Purification and characterization fo a phosvitin kinase from the thyroid gland

150

Biochimica et Biophysica A eta, 756 (1983) 150- 15~ Elsevier Biomedical Press

BBA 21404

PURIFICATION AND CHARACTERIZATION OF A P H O S V I T I N KINASE FROM T H E T H Y R O I D GLAND YVONNE MUNARI-SILEM, BERNARD ROUSSET and RENI~ MORNEX I N S E R M U. 197, Laboratoire de Mbdecine Expbrimentale, FacultO de Mbdecine Alexis Carrel, rue Guillaume Paradin, 69372 l~von Cedex 2 (France)

(Received June 18th, 1982) (Revised manuscript received November 10th, 1982)

K£V words: Protein phosphorylation; Phosvitin kinase; (Thyroid gland)

1. Two cyclic A M P independent protein kinases phosphorylating preferentially acidic substrates have been identified in soluble extract from human, rat and pig thyroid glands. Both enzymes were retained on DEAE-cellulose. The first enzyme activity eluted between 60 and 100 mM phosphate (depending on the species), phosphorylated both casein and phosvitin and was retained on phosphocellulose; this enzyme likely corresponds to a casein kinase already described in many tissues. The second enzyme activity eluted from DEAE-cellulose at phosphate concentrations higher than 300 mM, phosphorylated only phosvitin and was not retained on phosphocellulose. These enzymes were neither stimulated by cyclic AMP, cyclic G M P and calcium, nor inhibited by the inhibitor of the cyclic AMP dependent protein kinases. 2. The second enzyme activity was purified from pig thyroid gland by the association of affinity chromatography on insolubilized phosvitin and DEAE-cellulose chromatography. Its specific activity was increased by 8400. 3. The purified enzyme (phosvitin kinase) was analyzed for biochemical and enzymatic properties. Phosvitin kinase phosphorylated phosvitin with an apparent K m of 100 /~g/mi; casein, histone, protamine and bovine serum albumin were not phosphorylated. The enzyme utilized ATP as well as G T P as phosphate donor with an apparent K m of 25 and 28/~M, respectively. It had an absolute requirement for Mg 2+ with a maximal activity at 4 mM and exhibited an optimal activity at pH 7.0. The molecular weight of the native enzyme was 110000 as determined by Sephacryl $300 gel filtration. The analysis by SDS-polyacrylamide gel electrophoresis revealed a major band with a molecular weight of 35000 suggesting a polymeric structure of the enzyme.

Introduction

Protein kinase activities (EC 2.7.1.37, ATP phosphotransferases) have been demonstrated during the last decade to be involved in the regulation of a variety of intracellular processes, including activation-deactivation of enzyme [1,2] and viral transformation [3,4]. Several types of protein kinases have been described. Some of them exhibit a cyclic nucleotide dependancy (cyclic AMP, cyclic GMP), while others are stimulated by specific effectors such as 0304-4165/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers

calcium via calmodulin, phospholipids or dsRNA [3]. But a number of protein kinase activities, without known specific activator, are still classified as messenger independent protein kinases. Little is known about the physiological involvement of these enzymes in the cell. Cyclic AMP independent protein kinases have been purified to various degree from cytosol [5 16], nuclei [17-20] and Golgi apparatus [21] from several species and tissues. Two distinct molecular species have been found and named I and II according to the nomenclature of Hathaway et al.

151

[13] or S and TS according to that of Clari [22]. We previously described various protein kinase activities in the human thyroid gland, including cyclic nucleotide independent activities [23-24]. As a prerequisite to the research of biological function of cyclic nucleotide independent protein kinases in the thyroid gland it appeared necessary to carry out a detailed analysis of their properties. The present report gives evidence for the presence in soluble thyroid extracts of different animal species of a protein kinase, strongly retained on DEAE-cellulose, which phosphorylates almost exclusively phosvitin. This enzyme activity has been purified by affinity chromatography. The enzymatic parameters (apparent K m for substrates, ion requirement) and the polypeptide composition (native and denaturated state) have been studied on a protein fraction enriched 8400 fold in enzyme activity.

Experimental procedures Materials [y-32p]ATP and [y-3zp]GTP were purchased from Amersham (spec. act. 2-4 Ci/mmol); unlabelled ATP, GTP, cyclic AMP, histone (calf thymus type II A), phosvitin, casein, protamine and bovine serum albumin were from Sigma; DEAE-cellulose (DE52) and phosphocellulose from Whatman; Sephacryl $300 from Pharmacia; Affigel 10 from Biorad. For comparison, a-casein from Sigma and casein from Merck were used in some experiments. All other chemicals used were from Merck and of the highest grade available. Tissue and enzyme preparations Normal human thyroid tissue was obtained from normal looking parts of thyroid glands from patients operated for 'cold' nodule. Tissue extracts were prepared within 1 h after surgery. Pig thyroid glands were obtained at a local slaughterhouse and kept on ice until homogenization which was performed within 1.5 h after removal. Rat thyroids were taken from adult animals (200-350 g) and immediately processed. Tissues were homogenized in 5 mM potassium phosphate, 2 mM EDTA, pH 7.0 (buffer A) or 50 mM potassium phosphate, 2 mM EDTA, pH 7.0 (buffer B), using a Waring Blendor and then an all-glass motor driven homo-

genizer. Homogenates were centrifuged at 50000 x g or 105000 X g for 1 h at +4°C and the supernatants were dialysed overnight against the homogenization buffer. Dialysed supernatants were stored frozen at - 2 0 ° C until use.

Methods Protein kinase assay. It was performed following the method of Miyamoto et al. [25] with small modifications [26]. The standard incubation mixture contained: 50 mM a-glycerophosphate, pH 6.5, 2 mM theophylline, 0.3 mM EGTA, 10 mM MgC12 (or 4 mM MgC12 for phosvitin kinase activity measurement), 0.1 mM ATP (800 000 cpm [~,-32p]ATP), 50 to 200 #g of protein substrate (phosvitin, dephosphorylated casein (Sigma), histone or bovine serum albumin) in a final volume of 200 #1. The reaction was initiated by addition of the enzyme and was stopped after 10 min at 34°C by addition of 2 ml of cold 7.5% trichloracetic acid. Sample washing and counting for 32p incorporated was as described elsewhere [26]. The concentration of substrate and enzyme for each experiment are designated in the legend of the figures. Each determination was made in triplicate. Blank values corresponding to 32p incorporated into bovine serum albumin in the absence of other substrate were subtracted from all values. DEAE-cellulose chromatography. DE52 cellulose was prepared according to the manufacturer's instructions and equilibrated at +4°C in 10 ml columns with buffer A. 50 000 X g supernatants or enzyme containing solutions were applied to the column and the ion-exchanger was washed with 10 ml of buffer A at a flow rate of 24 ml/h. The proteins were eluted by a linear 5-500 mM potassium phosphate gradient (2 x 30 ml) performed in buffer A. 2.5 ml fractions were collected. Each fraction was assayed for protein kinase activity in the standard conditions. Phosphocellulose chromatography. Phosphocellulose Pll was equilibrated with buffer B and 50 000 x g supernatants equilibrated with the same buffer were applied to the column (10 ml of gel) at a flow rate of 24 ml/h. The column was washed with 15 ml of buffer B and the proteins were eluted by a linear 0.1 to 1.5 M NaC1 gradient in buffer B (2 x 30 ml). Each fraction was assayed for protein kinase activity in the standard conditions.

152

Affinity chromatography. Phosvitin was covalently bound to an insoluble matrix Affigel 10 (N-hydroxysuccinimide ester of a succinylated aminoalkyl agarose gel). Affigel 10 was washed with water and suspended in 0.1 M N a i l CO 3, pH 8.0, at room temperature and mixed with a phosvitin solution at a ratio of 2 mg protein/ml of packed gel. The volume ratio between the ligand solution and packed gel was 0.7. The mixture was kept for 1 h at room temperature and overnight at + 4 ° C with continuous mixing. The suspension was then reacted with 0.1 volume of 1 M ethanolamine, pH 8.0, at room temperature for 1 h to block unreacted sites. The gel suspension was transferred to a column (3 x 10 cm) and washed with 10 bed volumes of 50 mM potassium phosphate, pH 7.0 (buffer C) and maintained at + 4°C until use. Gel filtration. Sephacryl $300 gel was packed in a column (1.5 × 5 0 cm) and equilibrated with buffer A containing 500 mM NaCI. Enzyme solutions (500 #1 containing 1 ~g phosvitin kinase and 2.5 mg bovine serum albumin) were applied to a column and elution was made with the starting buffer. Equilibration and elution were performed at a flow rate of 2.5 ml/h. Electrophoresis. Sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis was performed on slab gels as previously described [27]. Protein iodination. Proteins purified by affinity chromatography and DEAE-cellulose chromatography (phosvitin kinase) were iodinated by chemical oxidation. The reaction mixture (60 #1) contained 2 ktg purified protein, 200 p.Ci carrier-free ~25INa, 120 #g chloramine T in 5 mM potassium phosphate. The reaction was stopped after 1 min by addition of 100 p~l of sodium metabisulfite (2.4 m g / m l ) and the solution was supplemented with 1.2 mg bovine serum albumin. The labelled protein was separated from [~25I]iodide by gel filtration on Sephadex G-25 equilibrated with 10 mM sodium phosphate, 154 mM NaC1, pH 7.4 and the material in the void volume was analyzed by SDS-polyacrylamide gel electrophoresis. Protein determination. Protein was assayed either by the Coomassie blue method [29] using the Biorad reagent, or by the method of Lowry et al. [30] using bovine serum albumin as standard. The method used is given in the legend of figures.

Dephosphorylation of casein. Casein was dephosphorylated following the method described by Ashby et al. [28]. The concentration of dephosphorylated casein was adjusted to 30 m g / m l in distilled water and stored frozen at - 2 0 ° C . Results

Identification and separation of thyroid protein kinase activities DEAE-cellulose chromatography. The 50000 × g supernatants of human, rat and pig thyroid glands were chromatographed on DEAE-cellulose and protein kinase activities were identified by monitoring the incorporation of radioactive phosphate into histone, phosvitin or casein. The classical cyclic AMP-dependent histone kinase activities previously characterized in the thyroid gland by action of their specific inhibitor [23] (types I and II) eluted at 60 and 150 mM phosphate, respectively. A similar elution pattern was obtained with human, rat and pig thyroid extracts (Fig. 1A). Several peaks of cyclic AMP-independent protein kinase activities were observed (Fig. I B). A cyclic AMP-independent histone kinase was eluted at

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Fig. 1. DEAE-cellulose chromatography of thyroid soluble extracts. 10 ml of 5 0 0 0 0 × g supernatants from h u m a n (a,d), rat (b,e) and pig (c,f) thyroid glands (containing an identical protein concentration for all tissues) were applied to the colu m n and eluted. Aliquots of 50 ~1 from each fraction were assayed for protein kinase activity: (A) using histone (50 ,ug) in the absence ( x - - x ) or in the presence (O . . . . . . o) of cyclic A M P (10 5 M ) and (B) using phosvitin (200 #g) (A A) or casein (50 #g) (© ©). The dotted line indicates the change in phosphate concentration.

153 350 m M phosphate in all thyroid glands. A peak of casein kinase activity was eluted at 80, 60 or 100 m M phosphate in human, rat or pig thyroid tissue, respectively. Two peaks of phosvitin kinase activity were present in thyroid tissue: the first peak coeluted exactly with the casein kinase activity; the second peak which phosphorylated only phosvitin was tightly b o u n d to DEAE-cellulose and was eluted at 450 m M phosphate (human thyroid) and at 350 m M (pig and rat thyroid). This second peak never phosphorylated casein in any thyroid tissue so far examined and its phosvitin kinase activity was 2.5, 4.8 and 8.8 times higher than that of the phosvitin phosphorylating activity of the first peak in human, rat and pig thyroid tissue, respectively. In order to determine whether the casein kinase and phosvitin kinase activities which coeluted represented or not the same enzymatic entity, soluble thyroid extracts were fractionated by phosphocellulose chromatography. Phosphocelhdosechromatography. When thyroid 50000 × g supenatants were applied to a phosphocellulose column, a peak of protein kinase activity which phosphorylated both phosvitin and

casein was retained on the column and eluted at

800 mM NaC1 (Fig. 2). The major part of the phosvitin kinase and the total histone kinase activity were found in the flow though of the column. The material which was not adsorbed on phosphocellulose was subsequently c h r o m a t o g r a p h e d on DEAE-cellulose (Fig. 3). The cyclic AMP-dependent histone kinases (types I and II) eluted at 80 and 200 m M phosphate, and the phosvitin kinase activity that did not phosphorylate casein eluted at 350 m M phosphate. There was no peak of kinase activity which phosphorylated both casein and phosvitin. Since the casein kinase and the phosvitin kinase activities which coeluted between 60 and 100 m M phosphate (depending on the species) on DEAE-cellulose were retained together on phosphocellulose and since these kinase activities only used A T P as phosphate d o n o r (data not shown), it seems likely that these kinase activities represent a single enzyme component. This protein kinase likely corresponds to the casein kinase already described in cytosolic and nuclear fraction of various organs [5-7,14]. Thyroid glands would therefore contain a classical casein kinase phos-

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Fig. 3. DEAE-cellulose chromatography of pig thyroid soluble proteins not retained on phosphocellulose. The dialysed flowthrough of the phosphocellulose column was applied to a DEAE-cellulose column, prepared and eluted in the same conditions as in Fig. I. 2 ml fractions were collected. Aliquots of 50 #1 of each fraction were assayed for protein kinase activity using histone (50 /tg) in the absence (× ×) or in the presence (O- . . . . . O) of cyclic AMP (10 -'~ M), phosvitin (200 /~g) (A A) or casein (50 ~tg) (© O). The dotted line indicates the change in phosphate concentration.

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phorylating casein and phosvitin and a phosvitin kinase phosphorylating almost exclusively phosvitin (see below). We have tried to purify and characterize the latter enzyme activity.

Purification of the phosvitin kinase The total phosvitin kinase activity present in crude 105000 x g supernatants of thyroid tissue was retained on immobilized phosvitin and was eluted in a single peak at 1.0 M NaC1 (Fig. 4). Histone kinase activities were found in the flow through of the column. This purification step resulted in a 8400 fold increase in the specific activity of the phosvitin kinase activities which was of 6 ffmol phosphate transferred/fig of proteins, as measured in the standard conditions in the presence of 200 fig phosvitin. The recovery of the enzyme activity was about 100%. Since the purification procedure was based on the substrate specificity the eluted material contained both casein and phosvitin kinase activities. The two

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kinase activities were separated by DEAE-cellulose and the second peak was concentrated by a m m o n i u m sulfate precipitation. This purification step did not lead to an increase in the specific activity of the phosvitin kinase as compared to that obtained after the affinity chromatography. The phosvitin kinase devoid of any casein kinase activity, was used for further biochemical studies. The purified enzyme was kept frozen at - 20°C for several months without any loss of activity either in the presence or the absence of bovine serum albumin and even after several thawingfreezing cycles.

Biochemical and enzymatic properties of the phosvitin kinase Enzymatic parameters. Time-course experiments showed that the phosphorylation of phosvitin was linear with time for up to 15 min of incubation, at optimal substrate concentrations. The Mg 2 + requirement curve of the purified enzyme was shown in Fig. 5 and its maximal activity was obtained at 4 mM MgCI2. Similar phosvitin phosphorylation activities were obtained when ATP and G T P were used as phosphate donors (Fig. 6). Lineweaver-Burk plots

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Fig. 4. Filtration of pig thyroid soluble protein on immobilized phosvitin. 105000x g supernatants (160 ml) was applied to a column of phosvitin-affigel at a flow rate of 24 m l / h and the gel was washed by 100 ml of buffer C. Elution was performed by a 0 to 2 M NaC1 gradient (2 x 75 ml). Fractions of 4 ml were collected before the beginning of the gradient and 2.0 ml fractions were collected during the development of the gradient. Aliquots of 50 ,ul of each fraction were assayed for protein kinase activity in the presence of histone (50 #g) in the absence (x × ) or in the presence ( I . . . . . . I ) of cyclic AMP (10 -5 M), phosvitin (200 p,g) (A A) or casein (50 ,ug) (O O). Proteins ( ~ . . . . . . ~ ) were assayed by the Coomassie blue method. The dotted line indicates the change in NaC1 concentration.

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values of 25 and 28/~M for ATP and GTP, respectively. Fig. 7 shows the substrate specificity of the enzyme. The apparent g m of

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phosphorylation of phosvitin as determined by Lineweaver-Burk plot was 100 /~g/ml. Assuming an average molecular weight of 40000 for phosvitin, the apparent K m value would be in the micromolar range (2.5 /~M). The purity of phosvitin used in these experiments was controlled by SDS-polyacrylamide gel electrophoresis. Using Toluidine blue or Coomassie blue for gel staining, no other protein than phosvitin (38000-40000 molecular weight) was observed on gel loaded with up to 75/tg phosvitin. Accordingly the 32p-labelled products obtained by phosphorylation of phosvitin was a 38 000-40 000 molecular weight species (data not shown). Several other proteins were examined for their ability to serve as acceptor substrate. Casein (dephosphorylated), histone, protamine and bovine serum albumin were not phosphorylated at pH 6.5 in 50 mM glycerophosphate (bottom of Fig. 7). In order to ascertain that the phosvitin kinase exhibits a substrate specificity whatever the pH and the ionic strength of the incubation medium, we have measured its phosphorylating activity at various pH and NaC1 concentrations using casein or phosvitin as substrate. Results are presented in Fig. 8. At low ionic strength, the phosphorylation of phosvitin by the phosvitin kinase was maximum at pH 7.0 (Fig. 8A). At this

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Fig. 7. Substrate specificity of phosvitin kinase. Each reaction was carried out in the standard conditions of incubation in the presence of 0.02 /~g of enzyme and various concentrations of the following substrates: phosvitin (O O), histone (x x), casein (© ©), protamine ((~ O), bovine serum albumin (Lx zx). Inset: Lineweaver-Burk plot of phosvitin kinase activity versus phosvitin concentration.

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Fig. 8. (A) Effect of pH on phosvitin kinase activity. Substrates for pH activity were: phosvitin (Sigma) or dephosphorylated casein (Sigma). Activity was measured under optimal conditions: 4 mM MgCI2, 10 -4 M ATP in the presence of 0.02 #g of purified enzyme. Buffers were: 50 mM phosphate ( × ) and 50 mM Tris (e). (B) Effect of increasing concentration of NaCI on phosvitin kinase activity. The activity was measured in the same conditions as in (A), at pH 7.0. The value of NaCI represents the final concentration of NaCI in the assay.

156 p H , the activity was o p t i m a l in the presence of 100 m M NaCI (Fig. 8B). The similar salt d e p e n d e n c y was observed at p H 9.0 (data not shown). In contrast, the p h o s p h o r y l a t i n g activity of phosvitin kinase in the presence of casein was c o n s t a n t l y low. Therefore, the enzyme behaves as a phosvitin kinase whatever the p H and the ionic strength. The purified phosvitin kinase was insensitive to cyclic A M P , cyclic G M P a n d calcium (in the absence or in the presence of calmodulin). The en-

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zyme was also unaffected by the inhibitor of the cyclic A M P d e p e n d e n t histone kinases or by the i n h i b i t o r of the cyclic A M P i n d e p e n d e n t casein kinase described by Job et al. [31] (data not shown). Molecular weight determination. Based on the a p p a r e n t molecular weight of thyroglobulin, /~galactosidase, glycerokinase a n d bovine serum alb u m i n , the estimated molecular weight of the native enzyme was 110000 as d e t e r m i n e d by Sephacryl $300 gel filtration (Fig. 9). N o modification of this value was observed in the presence of 10 m M mercaptoethanol. W h e n s u b m i t t e d to a sedim e n t a t i o n velocity analysis on sucrose gradient (11-33%), the purified enzyme gave rise to aggregate, even in the presence of 500 m M NaCI a n d were u n a b l e to o b t a i n any estimate of the molecu-

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Fig. 9. Determination of the molecular weight of the native phosvitin kinase by Sephacryl $300 gel filtration. • • phosvitin kinase (1 #g) diluted in 500 #1 of buffer A containing 500 mM NaCI was applied to the column and eluted with the same buffer. Fractions of 0.5 ml were collected and aliquots of 50 #1 from each fraction were assayed for phosvitin kinase activity. • •: the enzyme pre-incubated for 30 min in buffer A containing 500 mM NaCI and 10 mM mercaptoethanol was applied to the same column equilibrated in buffer A containing 500 mM NaCI and 10 mM mercaptoethanol and eluted with the same buffer. For column calibration, markers were run separately or together with the enzymatic material in the absence (• •) or in the presence (• •) of mercaptoethanol; the markers (in order of decreasing kay) were bovine serum albumin, glycerokinase, fl-galaetosidase and thyroglobulin. Inset: plots of molecular weight of marker proteins versus the elution fraction number.

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157

lar by this method. Since the purified enzyme resulting from the second purification step (DEAE-cellulose chromatography) was obtained in relatively low amounts, the analysis of the polypeptide composition by SDS-polyacrylamide gel electrophoresis was performed after iodination of small quantities (2 ttg) of the purified material. SDS-polyacrylamide gel electrophoresis of purified t25I-labelled enzyme revealed a major band of 35 000 daltons as shown in Fig. 10.

Discussion The present study reports the purification and the characterization of a cyclic AMP independent protein kinase from thyroid gland that specifically phosphorylated phosvitin in vitro. The purification scheme involves an affinity chromatography procedure that provides reproducible high purification yield directly from 105000 × g supernatants. The high recovery (>~ 100%) could indicate that an inhibitor was removed during the purification. The absence of increase in specific activity after the DEAE-celhilose chromatography step is likely related to some loss of activity during this purification step. This has already been observed during the purification of other protein kinases [5,13] when the protein concentration becomes low. Our phosvitin kinase differs from a casein kinase also present in the same tissue, in several respects: (a) it phosphorylated only phosvitin while the casein kinase phosphorylated casein and phosvitin. (b) The casein kinase was retained on phosphocellulose and the phosvitin kinase was not. (c) Contrary to the casein kinase, the phosvitin kinase utilized ATP and GTP as phosphate donors. These characteristics indicated that the casein kinase present in thyroid tissue was rather similar to the casein/phosvitin kinase I (or S) described respectively in the cytosolic [5,8,9,13] and in the nuclear [ 17,19] fractions of various tissues. Although our phosvitin kinase shares some properties with the casein/phosvitin kinase II (or TS) already described [6,8,11,15,16,18]: MgC12 r e q u i r e m e n t , K m values for ATP and GTP, tendancy to aggregate, it differs by its inability to phosphorylate casein and to bind to phosphocellulose. Protein kinases phosphorylating preferentially phosvitin have been described in several

tissues [6,15] but they always phosphorylated also casein to a large extent. We have envisaged an artefactual absence of phophorylation of casein which could be related to the origin of casein or to the conditions of assay. The phosphorylation of casein from different commercial sources (dephosphorylated or not) was similar and constantly low as compared to that obtained with phosvitin. The specificity of our enzyme for phosvitin has been demonstrated in a wide range of pH and ionic strength. Hathaway et al. [13] reported a study on a casein kinase from rabbit reticulocytes that was not adsorbed on phosphocellulose in the presence of low monovalent ion concentrations but retained at 250 mM NaC1. The phosvitin kinase described here did not bind to phosphocellulose in any of these conditions. The purified phosvitin kinase behaves as a 110000 molecular weight protein in gel filtration experiments. The main polypeptide appeared to be a species of 35 000 daltons. This indicates that it could have a polymeric structure. The absence of effect of mercaptoethanol on the apparent molecular weight of the native enzyme suggests the absence of intersubunit disulfide bonds. The molecular weight of the holoenzyme is in the range of that reported for type I (or S) casein/phosvitin kinases from calf thymus [8], rabbit reticulocytes [13] and rat liver [5], and type II (or TS) enzyme found in Novikoff ascite tumor cells [16], rat liver [18] and rabbit reticulocytes [ 13]. In conclusion, the phosvitin kinase we described in the thyroid gland is different in many respects from the casein/phosvitin kinases already described in other tissues. The question as whether this enzyme is present in other mammalian tissues is presently under investigation.

Acknowledgments This work was supported by a grant from Biologie Humaine, Universit~ Claude Bernard, Lyon. We wish to thank A. Stefanutti for technical assistance and S. Terfous for secretarial work.

References 1 Rubin, C.S. and Rosen, O.M. (1975) Annu. Rev. Biochem. 44, 831-887

158 2 Krebs, E.G. and Beavo, J.A. (1979) Annu. Rev. Biochem. 48, 923-959 3 Erikson, R.L., Collett, M.S., Erikson, E. and Purchio, A.F. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 6260-6264 4 Presek, P., Glossmann, H., Eigenbrodt, E., Schoner, W., Rubsamen, H., Friis, R.R. and Bauer, H. (1980) Cancer Res. 40, 1733-1741 5 ltarte, E., Mor, M.A., Salavert, A., Pena, J.M., Bertomeu, J.F. and Guinovart, J.J. (1981) Biochim. Biophys. Acta 658, 334-347 6 Goldstein, J.L. and Hasty, M.A. (1973) J. Biol. Chem. 248, 6300-6307 7 Walinder, O. (1972) Biochim. Biophys. Acta 258, 411-421 8 Dahmus, M.E. (1981) J. BioL. Chem. 256, 3319-3325 9 Cochet, C., Job, D., Pirollet, F. and Chambaz, E.M. (1980) Endocrinology 106, 750-757 10 Bingham, E.W. and Groves, M.L. (1979) J. Biol. Chem. 254, 4510-4515 11 Boivin, P. and Galand, C. (1979) Biochem. Biophys. Res. Commun. 89, 7-16 12 lssinger, O.G. (1977) Biochem. J. 165, 511-518 13 Hathaway, G.M. and Traugh, J.A. (1979) J. Biol. Chem. 254, 762-768 14 Kemp, B.E., Froscio, M., Rogers, A. and Murray, A.W. (1975) Biochem. J. 145, 241-249 15 De Paoli-Roach, A.A., Ahmad, Z. and Roach, P.J. (1981) J. Biol. Chem. 256, 8955-8962 16 Dahmus, M.E. and Natzle, J. (1977) Biochemistry 16, 1901-1907

17 Desjardins, P.R., Lue, P.F., Liew, C.C. and Gornall, A.G. (1972) Can. J. Biochem. 50, 1249-1259 18 Thornburg, W. and Lindell, T.J. (1977) J. Biol. Chem. 252, 6660-6665 19 Thornburg, W., O'Malley, A.F. and Lindell, T.J. (1978) J. Biol. Chem. 253, 4638-4641 20 Kranias, E.G. and Jungmann, R.A. (1978) Biochim. Biophys. Acta 517, 447-456 21 Bingham, E.W. and Farrell, H.M. (1974) J. Biol. Chem. 249, 3647- 3651 22 Clari, G., Pinna, L.A. and Motet, T.V. (1976) Biochim. Biophys. Acta 451, 484-490 23 Munari-Silem, Y., Orgiazzi, J. and Mornex, R. (1979) Biochem. Biophys. Res. Commun. 89, 1314-1321 24 Munari-Silem, Y., Orgiazzi, J. and Mornex, R. (1981) Biochimie 63, 527-533 25 Miyamoto, E., Kuo, J.F. and Greengard, P. (1969) J. Biol. Chem. 244, 6395-6402 26 Munari, Y., Orgiazzi, J. and Mornex, R. (1978) FEBS Lett. 88, 211-214 27 Rousset, B. and Wolff, J. (1980) J. Biol. Chem. 255, 11677-11681 28 Ashby. D. and Walsh, D.A. (1974) Methods Enzymol. 38, 350-358 29 Bradford, M.M. (1976) Annal. Biochem. 72, 248-254 30 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 31 Job, D., Pirollet, F., Cochet, C. and Chambaz, E.M. (1980) FEBS Lett. 108, 508-512