220 Biochimica et Biophysica A cta, 702 (1982) 220-232 Elsevier Biomedical Press

BBA 31105

PURIFICATION AND CHARACTERIZATION OF TROPOMYOSIN FROM BOVINE THYROID

RYOJI KOBAYASHI a, MASATO TAWATA a, MYLES L. MACE, Jr. b, WILLIAM A. BRADLEY c and JAMES B. FIELD a ~Diabetes Research Center, St. Luke's Episcopal Hospital, and Department of Medicine, Bavlor College of Medicine, h Department of Cell Biology, Baylor College of Medicine and CDivision of Atherosclerosis and Eipoprotein Research, Department of Medicine, Bavlor College of Medicine, Houston, TX 77030 (U.S.A.)

(Received August 3rd, 1981) (Revised manuscript received November 16th, 1981)

Key words: Tropomyosin; A'ctin binding," Troponin; (Bovine thyroid)

A tropomyosin has been purified from bovine thyroid and its properties compared with those of rabbit skeletal muscle tropomyosin. Thyroid tropomyosin was separated from contaminating vascular smooth muscle tropomyosin by hydroxyapatite chromatography. Thyroid tropomyosin resembles tropomyosin from other non-muscle cells in regard to subunit size, mobility on SDS-polyacrylamide gels in the presence and absence of 6 M urea, amino acid composition and morphology. Thyroid tropooo myosin has a subunit molecular weight of 30000 and forms Mg 2+ paracrystals with an axial period of 345 A, while paracrystai periodicities of muscle tropomyosins are 400 ~,. The amino acid composition of thyroid tropomyosin is very similar to that of other non-muscle cell tropomyosins. However, thyroid tropomyosin differs from other non-muscle cell tropomyosins in its ability to bind to actin and troponin as described below. Although the binding of brain tropomyosin to F-actin is thought to be weaker than that of muscle tropomyosin (Fine et al. (1973) Nature New Biol. 245, 182-186), both thyroid and muscle tropomyosins bind to actin in a similar ratio of 1 tropomyosin/6-7 actin monomers at saturation. The binding of tropomyosin to F-actin is strongly dependent on the Mg 2+ concentration. With thyroid tropomyosin, binding begins at I mM and is complete at about 4-5 mM Mg 2+ while, with muscle tropomyosin, binding is initiated at 1 mM Mg 2+ and reaches saturation at 2-3 mM Mg 2+. At saturation, both thyroid and muscle tropomyosins bind to the same binding site(s) on actin filaments with similar affinity. In contrast to platelet tropomyosin (Cote et al. (1978) FEBS Lett. 94, 131-135), thyroid tropomyosin binds to skeletal muscle troponin and troponin T. One-dimensional peptide maps of thyroid and rabbit skeletal muscle tropomyosin are distinctly different from each other. The air oxidation of thyroid tropomyosin yields covalently linked dimers similar to skeletal muscle tropomyosin dimers. In contrast to muscle tropomyosins, [32P]phosphate is not incorporated into thyroid tropomyosin.

Introduction

Thyroglobulin, a precursor of thyroid hormones, is synthesized in the thyroid follicle cells. The protein backbone of thyroglobulin seems to be completed in the cisternae of the rough-surfaced endoplasmic reticulum and carbohydrates are ad-

Abbreviations: TSH, thyrotropin; TEMED, N,N,N',N'- tetramethylethylenediamine.

ded in a stepwise manner during the migration of the molecule through the endoplasmic reticulum and Golgi apparatus [1]. The molecule is then transferred through the apical cell region into vesicles which discharge their content into the follicular lumen by exocytosis [1]. Unlike most other hormones, thyroid hormones are stored ex- traceUularly as components of thyroglobulin in the lumen of the thyroid follicle. Secretion of thyroid hormones initially involves transport of the

0167-4838/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

221

thyroglobulin into the follicular cell as colloid droplets by the process of endocytosis. Subse- quently, the colloid droplets fuse with lysosomes, and thyroxine and triiodothyronine are released following hydrolysis of thyroglobulin. It has been proposed that exocytosis and endocytosis may re- quire contractile events and contractile proteins have been tentatively implicated in the mechanism of transport and fusion of colloid droplets and lysosomes in thyroid [2,3]. In the past few years, several cytoplasmic proteins related to the cyto- skeleton have been described in thyroid, which include tubulin, myosin, actin and a profilin-like protein, that interact with actin [4-6].

In addition to the structural proteins, actin and myosin, muscle cells contain tropomyosin, a pro- tein which helps to regulate the Ca 2+-dependent interaction of actin and myosin. Tropomyosin of skeletal and cardiac muscle is known to be an integral component of the thin filament of the sarcomere and to be intimately involved in the Ca 2+ regulatory system for contraction and re- laxation [7]. Situated in the two grooves of the double-stranded structure of filamentous actin (F- actin), tropomyosin forms a long filament by ag- gregation of individual molecules. Throughout its length each tropomyosin interacts with seven actin monomers on each of the two strands of F-actin. Each tropomyosin molecule also binds 1 mol of troponin complex. As a result of the binding of Ca 2+ to troponin C, tropomyosin may alter its position in the groove of the actin filament and permit interaction of myosin heads and actin monomers [7].

Non-muscle cell tropomyosin has been isolated from platelets, brain, pancreas and fibroblasts [8- 12]. These proteins are similar to the muscle tropomyosin in terms of amino acid composition, a-helical content and ability to bind to actin and form Mg 2÷ paracrystals. However, they were found to differ from the rabbit skeletal muscle tropomyosin in molecular weight [8-12], ability to form end-to-end aggregates [13], NH 2 - and COOH-terminal sequences [ 13] and ability to bind to troponin [14].

In this report, as a part of our endeavor to understand the molecular mechanism of thyroid hormone secretion, we describe the isolation and properties of tropomyosin from bovine thyroid.

Thyroid tropomyosin resembles other non-muscle cell tropomyosin in respect to amino acid com- position [8,9], molecular weight [8-12] and Mg 2+ paracrystals [8-11 ]. However, thyroid tropomyosin is different from other non-muscle cell tropomyo- sins in its ability to bind to actin [9] and~ troponin [14].

Methods and Materials

Bovine thyroids were obtained from a local abattoir and transported to the laboratory packed in ice. After trimming, the tissue was used im- mediately or stored at -20°C.

Purification of bovine thyroid tropomyosin Bovine thyroid glands (200 g) were sliced and

homogenized in 3 vol. of 6 mM 2-mercaptoethanol in a Waxing blender for 30 s at 4°C. All subse- quent steps were performed at 4°C unless stated otherwise. 2-Mercaptoethanol (6 raM) was present at all stages of purification. The homogenate was filtered through three layers of cheese cloth. The filtered homogenate was mixed with 20 vol. of absolute ethanol and re-homogenized in a Waxing blender for 15 s. The supernatant wasi discarded and the sediment was suspended in ether, washed with the same solvent and air dried. The dried residue was extracted overnight in 1 M KCI/10 mM Tris-HC1/6 mM 2-mercaptoethanol, pH 7.5 (buffer A). The insoluble residue was removed by centrifugation at 30000 X g for 20 rain. The super- natant was heated for 5 rain in a boiling water bath and then rapidly cooled in an ice bath. The coagulated proteins were removed by: centrifuga- tion at 30000 × g for 20 rain. The soluble, thermo- stable fraction was brought to 40% s~turation by addition of solid ammonium sulfate. After stand- ing for 60 rain, the precipitate was removed by centrifugation at 30000 × g for 20 rain and the supernatant was brought to 60% saturation by addition of solid ammonium sulfate. The precipi- tate was recovered by centrifugation at 30000 × g for 20 rain, redissolved in 100 ml of buffer A and dialyzed against the same buffer for 4 h. The dialyzed sample was further purified by acidifica- tion to pH 4.1 with l M HCI. The precipitate was dissolved in 1 M KCI, 6 mM 2-mer~aptoethanol and 5 mM potassium phosphate buffer, pH 7.0

222

(buffer B) and dialyzed exhaustively against the same buffer. The dialyzed sample was applied to a column (2.2 × 9 cm) of hydroxyapatite which had been equilibrated 'with buffer B. The column was eluted with a linear phosphate gradient generated from 300 ml of 5 mM phosphate and 300 ml of 250 mM phosphate in 1 M KC1/6mM 2- mercaptoethanol, pH 7.0. Fractions of 4.7 ml were collected at a rate of 30 ml per h. Coomassie brilliant blue binding assay for protein [15] and SDS-polyacrylamide gel electrophoresis were used to monitor the elution profile. A representative chromatographic profile is shown in Fig. 2.

Gel electrophoresis and densitometry SDS-polyacrylamide gel electrophoresis was

performed as described by Laemmli [16]. Urea/SDS-polyacrylamide gel electrophoresis was identical to normal SDS-polyacrylamide gel elec- trophoresis except that the running gel contained 6 M urea and the stacking gel and running buffer contained 3 M urea. After electrophoresis the gels were fixed and stained with Coomassie brilliant blue R according to the method of Fairbanks et al. [17]. In some experiments, the gels were stained with Fast green FCF as described by Potter [18]. Quantitative densitometric measurements were made with a Joyce-Loebl densitometer on gels stained in the linear range with either Fast green of Coomassie brilliant blue R as described by Potter [18]. The quantity of actin, thyroid and skeletal muscle tropomyosln was determined by densitometry of the Coomassie brilliant blue stained gels on the basis of standardization with highly purified proteins. Representative standard curves are shown in Fig. 5.

Determination of tropomyosin binding to F-actin by ultracentrifugation

Both actin and tropomyosin were dialyzed for 16h against 2mM Tris-HCl/0.5 mM CAC12/0.5 mM ATP/6 mM 2-mercaptoethanol, pH 8.0, prior to use. The two proteins were then mixed to bring the final concentrations of actin and tropomyosin to 1 mg/ml and 0.3 mg/ml, respectively. Aliquots (final volume of 100 ~1) were placed in microfuge tubes (Beckman) and various concentrations of KCI and MgC12 were added from appropriate

stock solutions. Following a 2-h incubation at 20°C, the samples were centrifuged at 24000 rev./min for 2 h at 20°C in a Sorval AH627 rotor by the method of Murthy and Bharucha [19]. Resulting precipitates were analyzed by SDS- polyacrylamide gel electrophoresis.

Interaction between tropomyosin and troponin Mixtures of tropomyosin and troponin were

dialyzed against 50 mM Tris-HCl, 50 mM MgC12, pH 8.0 for 16h. Tropomyosin paracrystals which formed during the dialysis were collected by centrifugation at 20000 X g for 30 min and analyzed by SDS-polyacrylamide gel electrophore- sis.

Interaction between tropomyosin and troponin T was analyzed by the method described by Drabikowski and Dabrowska [20]. Both tropomyosin and troponin T were dialyzed for 16h against 10 mM Tris-HCl/0.5 M KCI/6mM 2-mercaptoethanol, pH 7.5, prior to use. A mixture of the two proteins was made to bring the final concentration of each to 0.5 mg/ml. It was then dialyzed against 10 mM Tris-HCl/0.1 M KCI/6 mM 2-mercaptoethanol, pH 7.5, for 16 h. Resulting precipitates were collected by centrifu- gation at 20000 × g for 30 min and analyzed by SDS-polyacrylamide gel electrophoresis.

Tropomyosin paracrystals for electron microscopic examination

Tropomyosin paracrystals were formed from 2 mg/ml protein solutions by overnight dialysis against 50 mM Tris-HCl/50 mM MgCI 2, pH 8.0. The resulting precipitates were placed on carbon- and Formvar-coated microscope grids and nega- tively stained with 1% uranyl acetate and ex- amined by a Philips electron microscope.

Air oxidation studies Air oxidation of tropomyosin was performed as

described by Stewart [21], by stirring 0.5 mg/ml solution of tropomyosin in 1 M NaCI/25 mM CUC12/25 mM sodium borate, pH 9.3, at 20°C for 16h. The product was characterized by SDS- polyacrylamide gel electrophoresis in the presence and absence of 2-mercaptoethanol in the sample buffer.

Phosphorylation experiments Bovine thyroid slices (50-100 mg wet wt.) about

0.2 mm thick were prepared at room temperature using a Stadie-Riggs microtome and incubated (1.5 g of wet tissue) for 2 h at 37°C, under 95% 02 and 5% CO 2 in 15 ml of modified Krebs-Ringer bicarbonate buffer (without unlabelled KH 2PO 4) containing inorganic [32 P]phosphate (5 mCi/flask), 8 mM glucose, 1 mg/ml of bovine serum albumin and 100 mU/ml of bovine TSH. After the incuba- tion, the slices were rapidly rinsed in 100 ml of Krebs-Ringer phosphate buffer. Then, the slices were homogenized in buffer A in a glass tissue homogenizer. The homogenate was heated for 5 min in a boiling water bath and rapidly cooled in an ice bath. The samples were rehomogenized in a glass tissue homogenizer and incubated for 2 h at room temperature. After the incubation, the coagulated proteins were removed by centrifuga- tion at 30000 × g for 20 min. The soluble fraction was further purified by acidification as described above. The precipitate was dissolved in 5 ml of buffer B and dialyzed exhaustively against the same buffer. The dialyzed sample was applied to a small hydroxyapatite (10 ml) column and eluted as described above. A representative chromato- graphic profile is shown in Fig. 12. Resulting fractions were analyzed by SDS-polyacrylamide gel electrophoresis. Gels were dried on cellophane paper and were exposed to Kodak X-ray films (XS-I) for 3 days.

Treatment of tropomyosin with alkaline phosphatase 1 mg/ml of thyroid tropomyosin in 50 mM

Tris-HCl/10 mM MgC12/0.15 M NaC1, pH 8.0, was incubated with alkaline phosphatase (40 /~U/ml) at 37°C. Samples (15 /tl) were taken at various times and analyzed by SDS-polyacrylamide gel electrophoresis.

One-dimensional peptide mapping One-dimensional peptide mapping was carried

out according to Cleveland et al. [22]. 1 mg/ml of purified protein in 0.125 M Tris-HCl/0.5% SDS/10% glycerol and 0.0001% Bromophenol blue, pH 6.8, was digested with variable amounts of trypsin or Staphylococcus aureus Vg protease as indicated in the legend to Fig. 11.

Amino acid analyses were performed with a

223

Beckman amino acid analyzer as described previ- ously [23]. Samples were hydrolyzed with 6 M HC1 in evacuated, sealed tubes for 24h at 100°C. Pro- tein concentrations were dertermined by the method of Lowry et al. [24]. In some experiments, protein concentrations were estimated lby the dye binding assay as described by Bradford [ 15]. Bovine ),-globulin was used as a standard.

Highly purified rabbit skeletal musclg actin was purified according to the method of Spudich and Watt [25]. Rabbit skeletal muscle trop0nin was a gift of Dr. James Potter, Departmenlt of Phar- macology, University of Cincinnati iSchool of Medicine. Troponin T was purified f~om rabbit skeletal muscle as described previously! [26]. Rab- bit skeletal muscle and chicken gizzard tropomyo- sin were purified according to the method of Ei- senberg and Kielley [27].

Acrylamide, bisacrylamide, sodium dodecyl sulfate, TEMED, ammonium persulfate, Coomas- sie brilliant blue R and hydroxyapatit¢ (Bio-Gel HT) were purchased from Bio-Rad Laboratories. Trypsin, alkaline phosphatase and Coornassie bril- liant blue G-250 were obtained from Sig~na Chem- ical Company. S. aureus V 8 protease was~ a product of Miles Laboratories.

Results

Purification of tropomyosin from thyroid The SDS-polyacrylamide gel electrophoresis

pattern of the thyroid tropomyosin preparation at the stage of isoelectric precipitation is shown in Fig. 1. Crude bovine thyroid tropomyosin gives rise to a close-spaced doublet band of approx. 30000 molecular weight with a doublet band of approx. 35000 molecular weight as a minor con- taminant. Both the 30000-dalton and 35 000-dalton doublets co-electrophoresed with crude bovine brain tropomyosin preparations on SDS- polyacrylamide gels (Fig. 1). The 35000-dalton contaminant was also found in the preparation of bovine brain tropomyosin [12] and chicken em- bryo brain tropomyosin [9] and was attributed to smooth muscle tropomyosin from contaminating blood vessels. Further purification of thyroid tropomyosin by hydroxyapatite chromatography removed much of the smooth muscle tropomyosin and other minor contaminants. Thyroid

224

Fig. 1. SDS-polyacrylamide gel electrophoresis of crude tropomyosins, a and b, standard SDS-polyacrylamide gel elec- trophoresis; c and d, SDS-polyacrylamide gel electi'ophoresis containing 6 M urea; a and c, thyroid tropomyosin (50/~g); b and d, brain tropomyosin (10 txg).

tropomyosin was eluted in a single fraction prior to the elution of smooth muscle tropomyosin (Fig. 2). Small amounts of other contaminants with chain weight about 70000 and Coomassie brilliant blue G positive materials other than protein (Fig. 2A and B) in the dye binding assay [15] were removed in fractions prior to the elution of thyroid tropomyosin. A yield of approx. 5 mg tropomyosin with approx. 95% purity was recovered from the hydroxyapatite column starting with 200g of bovine thyroid.

The results of SDS-polyacrylamide gel electro- phoresis in the presence and absence of urea are shown in Fig. 3. In the absence of urea, the puri- fied thyroid tropomyosin consists of two bands with approximate molecular weight of 30000. Rabbit skeletal muscle tropomyosin migrates with an anomalously high apparent molecular weight on urea/SDS-polyacrylamide gels [28] (Fig. 3). Bovine thyroid tropomyosin also migrated as a closely-spaced doublet with an apparent molecular weight of 45 000 on urea/SDS-polyacrylamide gels. However, the electrophoresis patterns of both rab- bit skeletal muscle tropomyosin and chicken giz- zard tropomyosin were different from that of thyroid tropomyosin. The tropomyosin with a 35000-dalton doublet band which originated from contaminating blood vessels co-electrophoresed with chicken gizzard tropomyosin on both SDS- and SDS/urea-polyacrylamide gels (data not shown).

Electron micrograph of Mg 2+ paracrystais When the thyroid tropomyosin was dialyzed

against 50 mM Tris-HC1/50 mM MgC12, pH 8.0, paracrystalline tactoids were formed. When these paracrystals were negatively stained with 1% uranyl acetate and examined in the electron microscope, they had an average axial periodicity of about

I ~30

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Q20

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20 40 60 80 100 120 140 160 Fr'act ion number

C~25M I

O.20M ~_

O.15M

8 (~IOM u Q

o.osM o 0.

Fig. 2. A, Chromatographic purification of the crude thyroid tropomyosin on hydroxyapatite. Details of the conditions are given in the Methods and Materials section. B. SDS-polyacrylamid¢ gel electrophoresis of fractions obtained during the purification. Crude thyroid tropomyosin (gel 0), hydroxyapatite fractions 18 to 90 (gels 18 to 90).

Fig. 3. SDS-polyacrylamide gel electrophoresis of thyroid, chicken gizzard and rabbit skeletal muscle tropomyosin in the absence (A) and presence (B) of 6 M urea. Gels a and d. thyroid tropomyosin; gels b and e, chicken gizzard tropomyo- sin; gels c and f, rabbit skeletal muscle tropomyosin.

345 tk (Fig. 4). Mg 2+ paracrystals produced f rom rabbit skeletal muscle t ropomyosin, chicken giz- zard t r o p o m y o s i n and the smoo th muscle t ropomyos in f rom contaminat ing blood vessels had a periodicity of about 400,~ characteristic of all muscle t ropomyosin (Ref. 10 and Fig. 4).

Amino acid composition Table I indicates the amino composi t ion of

thyroid t ropomyosin together with the composi- t ions of other previously reported tropomyosins. T h e a m i n o acid c o m p o s i t i o n o f t h y r o i d t ropomyos in is similar to that of human platelet and chicken embryo brain t ropomyosins [8,9].

Thyroid and skeletal muscle tropomyosin binding to F-actin

The binding of thyroid and skeletal muscle t ropomyos in to F-act in was determined by the combina t ion of ultracentrifugation and SDS- polyacrylamide gel electrophoresis with quanti ta- tive densi tometry as described in the Methods and Materials section. In order to take into account possible differences in the staining intensities of the three proteins, densi tometry of Coomassie bril- liant blue stained gels was calibrated with the use

225

TABLE I

COMPARISON OF AMINO ACID COMPOSITIONS OF THYROID, PLATELET AND BRAIN TROPOMYOSIN

Amino Bovine Human Chick acid thyroid platelet ~ brain b

Lys 89 c 79 7 I His 17 4 12 Arg 53 61 56 1/2 Cys 6 5 6 Asp 89 88 84 Thr 26 26 30 Ser 25 25 38 Glu 209 256 224 Pro 0 0 5 Gly 40 33 32 Ala 90 105 84 Val 42 33 32 Met 19 20 15 Ile 37 37 33 Leu 96 106 72 Tyr 10 8 14 Phe 9 7 13

Values taken from the data of Cohen and Cohen [8]. b Values taken from the data of Fine et al. [9]. c Amino acid analysis was normalized to 105 g.

of the purified protein by the method of Potter [18]. As shown in Fig. 5, staining intensities of these purified proteins increased linearly over a wide range of concentrations. However, staining intensities relative to the weight of these proteins differed f rom each other. Densitome~ry of Fast green F C F stained gels gave similar results (data not shown).

In the absence of KC1, the binding between t ropomyos in and actin was profoundly !affected by Mg 2÷ concentra t ion (Fig. 6). For skeletal muscle t ropomyosin, no binding occurred until ia threshold Mg 2+ level of 1 m M was reached, and then a very small increment in Mg 2+ concentra t ion resulted in a sharp increase of the binding. When the Mg 2+ concentra t ion was 2 - 3 mM, the binding of t r o p o m y o s i n was c o m p l e t e . F o r t h y r o i d t ropomyosin, the threshold Mg 2+ le,~el was the same, 1 mM, but the transition was i less steep. Act in was saturated with the t ropomyosin only when the Mg 2÷ concentra t ion exceeded 4 - 5 mM. At saturation, both thyroid and skeletal muscle t ropomyosins bound to actin in a similar ratio of 1

226

100

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0 3 4 5 6 7 8 9 10 11 12 13 14

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Fig. 4. (left) Electron micrograph of tropomyosin Mg 2+ paracrystals. Details of the conditions are given in the Methods and Materials section, a, rabbit skeletal muscle tropomyosin; b, tropomyosin originating from contaminating blood vessels; c, thyroid tropomyosin; d, chicken gizzard tropomyosin.

Fig. 5. (right) Dependence of densitometric peak areas on the concentration of purified proteins. Highly purified protein standards were subjected to SDS-polyacrylamide gel electrophoresis. Details of the conditions are given in the text. • O, skeletal muscle tropomyosin; O O, thyroid tropomyosin; • • , actin.

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tropomyosin/6-7 actin monomers. In the pres- ence of 0.1 M KCI, the binding between both thyro id and muscle t ropomyos ins and act in was i n d e p e n d e n t of Mg 2+ concen t ra t ion but with a

s imi lar ra t io of 1 t r o p o m y o s i n / 6 , 7 act in mono- mers (Fig. 7).

In o rde r to assess the aff ini ty be tween thyro id t r opomyos in and F-ac t in , sa tura t ing amounts of thy ro id t r opomyos in and muscle t ropomyos in were mixed with act in in the presence of 6 m M Mg 2+ or 100 m M KC1. As shown in Fig. 8 and Tab le II ,

bo th thyro id and muscle t ropomyos ins b o u n d to F -ac t in in a s imilar fashion in compet i t ive experi- men t s such that 1 mol of t ropomyos in was b o u n d to 7 act in monomers .

Fig. 6. Mg 2+ dependent tropomyosin binding to actin in the absence of KC1. Details of the conditions are given in the text. Molar ratios were calculated with the use of calibration curves (Fig. 4) and corrected for chain weight as follows: actin, 42000; thyroid tropomyosin, 2)<30000; muscle tropomyosin, 2)< 35000. • • , skeletal muscle tropomyosin; 0 O, thyroid tropomyosin.

227

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i

0 • • • ,0 8 o o •

o • • o O

o

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I I I ! I I I 1 I 0 1 2 3 4 5 6 7 8 9 10

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Fig. 7. Tropomyosin binding to actin in the presence of 100 mM KC1. Details of the conditions are given in the Methods and Materials section and legends of Fig. 5,

Fig. 8. SDS-polyacrylamide analysis of competitive tropomyo- sin binding to actin. Details of the conditions are !described in the Methods and Materials section, legends of Fig i 5 and Table II. Gels 1, 2 and 3, tropomyosin binding to actin in the presence of 6 mM MgC12; gels 4, 5 and 6, tropom$osin binding to actin in the presence of 100 mM KC1. Gels 1 and 4, thyroid tropomyosin; gels 2 and 5, skeletal muscle tropomyosin; gels 3 and 6, skeletal muscle and thyroid tropomyosin.

T A B L E II

C O M P E T I T I V E B I N D I N G O F T H Y R O I D T R O P O M Y O S I N A N D R A B B I T S K E L E T A L M U S C L E T R O P O M Y O S I N TO ACTIN

The binding of tropomyosins to actin was measured as described in the Methods and Materials section. The protein concentrations were: actin, I mg/ml, thyroid tropomysoin, 0.3 mg/ml; skeletal muscle tropomyosin, 0.35 mg/ml. Molar ratios were calculated with the use of calibration factors (Fig. 5) and corrected for chain weight as follows: actin, 42 000, thyroid tropomyosin. 2 × 30 000; muscle tropomyosin, 2 × 35 000,

Incubations Slot No, Molar Ratio

Actin Skeletal muscle Thyroid Total tropomyosin tropomyosin tropomyosin

6 mM Mg 2+

100 mM KCI

1 7 0.49 0.49 0.98 2 7 1.03 - 1.03 3 7 1.02 1.02 4 7 0.61 0.39 1.00 5 7 1.02 - 1.02 6 7 1.03 1.03

228

S a : b c e:: f : : :

Fig. 9. A. Tropomyosin binding to skeletal muscle troponin. Details of the conditions are given in the Methods and Materials section. Slot a, thyroid tropomyosin binding to troponin; slot b, muscle tropomyosin binding to troponin; slot c, troponin as a standard; slot d, thyroid tropomyosin as a standard; slot e, muscle tropomyosin as a standard. B, Tropomyosin binding to troponin T. Details of the conditions are given in the Methods and Materials section. Slot a, troponin T precipitate; slot b, precipitate mixture of troponin T and thyroid tropomyosin; slot c, precipitate mixture of troponin T and muscle tropomyosin; slot d, precipitate mixture of the three proteins; slot e, thyroid tropomyosin as a standard; slot f, skeletal muscle tropomyosin as a standard.

Binding of thyroid and skeletal muscle tropomyosin to troponin

The in te rac t ion of thyro id and skeletal muscle t r opomyos in with skeletal muscle t ropon in was s tud ied by the me thod descr ibed in the Me thods and Mate r ia l s section. As sliown in Fig. 9A, skeletal muscle t ropon in was inco rpora t ed i n t o bo th thyro id and skeletal muscle t ropomyos in M g 2+- p a r a c r y s t a l s . T h y r o i d a n d ske l e t a l m u s c l e t ropomyos ins were also c o m p a r e d with respect to their b ind ing to skeletal muscle t ropon in T b y the m e t h o d of Drab ikowsk i and D a b r o w s k a [20]. As shown in Fig. 9B, bo th the t ropomyos ins were co-prec ip i t a t ed with skeletal muscle t ropon in T.

Air oxidation studies R a b b i t skeletal muscle t ropomyos in conta ins

one or two cyste ine residues pe r po lypep t ide chain [29] which can be air oxidized to form an in- t r amolecu la r d isulf ide l inkage of the two chains. A s shown in Fig. 10, the air ox ida t ion of thyro id t r opomyos in y ie lded covalent ly- l inked d imers sim- i lar to skeletal muscle t ropomyos in d imers (Ref. 12

a b c d e f::

Fig. I 0. SDS-polyacrylamide gel electrophoresis of air-oxidized tropomyosins. Details of the conditions are given in the text. Slots a-c, buffer without 2-mercaptoethanol; slots d-f, buffer with 2-mercaptoethanol; a and d, mixture of thyroid and muscle tropomyosin was oxidized; b and e, muscle tropomyo- sin was oxidized; c and f, thyroid tropomyosin was oxidized.

229

Fig. I1. One-dimensional peptide mapping of tropomyosins. Details of the conditions are described in the text. A. a and b, non-digested proteins; c and d, digested with 8 gg/ml trypsin; e and f, digested with 4 gg/ml trypsin; g and h, digested with 2 #g/ml trypsin, a, c, e and g, muscle tropomyosin; b, d, f and

h, thyroid tropomyosin. B. a and b, non-diges!ed proteins; c and d, digested with 8 #g/ml V 8 protease; e and f, digested with 2#g/ml V 8 protease, a, c and e, skeletal muscle tropomyosin; b, d and f, thyroid tropomyosin.

and Fig. 10). The component of oxidized tropomyosin running as monomer was probably due to its cysteines being oxidized further to cysteic acid as described by Stewart [21]. When both oxidized thyroid and skeletal muscle tropomyosins were reduced, they behaved similarly to reduced tropomyosins, confirming that the dimer forma- tions were due to disulfide bond formation.

One-dimensional peptide mapping The technique of Cleveland et al. [22] for one-

dimensional peptide mapping by limited proteoly- sis was used to determine whether any similarities existed between the thyroid and skeletal muscle tropomyosin. The digestion patterns of thyroid t ropomyos in and rabbi t skeletal muscle tropomyosin using S. aureus V 8 protease and tryp- sin were distinctly different from each other (Fig. ll).

Phosphorylation experiments Following the demonstration that frog, rabbit

and chicken skeletal muscle tropomyosin and rab- bit cardiac muscle tropomyosin exist, at least in part, in a phosphorylated form [30-32], we in- vestigated the possibility of phosphate incorpora- tion into thyroid tropomyosin by modification of

the methods of Barany et al. [30] and Ribolow and Barany [31]. As shown in Fig. 12, [32p]phosphate was not incorporated into thyroid trgpomyosin. Recently, Lewis and Smillie [33] suggested that the quickly migrating phosphorylated component of rabbit cardiac tropomyosin on Urea/SDS- polyacrylamide gels disappeared upon treatment with alkaline phosphatase. Treatment i of thyroid tropomyosin with alkaline phosphata~e did not alter the SDS-polyacrylamide gel pattern of the protein (data not shown).

Discussion

Cell motility, as well as more specialized cell functions such as pseudopod formatioN, exocyto- sis, endocytosis and granular movemer~ts, appears to be mediated by contractile proteins ha a variety of non-muscle cells [34]. Many, if not all, of these processes are essential in the synthesis, ~torage and secretion of thyroid hormones by the thyroid gland. Morphological studies have revealed abundant cy- toplasmic microfilaments in thyroid cells [35,36]. A possible involvement of microtubules and mi- crofilaments in the thyroid endocytoti~ processes has been demonstrated using drugs which interfere with these structures [2,3]. Since the microfilament

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0.02 0.

O.O1

230

B C D E F " ~ ' 1

~ ~ O , 2 1 ~ "~'1 O.1M

10 20 30 40 50 60 70 80 90 FractiOn number"

D E F

~ ~KX30

30OO

2OOO I

v v

IO(30 o-

G

C D E F G

Fig. 12. Lack of [32p]phosphate incorporation into thyroid tropomyosin. Details of the conditions are given in the text. A. Hydroxyapatite chromatography of the crude thyroid tropomyosin. Each fraction was assayed for protein ( 0 O) and radioactivity (0 0). B. SDS- polyacrylamide gel electrophoresis of fractions obtained during the purification (protein staining). Hydroxyapatite fractions A to G (slots A to (3). C. SDS-polyacrylamide gel electrophoresis of fractions obtained during the purification (autoradiography). Hydroxyapatite fractions A to G (slots A to G).

disrupting agent cytochalasin B inhibited thyro- globulin endocytosis in vitro [2,3], it is possible that actin is involved in thyroid hormone secre- tion. The presence of myosin and actin in the thyroid supports an important role for contractile protein in thyroid function [4]. These observations prompted us to purify and characterize tropomyosin as a part of an investigation of the contractile proteins in the thyroid.

The unusual resistance of tropomyosin to organic solvents and heat treatment provided the basis for the isolation procedure, which was based on the method of Cohen and Cohen [8] for the isolation of platelet tropomyosin and that of Fine et al. [9] for the isolation of chicken embryo brain tropomyosin. One of the major problems encoun- tered in the purification of tropomyosin from thyroid was separation of it from smooth muscle tropomyosin originating from contaminating blood vessels. Traditionally, the separation of non-muscle cell tropomyosin from smooth muscle tropomyo- sin required elution and renaturation of the pro- tein band from polyacrylamide electrophoresis gels. Although purified non-muscle cell tropomyosin was obtained by this technique, the yield was very low [8]. This separation was accomplished by the additional step of a hydroxyapatite chromatogra- phy which was originally described by Eisenberg and KieUey [27] for the separation of troponin and tropomyosin. This simple method provided a product, in good yield, essentially free of smooth muscle tropomyosin and other contaminants. The isolated bovine thyroid tropomyosin consists of two types of subunits of approx. 30000 molecular weight which can be resolved on SDS- polyacrylamide gel electrophoresis in Laemmli's discontinuous buffer system [16]. Brain tropomyosin [12] and platelet tropomyosin [13] also consist of two different polypeptide chains of approx. 30000 molecular weight and this may be a common feature of tropomyosins from all non- muscle cells. In addition, the amino acid composi- tion of thyroid tropomyosin is very similar to that of other non-muscle cell tropomyosins. It contains large amounts of polar residues but no proline. The ratio of lysine/arginine is about 1.68, similar to other non-muscle tropomyosins and in- vertebrate tropomyosins [8] and lower than those of vertebrate tropomyosins [37]. The digestion pat-

terns of thyroid tropomyosin and rabbit skeletal muscle tropomyosin are distinctly different from each other based on the elegant technique of Cleveland et al. [22] for one-dimensional peptide mapping.

The purified protein forms paracrystals in the presence of Mg 2+ , a property of all tropomyosins [10]. Thyroid tropomyosin paracrystals have an average axial periodicity of about 345 .~, which is similar to that of human platelet [8], chicken em- bryo brain [9] and other non-muscle cell tropomyosins [10,11] and significantly less than that of skeletal and smooth muscle tropomyosin paracrystals. The axial period is thought to repre- sent the length of the molecule. The ratio of axial periodicity to the subunit molecular weight of the thyroid tropomyosin is comparable to that of muscle tropomyos!n, so it is reasonable to con- clude that the 345 A period is close to the length of the thyroid tropomyosin molecule as the 400,h, period is close to the length of tropomyosin from muscle.

An important property of muscle tropomyosin is its binding to F-actin in a ratio of one tropomyosin to seven actin monomers [7]. How- ever, the binding of non-muscle cell tropomyosin to F-actin is thought to be much weaker than that of muscle tropomyosin [9]. Fine et al. [9] calcu- lated that the molar ratio of actin to brain tropomyosin was 7 to 0.47, while it was 7 to 1.52 for actin binding to muscle tropomyosin. How- ever, they did not consider possible differences in the staining intensities of the three proteins. Our results using Coomassie and Fast green FCF stained gels indicate that both thyroid tropomyo- sin and skeletal muscle tropomyosin bind to actin in a similar ratio of 1 mol /6-7 mol at saturation. The binding of thyroid tropomyosin to F-actin is strongly dependent on the Mg 2+ concentration. 4-5 mM Mg 2+ is required for binding or thyroid tropomyosin to actin, while muscle tropomyosin binds well at 1-2 mM Mg 2+. Similar Mg 2+- dependency has been reported for brain tropomyosin [9]. The competitive binding study indicates that the affinity of thyroid tropomyosin to F-actin is similar to that of muscle tropomyosin.

Fine et al. [9] reported that brain tropomyosin interacts with muscle troponin to confer Ca 2+

sensitivity on a Mg2+-activated actomyosin. The

231

amino acid sequence of platelet tropomyosin is also very similar to muscle alpha and beta tropomyosin at the hypothetical troponin binding site [14]. In spite of this evidence, Cote et al. [14] found no binding between platelet tr0pomyosin and skeletal muscle troponin. This conclusion was based on no increase in viscosity induced by troponin and no interaction between platelet tropomyosin and troponin T-Sepharose affinity gels. However, thyroid tropomyosin can interact with muscle troponin and troponin T. The reason for this discrepancy with the findings of Cote et al. [14] is not clear at present.

Rabbit skeletal muscle tropomyosin contains one or two cysteine residues per polypeptide chain which can be air-oxidized to form an intramolecu- lar disulfide bond [21]. These cysteine residues must therefore lie adjacent to each other in the native molecule, indicating that the ahains are associated in register [21,38,39]. Thyroid tropomyosin also contains one or two cysteine residues per chain and, after oxidation, it migrates as two bands in good agreement with the studies of Bretscher and Weber for brain tropomyosin [12]. Since no higher weight bands are ,observed. the cysteine residues which are cross-linked appear to lie adjacent to one another with the chains assembled in register.

Evidence has accumulated that frog [31], rabbit [33] and chicken [30] skeletal muscle tropomyosins and rabbit cardiac muscle tropomyosirt [33J un- dergo phosphorylation. Although the ph3~siological significance of this process is unknown, phos- phorylation of rat cardiac tropomyosin jis Ca 2+- sensitive [40]. Furthermore, Lewis and Smillie [33] reported that the more quickly moving minor com- ponent of rabbit cardiac tropomyosin On SDS- polyacrylamide gel electrophoresis was a phos- phorylated species. Our results do notl indicate that [32p]phosphate is incorporated into thyroid tropomyosin and the pattern of thyroid tropomyosin on SDS-polyacrylamide gels is not changed by alkaline phosphatase treatment. The reason for this difference between thyroid tropomyosin and muscle tropomyosin is~ not ap- parent. However, since a single phosph0rylation site is located at serine 283 (penultimate at the COOH-terminal end) of muscle a-tropomyosins, these observations may be related to differences in

232

NH 2- and COOH- te rmina l sequences of muscle and non-musc le cell t ropomyosins [13]. Al terna-

tively, there is the possibili ty that the prote in

kinase responsible for t ropomyosin phosphory- la t ion is absent in thyroid.

Al though the precise physiologic role of thyroid

t ropomyosin remains to be elucidated, its presence in the thyroid with actin and myosin could provide the cell with a contract i le and structural system essential for thyroid hormone synthesis and secre- t ion.

Acknowledgements

The secretarial assistance of Shelley Dear ing is gratefully acknowledged. This research was sup- ported by grant A M 26088 from the Nat iona l Inst i tutes of Health.

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