase activity. The amount of latent microsomal protein kinase activity which is activated by Triton X-100 is age dependent. The degree of activation can best be compared when the activity ratio (specific activity of protein kinase treated with Triton X-100 versus specific activity of untreated protein kinase) is calculated. As indicated in Table III microsomes obtained from livers of prenatal rats and rats up to the age of 11 days exhibit a considerable amount of latent inactive protein kinase activity which is released in an active form by treatment of microsomes with 0.1% Triton X-100. The highest percentage of latent protein kinase activity appears to be present 3 days before birth, at the time of birth, and during the following postnatal period to the age of 5 days. Addition of cyclic AMP at concentrations of 10 -s to 10 -6 M to the assays has no significant stimulatory effect on microsomal protein kinase activity before or after Triton X-100 treatment. Triton X-100 treatment of liver microsomes obtained from the various age groups results only in a minor increase of the cyclic [ 3H] AMP-binding activity (14--16% increase) in all microsomal preparations studied. The resulting minor increase of cyclic [ 3H] AMP-binding activity is independent of the age of the animal from which the liver microsomal preparation is obtained. Discussion
Prenatal and postnatal development of the rat liver is known to be accompanied by alterations of the activities and changing patterns of certain hepatic
274 enzymes. Our studies concerned with the ontogeny of the protein phosphokinase system in subcellular fractions of the rat liver reveal significant developmental changes. The affinity of liver subcellular fractions for cyclic AMP as well as the specific protein kinase activity of liver subcellular fractions undergo marked fluctuations particularly during the late prenatal and early postnatal periods (3--4 days before and up to 26 days after birth). The cyclic AMP dependency of the soluble cytosol protein kinase is markedly diminished in 2-day-old rats which is accompanied and reflected by a significantly decreased cytosol cyclic [ 3HI AMP-binding activity. The change of cyclic AMP dependency and of activity ratio in 2-day-old rats may reflect a higher state of activation of cyclic AMP-dependent protein kinase, or the presence of proportionally higher levels of cyclic AMP-independent protein kinase. Cyclic AMP-binding activity in the liver particulate fractions (nuclei, mitochondria-lysosomes, microsomes) undergoes significant alterations during development (see Fig. 1). These alterations are not accompanied and reflected by changes in the cyclic AMP dependency of the particulate protein kinase activities (Fig. 2). The particulate protein kinase activities (particularly the nuclear and microsomal protein kinase activity) are essentially insensitive to cyclic AMP tested over a wide range of concentrations (10-8--10 -s M cyclic AMP). This finding suggests that the protein kinase activity expressed by the particulate fractions is either due to the presence of dissociated compartmentalized catalytic subunits of cyclic AMP-dependent protein kinases or to cyclic AMP-independent protein kinases which are not subject to cyclic AMP regulation. Furthermore, the data indicate that alterations of the cyclic AMP-binding activity in particulate fractions do not necessarily reflect alterations of cyclic AMP-binding regulatory subunits of cyclic AMP-dependent protein kinase. It is conceivable that some of the cyclic AMP-binding actJvity measured is due to the presence of cyclic AMP-binding proteins other than the regulatory units of cyclic AMP-dependent protein kinases. Our data indicate that most of the hepatic cyclic [ 3H] AMP-binding activity resides in the cytosol fraction and that the highest affinity for cyclic AMP is reached 10--11 days after birth in the cytosol, lysosomal-mitochondrial, and microsomal fractions but not in nuclei (Fig. 1). Novak et al. [5] and Christoffersen et al. [ 4] have shown that rat liver has the greatest ability to accumulate cyclic AMP in 5--10-day-old rats. It is conceivable that the high level of cyclic AMP-binding activity present at that time may facilitate the retention and compartmentalization of the cyclic nucleotide. Comparative studies of the relative subcellular distribution of protein kinase activity reveal significant changes of the specific activities and levels of protein kinase activity in the particulate fractions during hepatic development (see Fig. 2 and Table II). It is conceivable that the changes in particulate protein kinase activity observed during hepatic development may be partly due to a cyclic AMP-mediated, coordinated intraceUular redistribution of protein kinase and to a translocation of cytosol protein kinase and its compartmentalization primarily in the microsomal and lysosomal-mitochondrial fractions [13]. The increase of microsomal protein kinase activity during hepatic development, however, may also be the consequence of an unmasking of the latent protein kinase activity during the early postnatal period or may simply be the
275 result of an increased de novo synthesis of the enzyme. The demonstration of latent hepatic microsomal protein kinase activity at the perinatal and early postnatal age of the rat and the possibility of its activation at later stages of hepatic development may have some important physiological implications. Phosphorylation of ribosomal proteins has been reported to occur in a number of experimental systems [ 14--16]. Although the functional significance of ribosomal protein phosphorylation has yet to be uncovered, phosphorylative modification of ribosomal proteins may present an important regulatory step in the translational process. Activation of latent microsomal protein kinase activity during postnatal hepatic development might represent the initial step leading to the phosphorylative modification of specific ribosomal proteins whose functional modification is required during the developmental process. Additional indirect correlations between the observed developmental alterations of liver protein kinase and other known cellular hepatic processes may be established. Our studies show that the specific protein kinase activities of all liver subcellular fractions reach their highest values 2 days after birth of the rat (Fig. 2). Similar observations have been reported by Novak et al. [5] who measured the protein kinase activity in a 20 000 X g supernatant fraction of the liver. The increase of protein kinase activity in liver of 2-day-old rats coincides with the reported rapid decrease of liver glycogen levels [17], and increased protein kinase-mediated activation of glycogen phosphorylase [5] observed shortly after birth of the rat. The significant increase of the specific activity of liver nuclear protein kinase activity demonstrated in our studies 2 days after birth of the rat {Fig. 2) also correlates with the increased chromatin template activity observed in nuclei of 2-day-old rats [18]. Increased template activity may be the consequence of a changing composition or of a phosphorylative modification of the nuclear acidic protein component of chromatin. In view of the large body of evidence linking phosphorylative modification of nuclear proteins with gene activation and increased RNA synthesis [19,20], increased liver nuclear protein kinase activities in 2-day-old rats may be a prerequisite leading to an increased phosphorylation of nuclear proteins and consequently increased chromatin template activity. Acknowledgements The technical assistance of Mr David Radloff is greatly appreciated. This research was supported by Grant No. GB-41387 from the National Science Foundation, and in part by the Research and Education Fund, Northwestern Memorial Hospital. References 1 K n o x , W.E. ( 1 9 7 2 ) E n z y m e p a t t e r n s in fetal, a d u l t a n d neoplastic rat tissues, K a r g e r , Basel, S w i t z e r land 2 L a n g a n , T . A . ( 1 9 6 6 ) in Regulation of nucleic acid and protein biosynthesis, ( K o n i n g s b e r g e r , V . V . a n d Bosch, L.0 e d s ) , Vol. 10, pp. 2 3 3 - - 2 4 2 , B.B.A. L i b r a r y , Elsevier P u b l i s h i n g Co., Amsterdam
276 3 Langan, T.A. (1973) Advances in cyclic nucleotide research (Greengard, P. and Robison, G.A., eds), Vol. 3, pp. 99--153, Raven Press, New York 4 Christoffersen, T., M¢rland, J., Osnes, J.B. and ~ye, I. (1973) Biochim. Biophys. Aeta 313, 338--349 5 Novak, E., Drummond, G.I., Skala, J. and Hahn, P. (1972) Arch. Bioehem. Biophys. 150, 511--518 6 Fleiseher, S. and Kervina, M. (1974) Methods EnzymoL 31~ 6--41 7 Widnell, C.C. and Tara, J.R. (1967) Biochem. J. 92, 313--317 8 Hiestand, P.C., Eppenberger, U. and Jungmann, R.A. (1973) Endocrinology 93, 217--230 9 Lowry, O.H., Rosebrough~ N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 10 Burton, K. (1956) Biochem. J. 62, 315--323 11 Lemaire, S., Pelletier, G. and Labrie, F. (1971) J. Biol. Chem. 246, 7 3 0 3 - - 7 3 1 0 12 Maeno, H., Johnson, E.M. and Greengard, P. (1971) J. Biol. Chem. 246, 134--142 13 Jungmann, R.A., Lee, S.G. and DeAngelo, A.B. (1975) Advances in cyclic nucleotide research (Drummond, G.I., Greengard, P. and Robison, G.A., eds), Vol. 5, pp. 281--306, Raven Press, New York 14 Bazden, N. and Labrie, F. (1973) Biochemistry 12, 3096--3102 15 Eil, C. and Wool, I.G. (1973) J. Biol. Chem. 248, 5122--5129 16 Traugh, J.A., Mumby, M. and Traut, R.R. (1973) Proc. Natl. Acad. Sci. U.S. 70, 373--376 17 Shelley, H.J. (1961) Br. Med. Bull. 17, 137--143 18 Chytil, F., Glasser, S.R. and Spelsberg, T.C. (1974) Dev. Biol. 37, 295--305 19 Allfrey, V.G. (1970) Fed. Proc. 29, 1447--1460 20 Stein, G.S., Spelsberg, T.C. and Kleinsmith, L.J. (1974) Science 183, 817--824
°Biochimica et Biophysica Acta, 399 (1975) 265--276 © Elsevier Scientific Publishing Company, A m s t e r d a m -- Printed in The Netherlands
BBA 27699
ONTOGENY OF CYCLIC AMP-DEPENDENT PROTEIN PHOSPHOKINASE DURING HEPATIC DEVELOPMENT OF THE RAT
P.C. LEE and RICHARD A. J U N G M A N N
Department of Biochemistry, Northwestern University Medical School, Chicago, Ill. 60611
(U.S.A.)
(Received February 18th, 1975)
Summary The ontogeny of protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) and cyclic AMP-binding activity in subcellular fractions of liver was examined during prenatal and postnatal development of the male rat. 1. Protein kinase activity and cyclic AMP-binding activity were found in the nuclear, microsomal, lysosomal-mitochondrial, and soluble liver fractions. 2. The protein kinase activity of the soluble (105 000 X g SUlSernatant) fraction measured with histone F1 as substrate was stimulated by cyclic AMP. Cyclic AMP did not stimulate the protein kinase activity of the particulate fractions. 3. The protein kinase activity of all subcellular fractions increased rapidly from the activity observed in prenatal liver (3--4 days before birth) to reach maximal activity in 2-day-old rats. Thereafter, the protein kinase activity declined more slowly and regained the prenatal levels at 10 days after birth. 4. Considerable latent protein kinase activity was associated with liver microsomal fractions which could be activated by treatment of microsomes with Triton X-100. The latent microsomal protein kinase activity was highest in prenatal liver, at the time of birth, and 2 days after birth. During the subsequent postnatal development the latent microsomal protein kinase activity gradually declined to insignificantly low levels. " 5. During the developmental period examined (4 days before birth to age 60--90 days) marked alterations of the cyclic AMP-binding activity were determined in all subcellular fractions of rat liver. In general, cytosol, microsomal, and lysosomal-mitochondrial cyclic AMP-binding activity was highest in 10--11 day-old rats. Nuclear cyclic AMP-binding activity was highest 3--4 days before birth and declined at birth and during the postnatal period. There was no correlation between the developmental alteration of cyclic AMP-binding activity and cyclic AMP dependency of the protein kinase activity in any of the subceUular fractions. This suggests that the measured cyclic AMP-binding activi-
266 ty does not reflect developmental alterations of the cyclic AMP-binding regulatory subunit of cyclic AMP-dependent protein kinase.
Introduction Alterations of the activities of certain enzymes in vertebrate livers are known to form an integral part of the developmental process [1,2]. Cyclic AMP-dependent protein phosphokinase has been recognized in recent years to constitute a key hepatic enzyme mediating the phosphorylative and functional modification of a number of liver proteins [3]. Cyclic AMP (adenosine 3',5'monophosphate) is known to regulate the activity of the protein kinase in response to hormonal stimulation. A recent report by Christoffersen et al. [4] has demonstrated that hepatic enzymes involved in the metabolism of cyclic AMP undergo ontogenetic changes during the prenatal and early postnatal age of the rat. In addition, developmental changes of the activity of cyclic AMPdependent protein kinases coincide and correlate with changes of the hepatic glycogen metabolism in the rat [ 5]. In view of the importance of the cyclic AMP-dependent protein kinase system in mediating the hormonal control of hepatic function, we have examined the subcellular distribution and certain properties of hepatic protein kinase and cyclic AMP-binding activities at distinct stages of prenatal and postnatal development of the rat. Materials and Methods
Chemicals and animals. All biochemicals reagents were obtained from Sigma Chemical Co., St. Louis, Mo. [7 -32p] Adenosine 5'-triphosphate, ammonium salt (77.7 Ci/mmol), and [8-3H]adenosine 3',5'-monophosphate (27.5 Ci/mmol) were purchased from I.C.N. Isotope and Nuclear Division, Irvine, Calif., and Amersham Searle Corp., Arlington Heights, Ill. Male rats and pregnant rats at various times of gestation were obtained from Holtzman Co., Madison, Wisc. Preparation o f liver homogenate. Fetuses of both sexes were delivered by Caesarean section. Newborn and adult rats were sacrified by decapitation. The liver of rats older than 10 days was profused with a solution of 0.14 M NaC1/10 mM sodium citrate to rid of excess blood. Excised and profused livers were placed in 2vol. of ice-cold 0.01 M Tris buffer, pH 7.4, containing 0.25 M sucrose, 4 mM CaCl2, 5 mM MgC12 and homogenized in a Dounce homogenizer. The homogenate was filtered through gauze and again homogenized in a glass-to-glass Dual tissue grinder (Kontes, Vineland, N.J.). The resulting homogenate was used for the isolation of the subcellular fractions which were prepared using procedures described by Fleischer and Kervina [6]. The purity of the subcellular fractions and the degree of cross-contamination were judged according to the criteria given by Fleischer and Kervina [6]. Applying these criteria only a slight degree of cross-contamination was observed. Preparation of nuclei. Nuclei were prepared by the method of Widnell and Tata [7]. Accordingly, the homogenate was centrifuged at 800 × g for 15 min. The crude nuclear pellet was resuspended in 0.01 M Tris buffer, pH 7.4, con-
267 taining 2.3 M sucrose/5 mM MgC12 and sedimented by centrifugation at 105 000 X g for 60 min. The nuclei were again suspended in 0.01 M Tris buffer, pH 7.4, containing 2.3 M sucrose/5 mM MgC12 and sedimented by centrifugation at 105 000 X g for 60 min. The resulting purified nuclear pellet was suspended by homogenization with a glass-to-glass Potter-Elvehjem tissue grinder in 0.01 M potassium phosphate buffer (pH 7.0) to yield the nuclear fraction. Microscopic examination of these purified nuclei stained with hematoxylin and eosin showed no or relatively little contamination with cytoplasmic particles. The protein to DNA ratio of purified nuclei was 2.5 and did not change markedly with age of the rat. Comparison of the DNA content of the homogenate with that present in isolated nuclei revealed a routine recovery of 45--50%. The recovery of nuclei from the homogenate did not change with the age of the rat. Preparation of lysosomes-mitochondria. The 800 X g supernatant fraction was centrifuged at 13 000 X g for 15 min to yield the 13 000 X g pellet which was resuspended in 0.01 M Tris, pH 7.4, containing 0.25 M sucrose and sedimented by centrifugation at 13 000 X g for 15 min. The resulting 13 000 X g pellet was suspended by homogenization with a glass-to-glass Potter-Elvehjem tissue grinder in 0.01 M potassium phosphate buffer (pH 7.0), and designated lysosomal-mitochondrial fraction. The recovery of this fraction from the homogenate was routinely about 60% based on the recovery of succinate-cytochrome c reductase from the homogenate [6]. The percent recovery did not change with the developmental stage of the rat. Preparation of microsomes and cytosol. The 13 000 X g supernatant fraction was centrifuged at 105 000 X g for 60 min. The resulting supernatant, designated cytosol, was used for further experimentation. To achieve further purification the 105 000 X g pellet was suspended in 0.25 M sucrose and layered onto a 6.0 ml cushion of 1 M sucrose/0.01 M potassium phosphate buffer, pH 7.0. Centrifugation was carried out at 105 000 X g for 60 min. The sedimented pellet was homogenized in 0.01 M potassium phosphate buffer and designated microsomal fraction. The microsomal fraction isolated in this way contained both the rough and smooth microsomes. The recovery of this fraction was about 40% based on the recovery of glucose-6-phosphatase activity from the homogenate [6]. The percent recovery did not change with changing age of the rat. Determination of protein phosphokinase activity. The protein kinase assay was carried out as described previously [8] in a total incubation volume of 0.2 ml containing: 8--10 #g of protein to be assayed for protein kinase activity; 100 pg of histone F1; 10 #mol of sodium glycerol phosphate buffer, pH 7.0; 4 nmol of [7 -32p] ATP (0.15 pCi); 2 pmol of magnesium acetate; 2 pmol of NaF; 0.4 pmol of theophylline; 0.2 pmol of dithiothreitol; with or without cyclic AMP as indicated in the text. Incubation was started by the addition of [7_32p] ATP and carried out for 15 min at 37°C. The reaction was terminated by the addition of 2 ml of 20% trichloroacetic acid containing 1% sodium dodecyl sulfate. The samples were filtered using Millipore filters (0.3 pM). The filters were washed four times with 3 ml of 20% trichloroacetic acid/l% sodium dodecyl sulfate, dried, dissolved in 10 ml PCS (Amersham/Searle), and analyzed for radioactivity. Under the experimental conditions incorporation of
268 32 p into substrate protein proceed linearly with respect to incubation time and with increasing concentrations of protein exhibiting kinase activity. Assay o f cyclic AMP-binding activity. Binding of cyclic [ 3H] AMP to protein was determined by membrane filter assay as described previously [8] in a total incubation volume of 0.25 ml containing: 25 pmol of cyclic [ 3H] AMP; 16.5 pmol of Tris buffer, pH 7.4; 1.7 pmol of theophylline; 2.5 pmol of MgCI~ ; and 20--25 pg of protein to be assayed for cyclic AMP-binding activity. Incubation was carried out for 60 min at 2° C. The samples were filtered using Millipore filters (0.3 pm) which had been presoaked in ice-cold 0.25 mM Tris buffer, pH 7.4, containing 10 mM MgC12. Filters were washed five times each with 5 ml of 0.25 mM Tris/10 mM MgCI2, dissolved in 10 ml of PCS, and analyzed for radioactivity. Under the experimental conditions binding of cyclic [3H]AMP to protein was linear with protein concentrations up to 0.1 mg. Under the conditions no marked change of cyclic AMP-binding activity occurred after 60 min incubation. Protein concentration. Protein concentration was determined by the method of Lowry et al. [9] with bovine serum albun~i'n, fraction V, as reference standard. DNA concentration. DNA concentration was determined by the diphenylamine method of Burton [10] with calf thymus DNA as reference standard. Determination of radioactivity. Samples collected on Millipore filters were dissolved in 10 ml of PCS (Amersham/Searle) which was used as the scintillation fluid. All samples were counted in a model 3375 Packard Tri-Carb liquid scintillation spectrometer as previously reported [8]. Results It appears that protein phosphokinases are highly compartmentalized and found in close proximity to their phosphoprotein substrates. The present study not only indicates the degree of rat liver protein kinase compartmentalization but demonstrates alterations of the specific activities and relative subcellular distribution as a function of the age of the rat.
Cyclic [ 3H] AMP-binding activity in rat liver subcellular fractions A comparison of the affinity of rat liver subcellular fractions to bind cyclic. [ 3H] AMP reveals that throughout the age periods investigated cytosol exhibits the highest cyclic [ 3H] AMP-binding activity of the subcellular fractions assayed (Fig. 1). The specific cyclic [3H]AMP-binding activity ranges from 3 to 7.5 pmol of cyclic [ 3H] AMP bound per mg of protein in the cytosol fractions, about 1--2 pmol of cyclic [ 3H] AMP bound per mg of protein in the microsomal and lysosomal-mitochondrial fractions, to relatively low binding activities in the nuclear fractions which bind 0.08--0.22 pmol of cyclic [ 3H]AMP per mg of nuclear protein. A correlation of the cyclic [ 3H] AMP-binding activity of individual liver subcellular fractions as a function of age reveals a complex changing pattern which differs depending on the subcellular fraction investigated. The specific cyclic [ 3H] AMP-binding activity of cytosol appears to be similar in prenatal rats (3--4 days before birth) and in rats 25 days old, and older {Fig. 1). There is a distinct decline of the cytosol specific cyclic
269 SO CYTOSOL
~0
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C
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1
LYSOSOMESMITOCHONDRIA
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0
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55-~
60.90
0
~.4 Z ~ 4-5
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,
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Fig. 1. D e v e l o p m e r i t a l a l t e r a t i o n s of c y c l i c [ 3 H] A M P - b i n d i n g a c t i v i t y in s u b c e U u l a r f r a c t i o n s of r a t liver. F o r p r e p a r a t i o n of s u b e e l l u l a r f r a c t i o n s a n d d e t e r m i n a t i o n o f cyclic [3 H] A M P - b i n d i n g a c t i v i t y see Materials a n d M e t h o d s . E a c h v a l u e r e p r e s e n t s t h e m e a n +- S.E. of t w o d e t e r m i n a t i o n s on five s e p a r a t e groupS of a n i m a l s . Significant c h a n g e s f r o m t h e v a l u e s o b t a i n e d w i t h p r e n a t a l rats ( 3 - - 4 d a y s b e f o r e b i r t h ) are d e n o t e d b y s y m b o l s (* = p ,~ 0 . 0 1 ; ** = p < 0 . 0 0 5 ; ~ = p ~ 0 . 0 0 1 ) .
[ 3H] AMP-binding activity during the prenatal period and at the time of birth reaching its lowest activity 2 days after birth. At this age liver cytosol cyclic [ 3H] AMP-binding activity is approx. 60% lower than the average activity observed 3--4 days before birth or in rats 4 days old, and older. No change of the cyclic [ 3H] AMP-binding activity of the lysosomal-mitochondrial fraction is apparent during the early prenatal and postnatal period {Fig. 1) but the activity increases in 10--11- and 25--26-day-old rats. At 19 days of gestation (3--4 days before birth} nuclear cyclic [ 3H] AMPbinding activity is significantly higher than during the postnatal period. The lowest nuclear cyclic [ 3H] AMP-binding activity is measured at birth. It increases slightly during the postnatal period but remains lower than the nuclear cyclic [ s HI AMP-binding activity observed in fetal liver. Marked fluctuations of the microsomal cyclic [ 3H] AMP-binding activity are observed as a function of age culminating in an approx. 2-fold increase of cyclic [ 3H] AMP-binding activity in 10--11- and 25--26-day-old rats as compared to the binding activity at the other age group examined (Fig. 1). From the specific cyclic [ 3H] AMP-binding activities and the protein concentration in each of the purified subcellular fractions the amount of cyclic AMP-binding activity of the subcellular fractions and the relative subcellular distributions can be calculated. The relative subcellular distribution of the cyclic [ 3H] AMP-binding activity in rat liver during hepatic development is shown in Table I. About 90--95% of the total liver cyclic [ 3H] AMP-binding activity is present in the cytosol, with lesser proportional activity in the particulate fractions. Although there are marked fluctations of the cyclic AMP-binding activities in the particulate subcellular fractions as a function of the age of the rat,
270 TABLE I S U B C E L L U L A R D I S T R I B U T I O N O F C Y C L I C A M P - B I N D I N G A C T I V I T I E S IN R A T L I V E R D U R I N G PRENATAL AND POSTNATAL DEVELOPMENT T o t a l a c t i v i t y r e p r e s e n t s t h e s u m of t h e activities o f all p u r i f i e d s u b c e l l u l a r f r a c t i o n s isolated f r o m t h e s a m e h o m o g e n a t e . F o r p r e p a r a t i o n and r e c o v e r y o f p a r t i c u l a t e f r a c t i o n s see Materials a n d M e t h o d s . No c o r r e c t i o n s h a v e b e e n m a d e for r e c o v e r y o f p a r t i c u l a t e f r a c t i o n s . Since t h e r e c o v e r y of t h e p a r t i c u l a t e f r a c t i o n s is n o t q u a n t i t a t i v e , t h e c a l c u l a t e d values for c y t o s o l are c o r r e s p o n d i n g l y high.
Age ( d a y s )
-3 Birth 2 4-- 5 10--11 25--26 35--36 60--90
Cyclic [ 3 H ] A M P - b i n d i n g a c t i v i t y (% of t o t a l ) Cytosol
Lysosomes-mitochondria
Microsomes
Nuclei
92.2 93.0 90.3 94.2 94.8 95.7 93.0 94.7
3.9 4.0 5.2 2.8 2.6 2.1 2.6 2.2
2.3 2.3 4.1 2.8 2.5 1.9 4.2 2.8
1.55 0.71 0.30 0.21 0.14 0.17 0.25 0.20
cyclic AMP-binding activity in the cytosol remains relatively constant throughout the prenatal and postnatal developmental period. The relative proportion of cyclic AMP-binding activity decreases markedly in the lysosomal-mitochondrial, and in the nuclear fractions during the developmental period investigated. A temporary increase of the relative amount of microsomal cyclic AMP-binding activity is seen in 2--3-day-old rats and again in 35--36-day-old rats. Protein phosphokinase activity in rat liver subcellular fractions Fig. 2 graphically summarizes a comparative study of the protein kinase activity in liver subcellular fractions during prenatal and postnatal hepatic development. Several important features distinguishing the soluble protein kinase activity in the cytosol from the particulate protein kinase activities in the nuclear, microsomal, and lysosomal-mitochondrial fractions can be noted: (1) Among the subcellular fractions examined the range of the specific activities of cytosol protein kinase is generally higher (150--350 pmol of 32 p incorporated/ mg protein per min) than in the particulate fractions. (2) Under the conditions of the assay protein kinase activity in intact nuclei is unresponsive to stimulation by cyclic AMP tested at various concentrations of cyclic AMP (10 - 8 10 -s M). Microsomal and lysosomal-mitochondrial protein kinase activity is only marginally stimulated by cyclic AMP (10 -6 M). Responsiveness of the particulate protein kinase activities to cyclic AMP does not change markedly during prenatal and postnatal development. (3) Soluble cytosol protein kinase activity possesses considerable cyclic AMP dependency. Addition of cyclic AMP at a concentration of 10 -6 M to the assays results in a maximal 1.5--2-fold stimulation of protein kinase activity in the various cytosol fractions. The cyclic AMP dependency is reduced in liver cytosol obtained from rats at the time of birth and in 2-day-old rats which is expressed by a decline of the activity ratio (protein kinase specific activity measured in the presence of cyclic
271 t5 1 7 . * U)
MICROSOMES
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II
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o _= O.
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nln .... i
~-4 ~ Z 4 4
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~-tt
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Fig. 2. D e v e l o p m e n t a l a l t e r a t i o n s of p r o t e i n kinase a c t i v i t y in subceUulax f r a c t i o n s of r a t liver. F o r p r e p a x a t i o n of s u b c e l l u l a r f r a c t i o n s a n d p r o t e i n kinase assay see Materials a n d M e t h o d s . P r o t e i n kinase a c t i v i t y was a s s a y e d w i t h h i s t o n e F1 as s u b s t r a t e w i t h o u t a n d w i t h 1 " 10 -6 M cyclic AMP. O p e n bars, p r o t e i n kinase a c t i v i t y a s s a y e d w i t h o u t cyclic AMP; h a t c h e d bars, p r o t e i n kinase a c t i v i t y a s s a y e d in t h e p r e s e n c e o f 1 • 1 0 - 6 M cyclic AMP. N u m b e r at t h e t o p of e a c h b a r r e p r e s e n t s t h e p r o t e i n kinase a c t i v i t y r a t i o ( p r o t e i n kinase a c t i v i t y a s s a y e d in t h e p r e s e n c e of cyclic AMP versus p r o t e i n kinase a c t i v i t y a s s a y e d w i t h o u t cyclic AMP). E a c h v a l u e r e p r e s e n t s t h e m e a n -+ S.E. of t w o d e t e r m i n a t i o n s o n five s e p a r a t e g r o u p s o f animals. Significant c h a n g e s f r o m t h e values o b t a i n e d w i t h p r e n a t a l rats ( 3 - - 4 d a y s b e f o r e b i r t h ) are d e n o t e d b y s y m b o l s (see l e g e n d of Fig. 1).
AMP versus protein kinase specific activity measured in the absence of cyclic AMP) from a value of about 2.0 (in prenatal rats and in rats of the various age groups from age day 4--90) to 1.5. The specific activities of the soluble and of all particulate protein kinase activities increase markedly in 2-day-old rats from the levels found in prenatal animals but return to the prenatal levels in liver from rats 10 days old, and older. Cytosol and lysosomal-mitochondrial protein kinase exhibit additionally increased specific activities at the time of birth and in 4--5-day-old rats. Table II lists the calculated relative distribution of the total protein kinase activity in each subcellular fraction at distinct times during hepatic development. Under the experimental conditions cytosol protein kinase activity represents about 71--83% of the total activity present in the various liver homogenates. The relative level of cytosol protein kinase activity increases to the highest levels measured in liver from newly born, 4--5-, and 10--11-day-old rats. Thereafter, the relative amount of cytosol protein kinase declines again to about 71% of the total liver activity. The levels of lysosomal-mitochondrial protein kinase activity increase gradually from 8.5% of the total liver activity in prenatal rats to 17.5% of the total activity in 35--36-day-old rats and drop slightly to 13% in 60--90-day-old rats. The relative amount of protein kinase activity in the microsomal and nuclear fractions exhibits the most pronounced changes. Whereas the levels of microsomal protein kinase activity increase
272 T A B L E II SUBCELLULAR DISTRIBUTION OF PROTEIN PRENATAL AND POSTNATAL DEVELOPMENT
K I N A S E A C T I V I T I E S IN R A T L I V E R D U R I N G
T o t a l a c t i v i t y r e p r e s e n t s t h e s u m o f t h e activities of all p u r i f i e d s u b c e l l u l a r f r a c t i o n s i s o l a t e d f r o m the s a m e h o m o g e n a t e . P r o t e i n kinase a c t i v i t y w a s a s s a y e d w i t h o u t a d d i t i o n o f cyclic AMP using h i s t o n e F1 as s u b s t r a t e . F o r p r e p a r a t i o n a n d r e c o v e r y of p a r t i c u l a t e f r a c t i o n s see Materials a n d M e t h o d s . N o c o r r e c t i o n s h a v e b e e n m a d e for r e c o v e r y o f p a r t i c u l a t e f r a c t i o n s . Since t h e r e c o v e r y o f p a r t i c u l a t e f r a c t i o n s is n o t q u a n t i t a t i v e , t h e c a l c u l a t e d values for c y t o s o l are c o r r e s p o n d i n g l y high. Age ( d a y s )
-3 Birth 2 4-- 5 10--11 25--26 35--36 60-90
P r o t e i n kinase activity (% of t o t a l ) Cytosol
Lysosomes-mltochondria
M~erosomes
Nuclei
72.2 83.0 73.0 78.0 77.9 75.3 71.1 71.6
8.5 7.2 10.2 7.6 13.0 11.1 17.5 13.0
4.3 4.8 9.2 4.2 6.6 10.0 9.6 12.9
15.0 5.1 7.6 10.2 3.1 3.6 3.5 2.5
gradually from 4.3% of the total liver activity in prenatal rats to 12.9% in 60--90-day-old rats, with an intermittent decrease in 4--5-day-old rats, the relative levels of nuclear protein kinase activity decline drastically from 15% of the total activity in prenatal rats to only 5.1% at the time of birth, recover to 10.2% in 4--5-day-old rats, and finally decline to only 2--3% of the total activity in 10--1l-day-old rats, and older animals.
Effect of Triton X-I O0 treatment on microsomal cyclic AMP-binding activity and protein kinase activity A special feature of the microsomal protein kinase activity during the developmental period examined was its characteristic response to Triton X-100 treatment. It is well established that microsomes contain considerable latent protein kinase activity when assayed in vitro [11,12]. Treatment of microsomes with detergents, particularly the non-ionic detergent Triton X-100, unmasks and activates latent endogenous microsomal protein kinase activity. We have examined the effect of Triton X-100 on the protein kinase activity of the various liver subcellular fractions obtained from rats at distinct prenatal and early postnatal age periods. When protein kinase activity is assayed in vitro, Triton X-100 at concentrations of 0.5% and higher inhibits both protein kinase and cyclic [ 3H] AMP-binding activity in all subcellular fractions. Lower concentrations of Triton X-100 (0.1%) have no effect on cytosol, nuclear, and lysosomal-mitochondrial protein kinase activities b u t microsomal protein kinase activity is significantly increased. The degree of increase of microsomal protein kinase activity after Triton X-100 treatment appears to depend upon the age of the animal from which the liver microsomal fraction is obtained. Table III summarizes the results of several experiments in which the effect of Triton X-100 treatment on liver microsomal protein kinase activity was investigated during prenatal and early postnatal hepatic development. In agree-
273 TABLE III EFFECT OF TRITON X-100 ON RAT LIVER MICROSOMAL PROTEIN KINASE ACTIVITIES DURING PRENATAL AND POSTNATAL DEVELOPMENT T r i t o n X - 1 0 0 w a s a d d e d to m i c r o s o m a l f r a c t i o n s at a f i n a l c o n c e n t r a t i o n o f 0.1%. The f r a c t i o n s w e r e inc u b a t e d f o r 10 m i n a t 2°C a n d a l i q u o t s w e r e u s e d f o r p r o t e i n k i n a s e assay w h i c h w a s c a r r i e d w i t h h i s t o n e F1 b u t w i t h o u t c y c l i c A M P . T h e final c o n c e n t r a t i o n o f T r i t o n XolO0 in t h e a s s a y m i x t u r e w a s 0 . 0 0 5 % . F o r e x p e r i m e n t a l d e t a i l s see M a t e r i a l s a n d M e t h o d s . E a c h v a l u e r e p r e s e n t s t h e m e a n ± S.E. o f f o u r d e t e r minations on two separate preparations. Significance of changes from the values obtained without Triton X - 1 0 0 a r e d e n o t e d b y s y m b o l s (* P ~ 0 . 0 1 ; ** P ~ 0 . 0 0 5 ; * * * P ~ 0 . 0 0 1 ) .
Age (days)
P r o t e i n k i n a s e a c t i v i t i e s ( p m o l 32p i n c o r p o r a t e d / m i n p e r m g ) - Triton X-100
+ Triton X-100
+ Triton X-100 Ratio
-3 Birth 2 4-- 5 10--11 25--26 35--36 60---90
114+- 6 . 0 1 1 9 +- 3.7 152± 3.2 1 0 8 ± 10.9 1 2 9 -+ 1 0 . 0 101 ± 4.2 75-+ 5.2 99 -+ 1 1 . 5
189 + 1 4 . 4 " * 201 + 4 . 9 * * 271+ 12.7"** 151 -+ 8.9* 153 • 13.2 1 2 5 -+ 1 6 . 0 83 + 6 . 2 106 + 10.6
- Triton Xol00
1.7 1.7 1.8 1.4 1.2 1.2 1.1 1.1
ment with the data of Fig. 2 microsomal protein kinase activity reaches its highest specific activity in liver from 2-day-old rats. With the exception of microsomes from 35--36- and 60--90-day-old rats, treatment of microsomes with 0.1% of Triton X-100 prior to the protein kinase assay results in varying degrees of activation of latent microsomal protein kinase activity. The amount of latent microsomal protein kinase activity which is activated by Triton X-100 is age dependent. The degree of activation can best be compared when the activity ratio (specific activity of protein kinase treated with Triton X-100 versus specific activity of untreated protein kinase) is calculated. As indicated in Table III microsomes obtained from livers of prenatal rats and rats up to the age of 11 days exhibit a considerable amount of latent inactive protein kinase activity which is released in an active form by treatment of microsomes with 0.1% Triton X-100. The highest percentage of latent protein kinase activity appears to be present 3 days before birth, at the time of birth, and during the following postnatal period to the age of 5 days. Addition of cyclic AMP at concentrations of 10 -s to 10 -6 M to the assays has no significant stimulatory effect on microsomal protein kinase activity before or after Triton X-100 treatment. Triton X-100 treatment of liver microsomes obtained from the various age groups results only in a minor increase of the cyclic [ 3H] AMP-binding activity (14--16% increase) in all microsomal preparations studied. The resulting minor increase of cyclic [ 3H] AMP-binding activity is independent of the age of the animal from which the liver microsomal preparation is obtained. Discussion
Prenatal and postnatal development of the rat liver is known to be accompanied by alterations of the activities and changing patterns of certain hepatic
274 enzymes. Our studies concerned with the ontogeny of the protein phosphokinase system in subcellular fractions of the rat liver reveal significant developmental changes. The affinity of liver subcellular fractions for cyclic AMP as well as the specific protein kinase activity of liver subcellular fractions undergo marked fluctuations particularly during the late prenatal and early postnatal periods (3--4 days before and up to 26 days after birth). The cyclic AMP dependency of the soluble cytosol protein kinase is markedly diminished in 2-day-old rats which is accompanied and reflected by a significantly decreased cytosol cyclic [ 3HI AMP-binding activity. The change of cyclic AMP dependency and of activity ratio in 2-day-old rats may reflect a higher state of activation of cyclic AMP-dependent protein kinase, or the presence of proportionally higher levels of cyclic AMP-independent protein kinase. Cyclic AMP-binding activity in the liver particulate fractions (nuclei, mitochondria-lysosomes, microsomes) undergoes significant alterations during development (see Fig. 1). These alterations are not accompanied and reflected by changes in the cyclic AMP dependency of the particulate protein kinase activities (Fig. 2). The particulate protein kinase activities (particularly the nuclear and microsomal protein kinase activity) are essentially insensitive to cyclic AMP tested over a wide range of concentrations (10-8--10 -s M cyclic AMP). This finding suggests that the protein kinase activity expressed by the particulate fractions is either due to the presence of dissociated compartmentalized catalytic subunits of cyclic AMP-dependent protein kinases or to cyclic AMP-independent protein kinases which are not subject to cyclic AMP regulation. Furthermore, the data indicate that alterations of the cyclic AMP-binding activity in particulate fractions do not necessarily reflect alterations of cyclic AMP-binding regulatory subunits of cyclic AMP-dependent protein kinase. It is conceivable that some of the cyclic AMP-binding actJvity measured is due to the presence of cyclic AMP-binding proteins other than the regulatory units of cyclic AMP-dependent protein kinases. Our data indicate that most of the hepatic cyclic [ 3H] AMP-binding activity resides in the cytosol fraction and that the highest affinity for cyclic AMP is reached 10--11 days after birth in the cytosol, lysosomal-mitochondrial, and microsomal fractions but not in nuclei (Fig. 1). Novak et al. [5] and Christoffersen et al. [ 4] have shown that rat liver has the greatest ability to accumulate cyclic AMP in 5--10-day-old rats. It is conceivable that the high level of cyclic AMP-binding activity present at that time may facilitate the retention and compartmentalization of the cyclic nucleotide. Comparative studies of the relative subcellular distribution of protein kinase activity reveal significant changes of the specific activities and levels of protein kinase activity in the particulate fractions during hepatic development (see Fig. 2 and Table II). It is conceivable that the changes in particulate protein kinase activity observed during hepatic development may be partly due to a cyclic AMP-mediated, coordinated intraceUular redistribution of protein kinase and to a translocation of cytosol protein kinase and its compartmentalization primarily in the microsomal and lysosomal-mitochondrial fractions [13]. The increase of microsomal protein kinase activity during hepatic development, however, may also be the consequence of an unmasking of the latent protein kinase activity during the early postnatal period or may simply be the
275 result of an increased de novo synthesis of the enzyme. The demonstration of latent hepatic microsomal protein kinase activity at the perinatal and early postnatal age of the rat and the possibility of its activation at later stages of hepatic development may have some important physiological implications. Phosphorylation of ribosomal proteins has been reported to occur in a number of experimental systems [ 14--16]. Although the functional significance of ribosomal protein phosphorylation has yet to be uncovered, phosphorylative modification of ribosomal proteins may present an important regulatory step in the translational process. Activation of latent microsomal protein kinase activity during postnatal hepatic development might represent the initial step leading to the phosphorylative modification of specific ribosomal proteins whose functional modification is required during the developmental process. Additional indirect correlations between the observed developmental alterations of liver protein kinase and other known cellular hepatic processes may be established. Our studies show that the specific protein kinase activities of all liver subcellular fractions reach their highest values 2 days after birth of the rat (Fig. 2). Similar observations have been reported by Novak et al. [5] who measured the protein kinase activity in a 20 000 X g supernatant fraction of the liver. The increase of protein kinase activity in liver of 2-day-old rats coincides with the reported rapid decrease of liver glycogen levels [17], and increased protein kinase-mediated activation of glycogen phosphorylase [5] observed shortly after birth of the rat. The significant increase of the specific activity of liver nuclear protein kinase activity demonstrated in our studies 2 days after birth of the rat {Fig. 2) also correlates with the increased chromatin template activity observed in nuclei of 2-day-old rats [18]. Increased template activity may be the consequence of a changing composition or of a phosphorylative modification of the nuclear acidic protein component of chromatin. In view of the large body of evidence linking phosphorylative modification of nuclear proteins with gene activation and increased RNA synthesis [19,20], increased liver nuclear protein kinase activities in 2-day-old rats may be a prerequisite leading to an increased phosphorylation of nuclear proteins and consequently increased chromatin template activity. Acknowledgements The technical assistance of Mr David Radloff is greatly appreciated. This research was supported by Grant No. GB-41387 from the National Science Foundation, and in part by the Research and Education Fund, Northwestern Memorial Hospital. References 1 K n o x , W.E. ( 1 9 7 2 ) E n z y m e p a t t e r n s in fetal, a d u l t a n d neoplastic rat tissues, K a r g e r , Basel, S w i t z e r land 2 L a n g a n , T . A . ( 1 9 6 6 ) in Regulation of nucleic acid and protein biosynthesis, ( K o n i n g s b e r g e r , V . V . a n d Bosch, L.0 e d s ) , Vol. 10, pp. 2 3 3 - - 2 4 2 , B.B.A. L i b r a r y , Elsevier P u b l i s h i n g Co., Amsterdam
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