35. Purification O F Actin Based Motor Protein from Chara Corallina
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No. 10] Proc. Japan Acad., 70, Ser. B (1994) 175 35. Purification of Actin Based Motor Protein from Chara corallina By Keiichi YAMAMOTO , *) Munehiro KIKUYAMA,**) Noriko SUTOH-YAMAMOTO,*) and Eiji KAMITSUBO***) (Communicated by Noburo KAMIYA, M.J. A., Dec. 12, 1994) Abstract: Actin based motor protein was purified from Chara corallina using high performance liquid chromatography. Sodium dodecyl sulfate gel electrophoresis showed that the molecular weight of the main band was about 230 kDa. Although the molecular weight was quite similar to that of the heavy chain of muscle myosin (myosin II), the motor protein was soluble at low ionic strength and antibody raised against the main band did not recognize smooth muscle myosin. The antibody also did not recognize the actin based motor protein from lily pollen tube. The motor protein translocated fluorescently labeled actin filaments at about 25 µm1s in the in vitro motility assay. The MgATPase activity of the motor protein was enhanced by F-actin about 150 fold. Calcium ion concentration had little effect on both the motor activity and the actin activated MgATPase activity. Key words: Cytoplasmic streaming; Chara corallina, motor protein. Introduction. As is well known that the cytoplasmic streaming in characean cells is quite fast (about 70 um/s at 20°C). This streaming is caused by a certain actin based motor protein which runs, with bound vesicle (Nagai and Hayama,1979) or membranous network (Kachar and Reese, 1988), on the track of actin bundles attached to the chloroplasts lying under the plasma membrane (Kamitsubo, 1966; Nagai and Rebhun, 1966; Palevitz et al., 1974; Williamson,1974; Nothnagel and Webb,1982; Kamiya,1986). Speed of the streaming is much faster than that of the sliding of the thin actin filaments past the thick myosin filaments in free shortening muscle. Difference in the speed is due mainly to the motor protein itself because polystyrene beads coated with rabbit muscle heavy meromyosin moved only at about 5 ,um/s on Nitella actin bundles while crude extract of characean cells can support fast movement of fluorescently labeled rabbit skeletal muscle actin (Sheetz and Spudich, 1983; Rivolta et al., 1990). To understand the reason of this high speed, it is essential to purify the motor protein from characean cells and compare it with muscle myosin. Kato and Tonomura reported the existence of muscle myosin like protein in the Nitella cells (1977). However, the protein has not been characterized well after that and its participation to the cytoplasmic streaming was not demonstrated. We, therefore, undertook the purification of this motor protein using in vitro motility assay to follow the activity. The in vitro motility assay uses fluorescently labeled actin and observes its sliding on coverslip coated with motor protein under the fluorescence microscope (Kron and Spudich, 1986). The assay requires very small amount of sample (about 20 ul) and is suitable to follow the motor activity of characean cells because it is not easy to obtain large *) Department of Bioengineering , Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192, Japan. **) Biological Laboratory , Faculty of Liberal Arts, The University of the Air, 2-11 Wakaba, Mihama-ku, Chiba 260, Japan. ***) Biological Laboratory , Hitotsubashi University, Kunitachi, Tokyo 186, Japan. 176 K. YAMAMOTO et al. [Vol. 70(B), quantity of endoplasm from the cells. Characean cell has gigantic size but more than 90% of the cell was occupied by the central vacuole which contains strong proteolytic enzymes. Materials and methods. (a) Plant material and collection of endoplasm. Chara corallina was cultured in plastic buckets filled with well water and containing some soil on the bottom. The buckets were illuminated with diffused sun light under natural conditions. Internodal cells were isolated from neighboring cells and kept in artificial pond water containing 0.1 mM each of KCI, NaCI, and CaCl2 for one day before use. The cells were placed on Parafilm and exposed to the air until the turgor pressure was lost through transpiration. Then both ends of each cell were cut and the vacuolar sap was removed through internal perfusion (Tazawa,1964) with about 100 , t1 of a solution containing 0.25 M sucrose, 30 mM 3-(N-morpholino)propane sulfonic acid (MOPS) buffer, pH 7.0, 3 mM MgCl2i 5 mM EGTA, 2 mM DTT, 3 mg/ml bovine serum albumin (BSA), 1 mM phenyl methyl sulfonyl fluoride (PMSF), 0.05 mg/ml leupeptin, and 0.01 mg/ml aprotinin. The endoplasm and chloroplasts which were left inside the cell were squeezed out together with the perfusion solution and collected in a test tube. In this process, care was taken not to contaminate the specimen with any organisms living on the outer surface of the cell. (b) Proteins and reagents. BSA, leupeptin, and pepstatin were purchased from Sigma Chemical Co. Aprotinin, MOPS, and PMSF were purchased from Wako Chemical Co., Dojin Chemical Co., and Nakarai Chemical Co., respectively. Actin was extracted from the acetone powder of rabbit skeletal muscle and purified according to Spudich and Watt (1971). Smooth muscle myosin was a generous gift of Dr. Shinsaku Maruta of Soka University. Prestained molecular weight marker protein mixture was purchased from BioRad Laboratories, Inc. (c) In vitro motility assay. F-actin was labeled with rhodamine-phalloidin as described by Yanagida et al. (1984). A flow cell was prepared as described by Kron et al. (1991) but coverslip was not coated with either nitrocellulose or silicone. Assay solution contained 25 mM imidazole buffer, pH 7.5, 4 mM MgC12, 3 mM ATP, 0.5 mM EGTA, and 5 mM DTT. An enzyme system to reduce oxygen in solution was also added to avoid photodynamic action of rhodamine-phalloidin on the motility (Harada et al., 1990). Movement of fluorescently labeled actin was observed under a microscope (Nikon Microphot-FX) equipped with epifluorescence optics. Images were taken by a microchan- nel plate-intensified CCD camera (CS2400-97, Hamamatsu Photonics), processed with Argus 10 (Hamamatsu Photonics), and recorded on videoptape with a video recorder (BR-5611, Victor). The average speed of actin filaments was measured as follows. A piece of plastic wrap was placed on the video screen and leading edges of moving actin filaments were marked at time zero. The displacement of these edges after several frames was measured and the speed calculated by dividing the displacement by the time (1/30 sec for 1 frame). (d) Antibodies. Main band of motor protein was cut out from polyacrylamide gel after electrophoresis and homogenized in Freund's complete adjuvant. The suspension was injected to a rabbit. Second injection of the antigen with Freund incomplete adjuvant was done 2 weeks after the first injection. The animal was bled 10 days after the second injection. Isolation of IgG from serum was done using ammonium sulfate fractionation and DEAE cellulose column chromatography. Anti-panmyosin monoclonal antibody which was raised against mouse 3T3 cell myosin was purchased from Amersham Co (RPN. 1169). (e) SDS polyacrylamide gel electrophoresis and immunoblotting. SDS polyacryla- mide gel electrophoresis was done according to Laemmli and Favre (1973) using slab gel of 1 mm in thickness. Protein bands in gels were visualized either with Coomassie Brilliant Blue or silver staining. Electroblotting of protein bands to nitrocellulose membrane was done using a semidry blotting apparatus at 1 mA/cm2 for 1.5 hr. Membrane was washed No. 10] Actin Based Motor Protein from Chara corallina 177 Fig. 1. SDS polyacrylamide gel electrophoresis of the motor protein from Chara corallina. Lanes A and B, smooth muscle myosin and the motor protein from Chara corallina, respectively. A gradient gel (4-20%) was used. The position and the molecular weight of prestained molecular weight marker proteins were indicated. The molecular weight marker kit contained phosphorylase b (130 K), bovine serum albumin (75 K), ovalbumin (50 K), carbonic anhydrase (39 K), soybean trypsin inhibitor (27 K), and lysozyme (17 K). Note that, due to the prestaining, these molecular weights are different from those of unstained proteins. Fig. 2. Results of immunoblotting. Lanes A and B, smooth muscle myosin and crude extract of Chara corallina, respectively, detected by anti-panmyosin. Lane C, the same amount of the crude extract of Chara corallina detected by the anti-motor protein. Prestained molecular weight marker proteins were also blotted, and their position and the molecular weight were indicated. A gradient gel (4-20%) was used. once with TBS-Tween (iris buffered saline, pH 7.5 plus 0.05% Tween-20) solution. Blocking was done in 1% gelatin in TBS-Tween solution for 1 hr. Anti-motor protein IgG was diluted with the blocking solution to 4 ,ug/ml. Anti-panmyosin monoclonal antibody was diluted 20 fold with the same solution. These antibodies were allowed to react for overnight. After thorough washing with TBS-Tween solution, bound anti-motor protein and anti-panmyosin were detected by goat anti-rabbit IgG and goat anti-mouse IgG conjugated with alkaline phosphatase (Promega Co.), respectively. Color development was done using 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazorium dissolved in 0.1 M Tris-HCI, pH 9.5, 0.1 M NaCI, and 5 mM MgC12. Results. (a) Purification of the motor protein. Characean endoplasm and chloro- plasts were squeeze out together with the perfusion solution as mentioned above. To the solution, KCl and MgATP were added to make 0.3 M and 5 mM, respectively. The solution was centrifuged at 17000 x g for 5 min to remove chloroplasts. Resulting supernatant was passed through 0.45 um membrane filter and applied to a Superdex 200 column (Pharmacia) equilibrated with 0.1 M KCI, 20 mM imidazole buffer, pH 7.5, 3 mM MgCl2i1 mM ATP, 1 mM EGTA, and 1 mM DTT.