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Isolation and characterization of a 29,000 Dalton protein from ascidian (Halocynthia roretzi) body wall muscle
Comp. Biochem. Physiol. Vol. 85B, No. 1, pp. 71-76, 1986 Printed in Great Britain
0305-0491/86 $3.00+ 0.00 Pergamon Journals Ltd
ISOLATION A N D CHARACTERIZATION OF A 29,000 D A L T O N PROTEIN FROM ASCIDIAN (HALOCYNTHIA RORETZI) BODY WALL MUSCLE MASAKI SH1RAKATA,TAKASHI TAKAGI* and KAZUHIKO KONISHI Biological Institute, Faculty of Science, Tohoku University, Scndai, 980 Japan
(Received 28 October 1985) Abstract--1. In ascidian (Halocynthia roretzi) body wall muscle, a large amount of protein which had m.w. of 29,000 Daltons (29 K protein) by SDS-PAGE is present. 2. The 29 K protein existed as an oligomer in the extract. 3. Its sedimentation coefficient S20.wwas 18.8 S. 4. The oligomer was observed as a globular aggregate, which had an average diameter of 16.6 nm. 5. By indirect immunofluorescence method, the 29 K protein was revealed to be localized close to the plasma membrane. 6. The protein, which cross-reacted with the antibodies against the 29 K protein, was found only in body wall muscles of ascidian family and not in other animals.
INTRODUCTION
In vertebrate skeletal muscle, m a n y proteins which constitute myofibrils have been found and characterized. These proteins are classified into three groups: (a) contractile proteins; actin and myosin, (b) regulatory proteins; troponin, tropomyosin and actinins, (c) scaffold (backbone) proteins; desmin, conectin and Z-protein (Obinata et al., 1981). Most of these proteins are also present in vertebrate smooth muscle and invertebrate muscles except troponin which is absent in vertebrate smooth muscle and some of invertebrate muscles. The body wall muscle of ascidian is regarded as a smooth muscle because no striation is observed by electron microscopy (Toyota et al., 1979). However, like a skeletal muscle, this muscle cell has multiple nuclei (Shinohara and Konishi, 1982) and has troponin for the regulation of contraction (Toyota et al., 1979). Thus ascidian body wall muscle has characters of both skeletal and smooth muscles. In this muscle, there is a protein which has a mol. wt of 29,000 Daltons by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS--PAGE). This protein is extracted from washed ascidian myofibrils with thin filaments and was suggested to be a degradation product of troponin T (Toyota et al., 1979). In the present study, we purified and characterized ascidian 29,000 dalton protein.
as previously described (Miyakawa and Konishi, 1984) with some modifications: EGTA was omitted from the washing solution and 0.1 mM diisopropyl fluorophosphate (DIFP) was added. The final pellet was obtained as washed myofibrils.
Isolation of the 29 K protein The washed muscle was suspended in 4 volumes of 10 mM Tris-HCl buffer, pH 8.0, containing 2 mM EDTA, 2 mM ATP and 0.1 mM DIFP with a Polytron (Kinematica, Switzerland) and extracted by gentle stirring for 15 rain at 4°C. The homogenate was centrifuged at 39,000g for 15 rain and the supernatant was collected. The same extraction was repeated again and the pooled supernatant was applied to a column (3 x 20 cm) of DEAE-cellulose (DE-52, Whatman, Inc.) equilibrated with 10raM Tris-HCl buffer, pH 8.0. The proteins in the flow through fraction were pooled and precipitated with ammonium sulfate (60% saturation). After centrifugation at 39,000g for 15 rain, the precipitate was dissolved with 4 M urea, containing 10 mM sodium acetate buffer, pH 5.5, 1 mM EDTA and 1 mM 2-mercaptoethanol (urea buffer) and dialyzed overnight against the same buffer in the cold room. This sample was applied to a column (3 x 18 era) of CM-ccllulose (CM-32, Whatman, Inc.) equilibrated with urea buffer. The column was eluted by increasing NaCI concentration linearly (0-0.3 M) in urea buffer. Absorbance at 280 nm was monitored. Fractions in peaks were examined by SDS--PAGE and the fractions contains only the 29 K protein were pooled. The 29 K protein was precipitated with ammonium sulfate (60% saturation) and dialyzed against 6 M urea, containing 5mMTris-HCl buffer, pHS.0 and 1 mM 2-mercaptoethanol and stored at 4°C until use. Polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) was performed according to the method of Laemmli (1970) using 12.5% slab gel. Preparative SDS-PAGE was carried out on 2mm thickness of 12.5% gel. Mol. wt was determined from relative mobilities of proteins having known subunit reel. wt (horseshoe crab hemocyanin, bovine serum albumin, ascidian actin, ascidian tropomyosin and sperm whale myoglobin).
MATERIALS AND METHODS
Preparation of myofibrils of ascidian body wall muscle Ascidian (Halocynthia roretzi) was obtained from the Marine Biological Station of Asamushi, Tohoku University and a seafood market in Sendal, Myofibrils were prepared *Correspondence address: Takashi Takagi, Biological Institute, Faculty of Science, Tohoku University, Aobayama, Scndai-shi, Miyagi-ken, 980 Japan. 71
MASAKI SHIRAKATAet al.
72
Analytical ultracentrifugation Analytical ultracentrifugation was performed at 47,300 rpm at 21.8°C by a Hitachi analytical ultracentrifuge with Schlieren optics. Phase plate angle was 70°. Observed sedimentation coefficientwas corrected to the conditions of water as a solvent and at 20°C ($2o.,).
with a CM-cellulose column in the presence of urea. Preliminary experiments had shown that the 29 K protein was highly aggregative and toe aggregates was dissociated into monomer in the presence of denaturants such as urea (data not shown). Thus urea was added to the elution buffer to prevent the formation of aggregates, which caused unsuccessful Electron microscopy separation of proteins. The column was eluted by A drop of diluted sample (0.05-0.1 mg/ml) was placed on a carbon-coated 200-mesh copper grid and negatively increasing NaC1 concentration linearly and separated stained with 1% aqueous uranyl acetate. After air-drying, into two peaks as shown in Fig. 2. SDS-PAGE the preparations were examined with a Hitachi H-500 analysis showed that peak 1 contained the 29 K protein and peak 2 contained actin, tropomyosin and electron microscope operated at 100 kV. the 29 K protein (inset in Fig. 2). Fractions in peak Immunochemical techniques 1 shown as a solid bar were pooled as the purified The 29 K protein was further purified by preparative 29 K protein. The yield of the 29 K protein was SDS-PAGE (Lazarides, 1975). The purified protein was 0.24 mg/g of the washed myofibrils. dialyzed against 50mM NH4HCO3 and lyophilized. The Ultracentrifugation analysis of the 29 K protein in antigen was dissolved in phosphate buffered saline (PBS) the absence of urea showed a symmetrical pattern and emulsified with an equal volume of complete Freund's adjuvant (DIFCO Laboratories). Blood was collected 1 (Fig. 3), and the $20., was calculated to be 18.8 S. The week after the last boost. Immunoglobulin G fraction was value of S20,windicates that the 29 K protein is present purified by ammonium sulfate fractionation and DEAE- as an oligomer but not as a monomer. To confirm cellulose chromatography. Western blotting was performed above indication, the 29 K protein wa~ examined by according to the method of Towbin et al. (1979). To prevent electron microscopy. As shown in Fig. 4, globular nonspecific binding to antibodies, the nitrocellulose was aggregates, 16.6 _ 3.3 nm (n = 135) in diameter, were incubated for 1 hr with 3% bovine serum albumin (BSA) seen. To eliminate the possibility that the aggregate and 4/~g/ml anti rabbit whole serum IgO (Sigma Chemicals was an artificial product of urea, the partially purified Co.) in PBS at 25°C, and washed with PBS for 10 rain. Then it was incubated with 2/~g/ml anti 29 K protein containing 29 K protein, obtained without the use of urea, was 3% BSA. After washing the stripe was incubated with examined by electron microscopy. As shown in Fig. 2/,g/ml peroxidase conjugated anti rabbit IgG (Sigma 5, globular aggregates were seen in the electron Chemical Co.) for 40 min. Peroxidase reaction was carried micrographs of the supernatant obtained by centrifuout in 0.05% 3,3'-diaminobenzidine tetrahydrochloride gation at 100,000g of the thin filament fraction and (Doujin), 0.01% H202, 50 mM Tris--HC1 buffer, pH 7.6. the protein purified by DEAE-cellulose. In the supernatant of thin filament fraction, thin filaments were Immunofluorescence observed other than globular aggregates (Fig. 5a), Small pieces of ascidian muscle were rinsed in artificial sea water, and fixed with 4% formaldehyde in PBS for 4 hr. Fixed tissues were embedded in O.C.T. Compound (Tissue 1 2 3 4 5 Tek II, Miles Laboratories), and frozen in liquid nitrogen. The tissues were cut into 8-10/~m sections and fixed in cold acetone. The sections previously incubated with anti whole rabbit serum IgG were immersed in 2/~g/ml anti 29K protein IgG or IgG fraction of preimmun© serum. After washing, the sections were reacted with in 2/zg/mi fluorescein isothiocyanate (FITC)-eonjugated goat anti rabbit IgG (Sigma Chemicals Co.). The stripes were observed with a Nikon phase/epifluorescence microscope.
A RESULTS
Isolation o f the 2 9 K protein Ascidian body wall muscle contained the protein which had the mol. wt 29,000 daltons in SDS-PAGE (Fig. 1, lane 1). The content of the 29 K protein in muscle was almost corresponding to that of actin by densitometry (29K protein; 16.5%, actin; 13.1%). When thin filaments were extracted from the washed myofibrils in the presence of EDTA and ATP, the 29 K protein was also extracted (Fig. 1, lane 2). The thin filament fraction was centrifuged at 100,000 g for 1 hr to remove glycogen and other proteins and yielded the clear supernatant (Fig. 1, lane 3). The supernatant was applied to a DEAE-cellulose column. Actin, tropomyosin and troponin were absorbed to the column and the 29 K protein was eluted in the flow through fraction (Fig. 1, lane 4). A small amount of actin and tropomyosin was also eluted in this fraction. The 29 K protein was further purified
Trn' 29K
I
Fig. 1. SDS-PAGE pattern of purification steps. Muscle homogenate (lane 1); extract of myofibril in the presence of EDTA and ATP (lane 2); supematant ofa centrifugation at 100,000g for 1 hr (lane 3); DEAE-cellulose column flow through fraction (lane 4); purified 29K protein by CM-colluiose column chromatography (lane 5). 29 K, the 29 K protein; A, aetin; Tm, tropomyosin.
Ascidian muscle protein
73
0.2 Z
1.0
0
Ol
0.1 0.5
30
40
50 60 70 Fraction number
80
90
Fig. 2. Purification of the 29 K protein by CM-cellulose chromatography. A column (3 x 18 cm) was equilibrated with 4 M urea, containing 10mM sodium acetate buffer, pH5.5 and l m M 2-mercaptoethanol, and eluted by increasing NaCI concentration linearly as indicated by the solid line. Fraction size = 7 ml/tube. Inset shows the SDS-PAGE pattern of eluted fractions. The numbers indicate fraction number. Fraction 1 indicated by a solid bar was pooled as a purified 29 K protein.
Fig. 3. Sedimentation velocity pattern of the 29 K protein in 10 mM Tris-HC1 buffer, pH 8.0, containing 0.5 mM EDTA and 1 mM 2-mercaptoethanol. Centrifugation was performed at 47,300 rpm at 21.8°C. The protein concentration was 2.3 mg/ml. Schlieren pattern was taken at 10 rain after reaching maximum speed. The calculated sedimentation coefficient, $20,, was 18.8 S.
Fig. 4. Electron micrograph of negatively stained the 29 K protein. The purified 29 K protein in 6 M urea was dialyzed against 10raM Imidazole-HC1 pH 7.0, containing I mM 2-mercaptoethanol and negatively stained with 1% uranyl acetate. The bar represents 0.1/tin.
M~AKI SHIRAKATAet al.
74
Fig. 5. Electron micrograph of negatively stained samples obtained at two stages of the purification procedure of the 29 K protein. Supernatant of thin filament fraction centrifuged at I00,000 g for 1 hr (a); fraction purified DEAE-cellulose (b). The bar represents 0.1 pm.
and the filaments were free from aggregates. These results showed that in the extract the 29 K protein existed as an oligomer, observed as a globular aggregate (16.6nm in diameter) by electron microscopy and also indicated that the 29 K protein had no interaction with thin filaments.
DISCUSSION
A new protein was isolated from ascidian body wall muscle, which had a molecular weight of 29,000 by SDS-PAGE. The 29 K protein exists as an oligomer (S20.w= 18.8 S), and dissociates in monomer in the presence of urea or SDS, and reassociates oli-
Localization of the 29 K protein To investigate the localization of the 29 K protein in the cell, we prepared rabbit anti 29 K protein antibodies. The specificity of the antibodies was assessed by immunoblotting method. Fig. 6 shows the antibodies reacted specifically with the 29 K protein. The localization of the 29 K protein was examined in frozen sections of ascidian body wall muscle by indirect immunofluorescence method. A cell of ascidian body wall muscle is spindle shaped and the size is 10-30/~m in diameter and about 200/zm in length (Shinohara and Konishi, 1982). As shown in Fig. 7, the cross section of inner muscle (upper left) and the longitudinal section of outer muscle (lower right) were seen. In longitudinal section, the plasma membrane was strongly stained with fluorescence and inside of the cell was stained weakly (Fig. 7b). In cross section, the plasma membrane was stained strongly with fluorescence (Fig. 7b). Thus the 29 K protein seems to exist close to the plasma membrane. To elucidate whether the 29 K protein is specific to ascidian muscle cell, we investigated the several muscles of vertebrate; rabbit skeletal muscle and chicken gizzard, and invertebrate; scallop (Patinopecten yessoensis) adductor muscle and swimming crab (Portunus trituberculatus) leg muscle by immunoblotting method. In none of them, did we find any crossreacted ones with anti 29 K protein antibodies. In other species of ascidian muscles (Halocynthia hilgendorfi and Pyura mirabilis), we found the crossreacted proteins with anti 29 K protein antibodies; however, the mol. wt and the contents were slightly different in species (Fig. 8).
a
1
2
b
1
2
Fig. 6. Characterization of anti 29 K protein antibodies. (a) Coomassie Brilliant Blue stained polyacrylamide slab gel of muscle homogenate (lane 1) and the 29 K protein (lane 2). (b) Immunoblotting pattern of(a), stained by the peroxidase procedure using anti 29 K protein antibodies.
Ascidian muscle protein
Fig. 7. Phase-contrast and fluorescence micrograph of frozen sections of ascidian body wall muscle stained indirectly with anti 29 K protein antibodies. The cross section of inner muscle: upper left; the longitudinal section outer muscle: lower right (a, b). The control was stained with preimmune IgG (c, d). The bar represents 50 pro.
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3
To know the function of the 29 K protein, we tried to find similar proteins of other animals, of which function has been known. In rabbit skeletal muscle and chicken gizzard smooth muscle, various proteins which constitute myofibrils have been known. Among these proteins actin and desmin polymerize into filaments. However any protein which makes globular aggregates has not been reported. Furthermore any 29,000 dalton protein which localizes under the plasma m e m b r a n e has not been found in vertebrate striated muscle and smooth muscle. As the content of the 29 K protein in ascidian body wall muscle was almost the same with that of actin, it is assumed that the 29 K protein is not an enzyme, but one of the components of structural protein of ascidian body wall muscle cell. Acknowledgements--We would like to thank Dr. Y. Numakunai (Marine Biological Station at Asamushi) for supplement of ascidian and Dr. H. Ishihara (Department of Biochemistry, Faculty of Agriculture) for ultracentrifugation analysis.
REFERENCES
Fig. 8. SDS-PAGE pattern of ascidian body wall muscles of several species. Halocynthta roretzi (lane 1), Halocynthia hilgendorfi (lane 2), Pyura mirabilis (lane 3). Asterisks indicate the cross-reacted proteins with anti 29 K protein antibodies. 29 K, the 29 K protein; A, actin.
gomer by removal of urea. The oligomer was seen as a globular aggregates which had an average diameter of 16.6 rim. Indirect immunofluorescence of the frozen section revealed the 29 K protein localized at the plasma membrane (Fig. 7). However, the 29 K protein was not thought to be integrated to the plasma membrane, since the 29 K protein was extracted without a detergent. Probably the 29 K protein is localized close to the plasma membrane. The 29 K protein was found only in ascidian family, thus this protein is specific for ascidian muscle.
Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.). 227, 680-684. Lazarides E. (1975) Tropomyosin antibody: The specific localization tropomyosin in nonmuscle cells. J. Cell Biol. 65, 549-561. Miyakawa I. and Koulshi K. (1984) Removal of troponin C and desensitization of myosin B from aseidian smooth muscle by treatment with ethylene diamine tetraacetate. J. Biochem. (Tokyo). 95, 57°65. Obinata T., Maruyama K., Sugita H., Kohama K. and Ebashi S. (1981) Dynamic aspects of structural proteins in vertebrate skeletal muscle. Muscle & Nerve. 4, 456-488. Shinohara Y. and Konishi K. (1982) Ultrastructure of the body-waU muscle of the ascidian Halocynthia roretzi smooth muscle cell with multiple nuclei. J. Exp. Zool. 221, 13%142. Towbin H., Staehelin T. and Gordon J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA. 76, 435(b4354. Toyota N., Obinata T. and Terakado K. (1979) Isolation of troponin-tropomyosin-containingthin filaments from acsidian smooth muscle. Comp. Biochem. Physiol. 62B, 433--441.
0305-0491/86 $3.00+ 0.00 Pergamon Journals Ltd
ISOLATION A N D CHARACTERIZATION OF A 29,000 D A L T O N PROTEIN FROM ASCIDIAN (HALOCYNTHIA RORETZI) BODY WALL MUSCLE MASAKI SH1RAKATA,TAKASHI TAKAGI* and KAZUHIKO KONISHI Biological Institute, Faculty of Science, Tohoku University, Scndai, 980 Japan
(Received 28 October 1985) Abstract--1. In ascidian (Halocynthia roretzi) body wall muscle, a large amount of protein which had m.w. of 29,000 Daltons (29 K protein) by SDS-PAGE is present. 2. The 29 K protein existed as an oligomer in the extract. 3. Its sedimentation coefficient S20.wwas 18.8 S. 4. The oligomer was observed as a globular aggregate, which had an average diameter of 16.6 nm. 5. By indirect immunofluorescence method, the 29 K protein was revealed to be localized close to the plasma membrane. 6. The protein, which cross-reacted with the antibodies against the 29 K protein, was found only in body wall muscles of ascidian family and not in other animals.
INTRODUCTION
In vertebrate skeletal muscle, m a n y proteins which constitute myofibrils have been found and characterized. These proteins are classified into three groups: (a) contractile proteins; actin and myosin, (b) regulatory proteins; troponin, tropomyosin and actinins, (c) scaffold (backbone) proteins; desmin, conectin and Z-protein (Obinata et al., 1981). Most of these proteins are also present in vertebrate smooth muscle and invertebrate muscles except troponin which is absent in vertebrate smooth muscle and some of invertebrate muscles. The body wall muscle of ascidian is regarded as a smooth muscle because no striation is observed by electron microscopy (Toyota et al., 1979). However, like a skeletal muscle, this muscle cell has multiple nuclei (Shinohara and Konishi, 1982) and has troponin for the regulation of contraction (Toyota et al., 1979). Thus ascidian body wall muscle has characters of both skeletal and smooth muscles. In this muscle, there is a protein which has a mol. wt of 29,000 Daltons by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS--PAGE). This protein is extracted from washed ascidian myofibrils with thin filaments and was suggested to be a degradation product of troponin T (Toyota et al., 1979). In the present study, we purified and characterized ascidian 29,000 dalton protein.
as previously described (Miyakawa and Konishi, 1984) with some modifications: EGTA was omitted from the washing solution and 0.1 mM diisopropyl fluorophosphate (DIFP) was added. The final pellet was obtained as washed myofibrils.
Isolation of the 29 K protein The washed muscle was suspended in 4 volumes of 10 mM Tris-HCl buffer, pH 8.0, containing 2 mM EDTA, 2 mM ATP and 0.1 mM DIFP with a Polytron (Kinematica, Switzerland) and extracted by gentle stirring for 15 rain at 4°C. The homogenate was centrifuged at 39,000g for 15 rain and the supernatant was collected. The same extraction was repeated again and the pooled supernatant was applied to a column (3 x 20 cm) of DEAE-cellulose (DE-52, Whatman, Inc.) equilibrated with 10raM Tris-HCl buffer, pH 8.0. The proteins in the flow through fraction were pooled and precipitated with ammonium sulfate (60% saturation). After centrifugation at 39,000g for 15 rain, the precipitate was dissolved with 4 M urea, containing 10 mM sodium acetate buffer, pH 5.5, 1 mM EDTA and 1 mM 2-mercaptoethanol (urea buffer) and dialyzed overnight against the same buffer in the cold room. This sample was applied to a column (3 x 18 era) of CM-ccllulose (CM-32, Whatman, Inc.) equilibrated with urea buffer. The column was eluted by increasing NaCI concentration linearly (0-0.3 M) in urea buffer. Absorbance at 280 nm was monitored. Fractions in peaks were examined by SDS--PAGE and the fractions contains only the 29 K protein were pooled. The 29 K protein was precipitated with ammonium sulfate (60% saturation) and dialyzed against 6 M urea, containing 5mMTris-HCl buffer, pHS.0 and 1 mM 2-mercaptoethanol and stored at 4°C until use. Polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) was performed according to the method of Laemmli (1970) using 12.5% slab gel. Preparative SDS-PAGE was carried out on 2mm thickness of 12.5% gel. Mol. wt was determined from relative mobilities of proteins having known subunit reel. wt (horseshoe crab hemocyanin, bovine serum albumin, ascidian actin, ascidian tropomyosin and sperm whale myoglobin).
MATERIALS AND METHODS
Preparation of myofibrils of ascidian body wall muscle Ascidian (Halocynthia roretzi) was obtained from the Marine Biological Station of Asamushi, Tohoku University and a seafood market in Sendal, Myofibrils were prepared *Correspondence address: Takashi Takagi, Biological Institute, Faculty of Science, Tohoku University, Aobayama, Scndai-shi, Miyagi-ken, 980 Japan. 71
MASAKI SHIRAKATAet al.
72
Analytical ultracentrifugation Analytical ultracentrifugation was performed at 47,300 rpm at 21.8°C by a Hitachi analytical ultracentrifuge with Schlieren optics. Phase plate angle was 70°. Observed sedimentation coefficientwas corrected to the conditions of water as a solvent and at 20°C ($2o.,).
with a CM-cellulose column in the presence of urea. Preliminary experiments had shown that the 29 K protein was highly aggregative and toe aggregates was dissociated into monomer in the presence of denaturants such as urea (data not shown). Thus urea was added to the elution buffer to prevent the formation of aggregates, which caused unsuccessful Electron microscopy separation of proteins. The column was eluted by A drop of diluted sample (0.05-0.1 mg/ml) was placed on a carbon-coated 200-mesh copper grid and negatively increasing NaC1 concentration linearly and separated stained with 1% aqueous uranyl acetate. After air-drying, into two peaks as shown in Fig. 2. SDS-PAGE the preparations were examined with a Hitachi H-500 analysis showed that peak 1 contained the 29 K protein and peak 2 contained actin, tropomyosin and electron microscope operated at 100 kV. the 29 K protein (inset in Fig. 2). Fractions in peak Immunochemical techniques 1 shown as a solid bar were pooled as the purified The 29 K protein was further purified by preparative 29 K protein. The yield of the 29 K protein was SDS-PAGE (Lazarides, 1975). The purified protein was 0.24 mg/g of the washed myofibrils. dialyzed against 50mM NH4HCO3 and lyophilized. The Ultracentrifugation analysis of the 29 K protein in antigen was dissolved in phosphate buffered saline (PBS) the absence of urea showed a symmetrical pattern and emulsified with an equal volume of complete Freund's adjuvant (DIFCO Laboratories). Blood was collected 1 (Fig. 3), and the $20., was calculated to be 18.8 S. The week after the last boost. Immunoglobulin G fraction was value of S20,windicates that the 29 K protein is present purified by ammonium sulfate fractionation and DEAE- as an oligomer but not as a monomer. To confirm cellulose chromatography. Western blotting was performed above indication, the 29 K protein wa~ examined by according to the method of Towbin et al. (1979). To prevent electron microscopy. As shown in Fig. 4, globular nonspecific binding to antibodies, the nitrocellulose was aggregates, 16.6 _ 3.3 nm (n = 135) in diameter, were incubated for 1 hr with 3% bovine serum albumin (BSA) seen. To eliminate the possibility that the aggregate and 4/~g/ml anti rabbit whole serum IgO (Sigma Chemicals was an artificial product of urea, the partially purified Co.) in PBS at 25°C, and washed with PBS for 10 rain. Then it was incubated with 2/~g/ml anti 29 K protein containing 29 K protein, obtained without the use of urea, was 3% BSA. After washing the stripe was incubated with examined by electron microscopy. As shown in Fig. 2/,g/ml peroxidase conjugated anti rabbit IgG (Sigma 5, globular aggregates were seen in the electron Chemical Co.) for 40 min. Peroxidase reaction was carried micrographs of the supernatant obtained by centrifuout in 0.05% 3,3'-diaminobenzidine tetrahydrochloride gation at 100,000g of the thin filament fraction and (Doujin), 0.01% H202, 50 mM Tris--HC1 buffer, pH 7.6. the protein purified by DEAE-cellulose. In the supernatant of thin filament fraction, thin filaments were Immunofluorescence observed other than globular aggregates (Fig. 5a), Small pieces of ascidian muscle were rinsed in artificial sea water, and fixed with 4% formaldehyde in PBS for 4 hr. Fixed tissues were embedded in O.C.T. Compound (Tissue 1 2 3 4 5 Tek II, Miles Laboratories), and frozen in liquid nitrogen. The tissues were cut into 8-10/~m sections and fixed in cold acetone. The sections previously incubated with anti whole rabbit serum IgG were immersed in 2/~g/ml anti 29K protein IgG or IgG fraction of preimmun© serum. After washing, the sections were reacted with in 2/zg/mi fluorescein isothiocyanate (FITC)-eonjugated goat anti rabbit IgG (Sigma Chemicals Co.). The stripes were observed with a Nikon phase/epifluorescence microscope.
A RESULTS
Isolation o f the 2 9 K protein Ascidian body wall muscle contained the protein which had the mol. wt 29,000 daltons in SDS-PAGE (Fig. 1, lane 1). The content of the 29 K protein in muscle was almost corresponding to that of actin by densitometry (29K protein; 16.5%, actin; 13.1%). When thin filaments were extracted from the washed myofibrils in the presence of EDTA and ATP, the 29 K protein was also extracted (Fig. 1, lane 2). The thin filament fraction was centrifuged at 100,000 g for 1 hr to remove glycogen and other proteins and yielded the clear supernatant (Fig. 1, lane 3). The supernatant was applied to a DEAE-cellulose column. Actin, tropomyosin and troponin were absorbed to the column and the 29 K protein was eluted in the flow through fraction (Fig. 1, lane 4). A small amount of actin and tropomyosin was also eluted in this fraction. The 29 K protein was further purified
Trn' 29K
I
Fig. 1. SDS-PAGE pattern of purification steps. Muscle homogenate (lane 1); extract of myofibril in the presence of EDTA and ATP (lane 2); supematant ofa centrifugation at 100,000g for 1 hr (lane 3); DEAE-cellulose column flow through fraction (lane 4); purified 29K protein by CM-colluiose column chromatography (lane 5). 29 K, the 29 K protein; A, aetin; Tm, tropomyosin.
Ascidian muscle protein
73
0.2 Z
1.0
0
Ol
0.1 0.5
30
40
50 60 70 Fraction number
80
90
Fig. 2. Purification of the 29 K protein by CM-cellulose chromatography. A column (3 x 18 cm) was equilibrated with 4 M urea, containing 10mM sodium acetate buffer, pH5.5 and l m M 2-mercaptoethanol, and eluted by increasing NaCI concentration linearly as indicated by the solid line. Fraction size = 7 ml/tube. Inset shows the SDS-PAGE pattern of eluted fractions. The numbers indicate fraction number. Fraction 1 indicated by a solid bar was pooled as a purified 29 K protein.
Fig. 3. Sedimentation velocity pattern of the 29 K protein in 10 mM Tris-HC1 buffer, pH 8.0, containing 0.5 mM EDTA and 1 mM 2-mercaptoethanol. Centrifugation was performed at 47,300 rpm at 21.8°C. The protein concentration was 2.3 mg/ml. Schlieren pattern was taken at 10 rain after reaching maximum speed. The calculated sedimentation coefficient, $20,, was 18.8 S.
Fig. 4. Electron micrograph of negatively stained the 29 K protein. The purified 29 K protein in 6 M urea was dialyzed against 10raM Imidazole-HC1 pH 7.0, containing I mM 2-mercaptoethanol and negatively stained with 1% uranyl acetate. The bar represents 0.1/tin.
M~AKI SHIRAKATAet al.
74
Fig. 5. Electron micrograph of negatively stained samples obtained at two stages of the purification procedure of the 29 K protein. Supernatant of thin filament fraction centrifuged at I00,000 g for 1 hr (a); fraction purified DEAE-cellulose (b). The bar represents 0.1 pm.
and the filaments were free from aggregates. These results showed that in the extract the 29 K protein existed as an oligomer, observed as a globular aggregate (16.6nm in diameter) by electron microscopy and also indicated that the 29 K protein had no interaction with thin filaments.
DISCUSSION
A new protein was isolated from ascidian body wall muscle, which had a molecular weight of 29,000 by SDS-PAGE. The 29 K protein exists as an oligomer (S20.w= 18.8 S), and dissociates in monomer in the presence of urea or SDS, and reassociates oli-
Localization of the 29 K protein To investigate the localization of the 29 K protein in the cell, we prepared rabbit anti 29 K protein antibodies. The specificity of the antibodies was assessed by immunoblotting method. Fig. 6 shows the antibodies reacted specifically with the 29 K protein. The localization of the 29 K protein was examined in frozen sections of ascidian body wall muscle by indirect immunofluorescence method. A cell of ascidian body wall muscle is spindle shaped and the size is 10-30/~m in diameter and about 200/zm in length (Shinohara and Konishi, 1982). As shown in Fig. 7, the cross section of inner muscle (upper left) and the longitudinal section of outer muscle (lower right) were seen. In longitudinal section, the plasma membrane was strongly stained with fluorescence and inside of the cell was stained weakly (Fig. 7b). In cross section, the plasma membrane was stained strongly with fluorescence (Fig. 7b). Thus the 29 K protein seems to exist close to the plasma membrane. To elucidate whether the 29 K protein is specific to ascidian muscle cell, we investigated the several muscles of vertebrate; rabbit skeletal muscle and chicken gizzard, and invertebrate; scallop (Patinopecten yessoensis) adductor muscle and swimming crab (Portunus trituberculatus) leg muscle by immunoblotting method. In none of them, did we find any crossreacted ones with anti 29 K protein antibodies. In other species of ascidian muscles (Halocynthia hilgendorfi and Pyura mirabilis), we found the crossreacted proteins with anti 29 K protein antibodies; however, the mol. wt and the contents were slightly different in species (Fig. 8).
a
1
2
b
1
2
Fig. 6. Characterization of anti 29 K protein antibodies. (a) Coomassie Brilliant Blue stained polyacrylamide slab gel of muscle homogenate (lane 1) and the 29 K protein (lane 2). (b) Immunoblotting pattern of(a), stained by the peroxidase procedure using anti 29 K protein antibodies.
Ascidian muscle protein
Fig. 7. Phase-contrast and fluorescence micrograph of frozen sections of ascidian body wall muscle stained indirectly with anti 29 K protein antibodies. The cross section of inner muscle: upper left; the longitudinal section outer muscle: lower right (a, b). The control was stained with preimmune IgG (c, d). The bar represents 50 pro.
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2
3
To know the function of the 29 K protein, we tried to find similar proteins of other animals, of which function has been known. In rabbit skeletal muscle and chicken gizzard smooth muscle, various proteins which constitute myofibrils have been known. Among these proteins actin and desmin polymerize into filaments. However any protein which makes globular aggregates has not been reported. Furthermore any 29,000 dalton protein which localizes under the plasma m e m b r a n e has not been found in vertebrate striated muscle and smooth muscle. As the content of the 29 K protein in ascidian body wall muscle was almost the same with that of actin, it is assumed that the 29 K protein is not an enzyme, but one of the components of structural protein of ascidian body wall muscle cell. Acknowledgements--We would like to thank Dr. Y. Numakunai (Marine Biological Station at Asamushi) for supplement of ascidian and Dr. H. Ishihara (Department of Biochemistry, Faculty of Agriculture) for ultracentrifugation analysis.
REFERENCES
Fig. 8. SDS-PAGE pattern of ascidian body wall muscles of several species. Halocynthta roretzi (lane 1), Halocynthia hilgendorfi (lane 2), Pyura mirabilis (lane 3). Asterisks indicate the cross-reacted proteins with anti 29 K protein antibodies. 29 K, the 29 K protein; A, actin.
gomer by removal of urea. The oligomer was seen as a globular aggregates which had an average diameter of 16.6 rim. Indirect immunofluorescence of the frozen section revealed the 29 K protein localized at the plasma membrane (Fig. 7). However, the 29 K protein was not thought to be integrated to the plasma membrane, since the 29 K protein was extracted without a detergent. Probably the 29 K protein is localized close to the plasma membrane. The 29 K protein was found only in ascidian family, thus this protein is specific for ascidian muscle.
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