Modification of protein kinase pattern in human placentae during gestation

Placenta (:984), 5, 443-454

Modification of Protein Kinase Pattern in Human Placentae during Gestation B. CEMERIKIC-JEKIC a'b& M. PAVLOVICHOURNAC Unitb de Recherche sur la Glande Thyroide et la REgulation Hormonale, 78, rue du G1 Leclerc, 9427o Bic~tre, France ~ Present address: Laboratory of Endocrinology, Department of Endocrinology, Immunology and Nutrition INEP, Banatska 3ib, iio8o Zemun, Yugoslavia To whom correspondence should be addressed

INTRODUCTION It is now widely established that the effects of many hormones are mediated by intracellular, second messengers, such as cyclic AMP (cAMP), cyclic GMP (cGMP) and Ca2+-calmodulin (Robison, Bucher and Sutherland, I971; Krebs and Beavo, 1979; Cohen, 1982). These mediators, in turn, are known to interact with specific protein kinases, resulting in phosphorylation of different cell proteins (Hussa, Story and Pattillo, x975; Lee et al, 1976; Cohen, I982; Taylor, I982). Numerous effects of cAMP on placental metabolism have been described (Hanwerger et al, I973; Chou, Wang and Robinson, I978), but protein kinases and their phosphorylation substrates regulated by these second messengers have not been studied in detail in the human placenta. A few data have appeared recently (Carpenter, Poliner and King, I98o) reporting the presence of protein kinase activity in human term placental membrane preparations that can be enhanced by the epidermal growth factor (EGF). A significant difference between the patterns of phosphorylated proteins from organ cultures of first-trimester and term placentae was also described. This last result suggested that protein kinase patterns may change in the placenta during gestation (Galski et al, I982). In this paper we describe the presence and some characteristics of protein kinases in the cytosol from placentae of different gestational age, and the changes in their activities throughout the period of gestation. The possibility of using in vitro incubation experiments for the study of placental protein kinases has also been shown.

MATERIALS AND METHODS Clinical material Fresh human placentae were obtained after legal abortions (six- to eight-week pregnancies), each experiment representing a pool of eight to ten individual placentae. Full term placentae were obtained from five women with uncomplicated pregnancies who delivered spontaneously at the Clinic of Gynaecology and Obstetrics, Faculty of Medicine, Belgrade. 443

444

B. Cemerikic-Jekic, M. Pavlovic-Hournac

Preparation o f cytosol and particulate fractions Immediately a f t e r removal, the tissue was rinsed and dissected free of connective tissue, major blood vessels a n d fetal membranes, frozen in liquid nitrogen and kept at - 8o~ until assayed. The subsequent cytosol fraction preparations were carried out as previously described (ToruDelbauffe et al, i982), except that the buffer used for homogenization was 5 mM Tris-HCl, pH 7'4, containing 3 mM EDTA and 6 mM/~-mercaptoethanol, and the weight: volume ratio was 1:3- After separation of cytosol, the high-speed sediment was washed twice and resuspended by homogenization. The suspension was treated for t h at 4~ with Nonidet P 4o, o.5 per cent final concentration, and proceeded with as described in detail eIsewhere (Toru-Delbauffe et al, 1982). The supernatant obtained after centrifugation represents the solubilized particulate enzyme fraction. DEAE-celluloSe chromatography of cytosol proteins DEAE-cellulose column (0.9 x 6 cm) was equilibrated with homogenization buffer. Cytosol proteins ( ,-- 2 rng in i ml) were applied and the column washed with io ml of the homogenization buffer. Elution was carried out with 4o ml linear concentration gradient made with o-35o mM NaCI in homogenization buffer. One-millilitre fractions were collected, and the osmolarity of each fraction determined by Radiometer conductivity meter. Fractionation o f protein phosphokinase activity by sucrose-gradient ultracentrifugation Samples ofcytosolic fraction (I-2 mg of proteins in i ml) were layered on the top of a t2 ml linear sucrose gradient, 11-22 per cent, prepared in the homogenization buffer and proceeded with as described previously (Toru-Delbauffe, Ohayon and Pavlovic-Hournac, 1979). For the estimation of sedimentation coefficients of different kinase peaks, myoglobin (S~o,w = 2) and glucose oxidase (S2o,W= 7.93) were used as internal markers. Estimation o f protein kinase activities Histone kinase activity was evaluated as previously described (Toru-Delbauffe, Ohayon and Pavlovic-Hournac, I979). Aliquots ofcytosolic and particulate enzymes (5-2o/~1) and of sucrose gradient or gel chromatography fractions (8o-ioo/zl) were incubated in a final volume of2oo/al in medium containing fl-glycerophosphate 5OmM; EGTA o.3 mM; theophylline 2 mM and magnesium acetate iomM in a potassium buffer i mM, pH 7-4; [Y-32P]ATP was added at a concentration of o. i mM, (specific activity about i oo cpm/pmole). Histones were at a concentration of 4 mg/rnl, and cAMP, when added, was 5/~M. Incubations were carried out at 3o~ for 5 min. At the end of the incubation 15o/11 of incubation mixture was spotted on 2-cm discs of Whatman 3 M M filter paper and precipitated with io per cent TCA containing carrier ATP o. i rnM. The subsequent procedure was as described previously (Toru-Delbauffe, Ohayon and Pavlovic-Hournac, 1979)" Both cAMP-dependent and cAMP-independent activities were estimated in parallel throughout this study. Cyclic AMP-independent histone kinase activity was evaluated in the presence of the heat-stable inhibitor of cAMP-dependent enzymes, previously described (ToruDelbauffe, ohayon and Pavlovic-Hournac, 1979). The cAMP-dep endent activity described in the present paper refers to the net activity which was calculated as the activity measured in the presence of cAMP minus the cAMP-independent activity. All results are expressed as cpm/g wet weight or as pmoles of 32p incorporated per gram of wet weight.

Human Placental Protein Kinases

445

Incubation o f placental tissue slices For incubation experiments, o.5 g of fresh, dissected placental tissues was weighed accurately and incubated in 3 ml of minimal essential Eagle medium (MEM) for 3 h in a Dubnoffshaker at 37~ in an atmosphere of 95 per cent 02 and 5 per cent CO2, as was described in detail by Genbacev et al (I977). RESULTS Protein kinase activity in h u m a n placental cytosol and particulate fraction The total histone kinase activity was estimated in the cytosol and particulate fractions of six- to eight-week old placentae and term placentae. Two types of histone kinase activity were present in both soluble and particulate fractions of the placentae studied: cAMP-dependent and cAMP-independent activity. The proportion of the total activity (cAMP--dependent plus cAMP-independent) present in the two subcellular compartments (cytosol and particulate fraction) was different, the protein kinase activity being predominant in cytosols from both young and term placentae. The particulate fraction contained 36.83 + 2. t4 and 25.85 + I.o3 per cent respectively for young and term placentae. It should be pointed out, however, that the procedure we used for the preparation of particulate fraction leads to the complete extraction of cAMP-dependent enzymes, but not of cAMP-independent ones. In the cytosol, the total histone kinase activity (cAMP--dependent plus cAMP-independent) was approximately four times higher in term placentae. As can be seen on Figure i, this is 3-

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Figure t. Cyclic AMP-dependent ( D ) and cAMP-independent (I~) histone kinase activity in the cytosol of placentae of different gestational age. Young (A) and term (B) placental tissues were homogenized and processed as described in Materials and Methods. Aliquots of 5-2o/al were incubated for 5 rain at 30 ~ Results are expressed in nmoles 32p incorporated into histories per gram of tissue per minute of incubation. The number of assays are given under each column.

446

B. Cemerikic-Jekic, M. Pavlovic-Hournac

due primarily to the selective increase of cAMP-dependent activity (4o6 per cent), while cAMP-independent enzymes are less stimulated (27o per cent). In both young and term placentae the cAMP-dependent histone kinases are predominant and represent about 7o per cent of the total cytosol kinase activity (Figure i). The cAMP-dependent kinase activity is about two times greater in the cytosol of young placentae as compared with the particulate fraction, and as much as four to five times greater in placentae at term (Table i). This is a consequence of different degrees of stimulation of soluble (4o6 per cent) and particulate (17o per cent) enzymes in term placentae. Table i. CyclicAMP-dependenthistonekinase activity in the cytosol and particulate fraction of the first- and third-trimester placentae

Weeks of gestation 6~8 38-4~

Cytosol (C)

Particulate fraction(P)

694 _+99a (34)b 2818 + I89 (28)

369 +_51 (x9) 6x8 + 65 (5)

C/P t.88 4.55

a Picomoles of 32p incorporated into histones per gram of tissue per minute. b Number of assays.

DEAE-cellulose c h r o m a t o g r a p h y In order to investigate the elution profile of placental cytosol, we fractionated both cytosol preparations of six- to eight-week old and term placentae by DEAE-cellulose chromatography. DEAE-ceUulose chromatography of young and term placental cytosol revealed two peaks of cAMP-dependent kinase activity corresponding to Type I (eluted at < ioo mM NaCI) and Type II (eluted at > lOO mM NaCI) isoenzymes. In young placentae the first peak was eluted at 20 + 2 m i NaC1 (n = 5) and in term placentae at 30 + 5 m i NaCI (n ----3)i while the second peak was eluted at 15o + 5 m i and 17o + 3 m i NaCI respectively (Figure 2, A and B). Young (six- to eight-week old) placentae contained 3o-35 per cent of Type I and 5o-6o per cent of Type II isoenzyme, whim placentae at term contained io-12 per cent of Type I and as much as 85-9o per cent of Type II isoenzyme of cAMP-dependent protein kinases. When cAMP-dependent kinase activity is expressed as pmoles 3zp per gram wet weight, the selective activation of Type II isoenzyme is observed in cytosol preparations of the term placentae. The kinase activity of peak I I is approximately four times higher in the cytosol of term placentae, when compared to the young ones (Table 2), while the activity of peak I remains unchanged throughout the period of gestation. Interestingly, one more peak of kinase activity was found after DEAE-cellulose chromatography in the soluble fraction of both first- and third-trimester placentae which was eluted at 215 + 5 mM NaCI (Figure 3, A and B). This kinase activity was cAMP-independent and was not affected by the heat-stable inhibitor of cAMP-dependent protein kinases. Sucrose-gradient ultracentrifugation After sucrose gradient ultracentrifugation of cytosol proteins, cAMP-independent kinase activity was separated into two distinct peaks. Their sedimentation coefficients are within the range 3.4 to 3.7 S for the first peak, and 6.7 to 7.o S for the second peak, in both young and term

Human Placental Protein Kinases

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~176176 I 400

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Fractions Figure z. DEAE--cellulose chromatography patterns of cAMP-dependent protein kinase activity from 6-8 week old (A) and term (B) placentae. Cytosol containing 2 mg proteins in i ml was applied on a column and eluted with a linear concentration gradient of NaCl as described in Materials and Methods. Fractions of i ml were collected and the cAMPdependent protein kinase activity was measured in 8o #1 aliquots. Results are expressed as net activity of cAMPdependent protein kinase activity.

Table 2. Selective activation of Type I I isoenzyme ofcytosolic cAMP-dependent protein kinases during gestation Weeks of gestation (~8 38-4 ~ Term/Young

Peak I

Peak II

143+ II a ] 37 4" 13 o.96

242+_23 io62 ___62 4.39

a Picomoles s2p incorporated into histones per gram of tissue per minute.

B. Cemerikic-Jekic, M. Pavlovic-Hournac

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Fract ions Figure 3. Elution profile after DEAE-cellulosechromatographyof cAMP-independent protein kinase activity of the

cytosoifrom first-trimester(A) and third-trimester (B) placentae.The cAMP-independenthistone kinase activitywas evaluatedin 8o #l aliquotsand in the presenceof the heat-stableinhibitor of cAMP-dependentenzymes.The results are expressed in cpm per gram of wet weight tissue. placentae (Figure 4, A and B). In the cytosol of both first- and third-trimester placentae, the first peak is predominant and contains almost 8o per cent of the total activity. E v a l u a t i o n o f p r o t e i n p h o s p h o k i n a s e a c t i v i t y in i n c u b a t e d p l a c e n t a e In young and term placental tissue slices incubated for 3 h, cAMP-dependent and c A M P independent histone kinase activities were tested in the total cytosol and after separation of kinase activities by either column chromatography or sucrose-gradient ultracentrifugation. T h e

Human Placental Protein Kinases

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449

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Figure 4. Sucrose gradient ultracentrifugation pattern of cAMP-independent histone kinases from 6 ~ week old

placentae(A) and term placentae(B). Cytosolscorrespondingto about 3oo mg of wet weight were layeredon the top of sucrose gradients and centrifuged as described in Materials and Methods. The cAMP-independent histone kinase activitywas estimatedin ioo #l aliquotsof coUectedfractions.The activityis expressedin cpm per gram of weighttissue.

estimation of histone kinase activity in the total cytosol of incubated placentae clearly demonstrated that this activity remains unchanged after 3 h of incubation of young and term placental tissue slices under our experimental conditions (Table 3). When applied to the DEAE-cellulose column and analysed by gel chromatography, the elution profile of young and term placental cytosol revealed the presence of two peaks of cAMP-dependent kinase activity, corresponding to those observed in non-incubated tissue. In young placentae, the first peak eluted at 22 + 1.4 mM NaCI (n = 4) and in term placentae at 35 + 3.7 mM NaCI (n = 3), while the second peak eluted at 146 + 4raM NaCI and I8o + 2.5 mM NaCI respectively. The proportions of the two peaks were 25-3o per cent of peak i and 6o-65 per cent of peak 2 in the first-trimester placental cytosol, and 8-1o per cent and 8o-85 per cent

B. Cemerikic-Jekic, M. Pavlovic-Hournac

450

Table 3. Cyclic AMP-dependent (PKa) and cAMP-independent (PKi) histone kinase activity in non-incubated and 3-hour incubated placentae of different gestational ages

Non-incubated

Weeks of gestation

Incubated

PKa

PKi

PKa

PKi

6~8

694+99 a (34)b

335_+9~ (22)

667_+63 (II)

298_+42 (19)

38-4 ~

2818_+189

915-1-66

2648+__219

865_+76

(28)

06)

(8)

(12)

o Picomolesof 3zp incorporated per gram of tissue per minute. b Number of assays.

Table 4. Sedimentationcoe~cients (S) of cAMP-independent histone kinase peaks from non-incubated and 3-hour incubated placentae

Non-incubated Incubated

Peak x

Peak 2

3.55 -----o.2I

6.85 _+0.23 6.9~ _+o.I8

3.71 ~-o.i 5

respectively in the placentae at term. T h e specific stimulation o f the peak II in the cytosol of term placentae, found in non-incubated tissue, was still observed after 3 h of incubation. T h e two peaks of cAMP-independent kinase activity identified after sucrose-gradient ultracentrifugation had the same sedimentation coefficients as those seen in the cytosol of placentae that were not submitted to incubation (Table 4). In other words, our results clearly demonstrate that there are no significant differences, in any studied parameter, between nonincubated and incubated placentae.

DISCUSSION Protein phosphorylation is considered to be one of the major mechanisms by which intracellular events respond to external physiological stimuli. Since the presence of protein kinases and their involvement in protein phosphorylation, and thus regulation of many cellular processes, have been demonstrated in nearly all mammalian tissues, it is reasonable to suppose that at least some hormones and some bioactive substances regulate placental metabolism through these specific protein kinases. Since protein kinases, as well as their phosphorylation substrates, have practically not been investigated in the human placenta, we started with the study of their occurrence in placental cytosol. Our experiments, using placentae of different gestational age, have shown the presence of cAMP-dependent and cAMP-independent activity in the soluble and particulate fractions of the placentae studied. T h e proportion o f the total histone kinase activity in the particulate fraction of young and term placentae was about 37 per cent and 26 per cent respectively. However, in spite o f its lower relative amount, the cAMP--dependent particulate activity in third-trimester placentae was approximately twice that of young placentae. T h e presence of

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451

protein kinase activity was demonstrated in cell membrane preparations of normal human term placentae; it was enhanced by epidermal growth factor and appeared to be cyclic nucleotideindependent (Carpenter, Poliner and King, i98o ). The results we are presenting here differ from those reported in the mentioned article; this is due primarily to different technical approaches, but the results are not conflicting. Our results concern the protein kinase activity from the total particulate fraction after solubilization by a detergent, while Carpenter et al studied the purified membrane preparations without solubilization and using only endogenous substrates. Under the same experimental conditions the absence of cAMP-dependent protein kinases was also observed in membrane preparations of thyroid glands. However, the cAMPdependent protein kinase was unmasked when protein kinase activity was evaluated in the presence of histones as exogenous substrates, or after solubilization of enzymes, as we did in the present work (Suzuky and Field, i978 ). DEAE-cellulose chromatography applied to cAMP-dependent kinase analysis revealed the presence of Type I and Type I I isoenzymes of cAMP-dependent protein kinases, corresponding to those described in other mammalian tissues (Corbin, Keely and Park, 1975). Under our experimental conditions, the high content in Type II isoenzyme of cAMPdependent protein kinases was demonstrated after gel chromatography in both young and term placental cytosol. The similar proportion of Type I and Type II cAMP-dependent protein kinase isoenzymes in the term placentae was recently reported by Moore et al at the 9th Rochester Trophoblast Conference, October 3rd-sth, 1982 (unpublished data). We also noted the selective stimulation of Type II cAMP--dependent isoenzyme activity in the cytosol of term placentae, non-incubated as well as incubated in vitro for 3 h. In spite of the fact that we still do not know the role of the two cAMP-dependent kinases in the placental tissue, we may postulate that they control different aspects of placental metabolism. There is evidence in the literature that the appearance of Type I and II isoenzymes is a function of differentiation and growth (Lee et al, i976; Haddock-Russell and Haddox, 1979; Salokangas et al, I981). However, there is not yet a definitive agreement between different authors in the correlation of Type I and Type II enzymes either with proliferation or with differentiation. In the placenta, similarly, the Type I and Type II kinases could be involved in proliferation and differentiation, since these two processes are very intensive during gestation. In this report we have shown the presence of a peak of cAMP-independent protein kinase activity after DEAE-cellulose chromatography of both young and term placental cytosol. Our further analysis on sucrose gradient revealed the presence of two peaks of cAMP-independent kinases, the first one being much more important in both placentae studied. A similar profile of two distinct peaks of cAMP-independent histone kinases was observed in thyroid and several other rat tissues after sucrose-gradient ultracentrifugation (Toru-Delbauffe, Ohayon and Pavlovic-Hournac, I983). After DEAE-cellulose chromatography a peak of cAMP-independent histone kinase, specific for HI histone and similar to the one we obtained, was found in neoplastic tissues, plasmocytoma and Morris hepatoma, and its involvement in cell proliferation has been postulated (Schmitt, Quirin-Stricker and Kempt, 198~). Although the results obtained on the placenta need more detailed studies to be explained, it is interesting to speculate about using the placenta as a model for neoplastic transformation studies. Our results with incubated placentae, showing that there are no significant differences between these and non-incubated placentae, are very encouraging, in our opinion, in providing the possibility of developing the study of placental protein phosphokinases into two domains: first, pharmacological study, i.e., the study of protein kinases after different 'in vitro' treatments with biologically active substances, such as drugs; secondly, biochemical studies that

452

B. Cemerikic-Jekic, M. Pavlovic-Hournac

could provide additional data for the better understanding of the regulation of placental metabolism, since protein phosphokinases are the intermediate link in the known chain of processes, starting on the cellular membrane and resulting in a given intracellular response.

SUMMARY The cAMP-dependent and cAMP-independent histone kinases have been studied in the two subcellular compartments (cytosol and particulate fraction) from placentae of different gestational age. The total protein kinase activity, as well as its distribution between the two compartments, changes during the period of gestation. The total activity is significantly increased in full-term placentae. The increase is much greater for the cAMP-dependent (4oo per cent), than for the cAMP-independent (27o per cent) protein kinases. It is much higher (4oo per cent) in the cytosol than in the particulate fraction (17o per cent); consequently, the particulate fraction of term placentae shows a relatively lower proportion of protein kinase activity (26 per cent of the total activity) than the corresponding fraction of young placentae (37 per cent). DEAE-cellulose chromatography revealed the presence of two cAMP-dependent protein kinase peaks which correspond to Type I and Type II isoenzymes described in many mammalian tissues (Corbin, Keely and Park, I975). The Type II isoenzyme is predominant in both first- and third-trimester placentae. The increase in protein kinase activity in term placentae is due to the selective activation of the Type ! I kinase only. The activity of the Type I isoenzyme remained unchanged throughout the period of gestation. The third peak eluted from the DEAE-.cellulose column corresponds to a cAMP-independent protein kinase activity. This cAMP'-independent protein kinase activity can be resolved by sucrose-gradient ultracentrifugation into two distinct peaks similar to those already observed in several rat tissues (Toru-Delbauffe, Ohayon and Pavlovic-Hournac, I983). The protein kinase patterns of both young and term placentae remain stable during the incubation of the tissues 'in vitro' for three hours.

ACKNOWLEDGEMENT The authors wish to thank Dr M. Movsesijanand Dr O. Genbacevof the Department of Endocrinology,Immunology and Nutrition, INEP, for their interest and generous help throughout this study. They also wish to thank Mrs A. Guedec for her excellent secretarial assistance. During this work, B. C.J. was the holder of a French Government fellowship. REFERENCES Carpenter, G., Poliner, L. & King, L. (I980) Protein phosphorylationin human placenta. Stimulation by epidermal growth factor. Molecular and Cellular Endocrinology, 28, 189-199. Chou, J. Y., Wang, S. S. & Robinson, J. C. (1978) Regulationof the synthesisof human chorionicgonadotropinby 5'-Bromo-z'-Deoxyuridineand DibutyrylCyclicAMP in trophoblasticand nontrophoblastictumor cells.Journal of Clinical Endocrinology and Metabolism, 47, 46-5 L Cohen, P. (i982) The roleof protein phosphorylationin neural and hormonalcontrolof cellular activity. Nature, ~96, 613~2o. Corbin, J. D., Keely, S. L. & Park, C. R. (1975) The distribution and dissociation of cyclic Adenosine 3'-5'-Monophosphate--dependentprotein kinases in adipose,cardiacand other tissues.Journal of BiologicalChemistry, 25o, 218-225. Galski, H., Weinstein, D., Gileadi, O. et al (I982) Phosphorylationof proteins in cultured human placenta. Biochemistry International, 5, I37-I43.

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Genbacev, O., Ratkovie, M., Kraineanic, M. & Sulovic, V. (1977) Effects of prostaglandin PGF2alphaon the synthesis of placental proteins and placental lactogen (HPL). Prostaglandins, I3, 723-733. Haddock-Russell, D. & Haddox, M. K. (I979) Cyclic AMP-mediated induction of ornitine decarboxylase in normal and neoplastic growth. Advances in Enzyme Regulation, I7, 61-87. Hanwerger, S., Barret, J., Tyery, L. & Schomberg, D. (1973) Differential effect of cyclic adenosine monophosphate on the secretion of human placental lactogen and human chorionic gonadotropin.JournalofClinicalEndocrinology and Metabolism, 36, 1268-127o. Hussa, R. D., Story, M. T. & Pattillo, R. A. (1975) Regulation of human chorionic gonadotropin (HCG) secretion by serum and dibutyryl cyclic AMP in malignant trophoblast cells in vitro. Journal of Clinical Endocrinology and Metabolism, 40, 4Ol-4O5. Krebs, E. G. & Beavo, J. A. (1979) Phosphorylation--dephosphorylation of enzymes. Annual Review of Biochemistry, 48, 923-959 9 Lee, P. C., Radloff, D., Schweppe, J. S. & Jungmann, R. A. (I976) Testicular protein kinases. Characterization of multiple forms and ontogeny. Journal of Biological Chemistry, 251, 914-921. Munro, H. N. (1979) Nucleic acid and placental development. In Placenta-A Neglected Experimental Animal (Ed.) Beaconsfield, P. & Villee, C., pp. 62-74. Oxford: Pergamon Press. Robison, G. A., Bucher, R. W. & Sutherland, E. W. (I971) Cyclic AMP and hormone action. In Cyclic AMP. pp. 17-47. New-York: Academic Press. Salokangas, A., Fabbro, D. Eppenberger, U. & Clu'ambach, A. (1981) cAMP-dependent protein kinases, their subunits, and cAMP-blnding protein from four sources: A comparison of physical characteristics determined by polyacrylamide gel electrophoresis, Archives of Biochemistry and Bwphysics, 2II, I58-I65. Schmitt, M., Quirin-Stricker, C. & Kempt, J. (I 982) Microsomal cAMP-independent histone HI kinase activity in plasmocytoma, Morris hepatoma and normal liver. Biochiraie, 63, I3-2o. Suzttky, S. & Field, J. B. (I978) Thyroid plasma membrane-associated protein kinases; Properties and substrates of solubilized and insoluble enzymes. Endocrinology, io3, I783-1793. Taylor, S. S. (1982) The in vitro phosphorylation ofchromatin by the catalytic subunit of cAMP-dependent protein kinase. Journal of Biological Chemistry, 257, 6o56-6o63. Toru-Delbauffe, D., Ohayon, R. & Pavlovie-Hournae, M. 0979) Hormonal regulation of thyroidal protein phosphokinase activities. I I differential sensitivity of type I and type II cAMP-dependent enzymes to the treatment of rats with thyroxine. Molecular and Cellular Endocrinology, I4, 141-155. Toru-Delbauffe, D., Ohayon, R. & Pavlovic-Hournac, M. (I983) Some characteristics and hormonal control of thyroidal cAMP-independent protein kinases. Molecular and Cellular Endocrinology, 29, I33-146. Toru-Delbauffe, D., Lognonne, J. L., Ohayon, R. et al (1982) Thyroidal cAMP-dependent protein kinases. Particular of the type I kinase, and compartmentalization of the two isoenzymes. EuropeanJournal of Biochemistry, I25, 267-275.