PARTIAL PURIFICATION AND CHARACTERIZATION OF A TOXIC SUBSTANCE FROM PEDICELLARIAE OF THE SEA URCHIN TOXOPNEUSTES PILEOLUS

Hideyuki NAKAGAWA and Akira KIM U RA Departmentof HealthScience, Facultyof Education, Universityof Tokushima,Tokushima 770, Japan Accepted June 26, 1982

A substance in the pedicellariae of the sea 7.1). The mast cells were obtained from the urchin Toxopneustes pileolus induces severe fluid by centrifugation and counted by breath shortening and dizziness in man (1). toluidine blue staining (5) and then resus Kimura et al. (2) partially purified the toxic pended in the buffer at approximately 106 substances from the pedicellariae of T.pileolus cells/ml. The reaction mixture was pre and studied their pharmacological actions. incubated at 37°C in a metabolic shaker for Recently, we found that the ammonium 5 min and the incubation was continued for sulfate fraction of the crude extract from the 10 min with or without the test compound in pedicellariae of T. pileolus caused a release of 2 ml final volume. The reaction was stopped histamine from isolated smooth muscle (3). by chilling the tubes in an ice-bath. The cells Therefore, in this study we have further were then removed from the medium by attempted to purify the ammonium sulfate centrifugation, and 0.5 ml of 2 N perchloric fraction with gel chromatography and acid was added. Histamine was measured characterized the toxic substance. fluorometrically by the method of Shore The crude extract from the pedicellariae of et al. (6). T. pileo/us was partially purified by am Two protein peaks were obtained at the monium sulfate precipitation (30-65% void volume (120 ml, measured by blue saturation) as previously described (3). The dextran) (Peak I, fractions 10-14) and as a precipitate was resuspended in 0.15 M subsequent peak (Peak II, fractions 22-28). sodium chloride (10 ml) and applied to a The activity of each fraction was bioassayed Sephadex G-200 column (2.2x80 cm) equili for the isotonic contraction of the longitudinal brated with 0.15 M sodium chloride. The muscle of the isolated guinea-pig ileum in column was eluted with the same solution at Tyrode solution at 37'C. The activity of the a flow rate of 16.8 ml per hr, and fractions of Peak I fraction was low, whereas that of the 10 ml each were collected. Protein concen Peak II fraction became higher in proportion tration of the effluent was measured by the to the protein concentrations. The results of method of Lowry et al. (4) using bovine purification steps 1-3 are shown in Table 1. serum albumin as the standard. For the The purities of the ammonium sulfate fraction experiment of histamine release, Wistar rats and Peak II fraction were increased 2 and (male or female) weighing 200 to 250 g were 5.8 times that of the crude extract, respec used. Animals were killed by decapitation and tively. The peak II fraction was acidic (pH exsanguinated. The peritoneum was washed 6.2). The submaximal contraction induced with Krebs-Ringer phosphate buffer contain by the Peak II fraction (10-5 g/ml) was ing 0.1% (w/v) bovine serum albumin (pH reduced to 80±4% (S.E.M., n=5) by 10-8 Table 1. Purification of a toxic substance in pedicellaria from Toxooneustes oileo/us

The biological activity (U) was expressed as the protein concentration which caused a con traction of the longitudinal muscle equivalent to 50% of the 40 mM KCI-induced maximum contraction. g/ml tripelennamine and was decreased by about 60% following the application of 3 x 10-8 g/ml tripelennamine. Furthermore, we have found that the ammonium sulfate fraction evoked release of histamine from isolated smooth muscle (3) and mast cells (in preparation). Therefore, the effect of the Peak II fraction on histamine release from rat peritoneal mast cells was studied. The peak I I fraction (10-4 to 2x10-3 g/ml) induced a dose-dependent release of hista mine from mast cells (ED50: 8.5 x 10-4 g/ml). The peak II fraction at a concentration of 10-3 g/ml caused the release of 61 +5% (S.E.M., n=4) of the total histamine content, and the release reached the maximum at a concentration of 2x`10-3 g/ml. The effect of the ammonium sulfate fraction was smaller than that of the Peak II fraction (ED50: 1.9 x 10-3 g/ml). Compound 48/80 caused almost a maximal release at a concentration of 1 ,ug/ml. The time course of histamine release induced by the Peak II fraction was similar to that induced by compound 48/8C (data not shown). Release was not detected by the Peak I fraction. At present, there are few investigations on histamine release from mast cells by the venom of sea urchins. Therefore, further study of the histamine Fig. 1. Polyacrylamide gel electrophoresis of the release of mast cells seems to be of sig Peak II fraction. Electrophoresis on 200 ug protein was carried out at pH 8.2 for 1 hr with a nificance in verifying the systemic actions of current of 2 mA per gel. The gel was stained with the pedicellarial toxin of sea urchins. Amido Black. A rough calibration of a Sephadex G-200 for her valuable advice on methods for column, performed by running bovine serum electrophoresis. albumin, ovalbumin, and cytochrome c, REFERENCES indicated that the Peak II fraction had a mol wt. of about 20,000. This corresponds to the 1) Fujiwara, T.: On the poisonous pedicellariae of Toxopneustes pileolus. Annotnes Zool. Japan. sedimentation coefficient of 2.1 S quoted by 15, 62-69 (1935) Kimura et al. (2) for their partially purified 2) Kimura, A., Hayashi, H. and Kuramoto, M.: toxin. It has also been reported that the Studies of urchi-toxins: Separation, purification active toxin in the pedicellaria of Tripneustes and pharmacological actions of toxinic sub gratilla had a sedimentation coefficient of stances. Japan. J. Pharmacol. 25, 109-120 2.6 S (7). (1975) 3) Kimura, A. and Nakagawa, H.: Action of an According to the method of Joh and extract from the sea urchin Toxopneustes Goldstein (8), disc electrophoresis in poly pileolus on isolated smooth muscle. Toxicon acrylamide gel was used to examine puri 18, 689-693 (1980) fication procedures. Electrophoresis was 4) Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.: Protein measurement with the carried out in 0.4 M glycine-Tris buffer (pH Folin phenol reagent. J. biol. Chem. 193, 265 8.2) with a 5.5% gel. Electrophoresis of the 275 (1951) Peak II fraction in the disc gel system showed 5) Bray, R.E. and Vanarsdel, P.P., Jr.: In vitro that several protein components were present histamine release from rat mast cells by chemical in this fraction (Fig. 1). Therefore, we further and physical agents. Proc. Soc. exp. Biol. Med. 106, 255-259 (1961 ) purified the Peak II fraction by DEAF-cellulose 6) Shore, P.A., Burkhalter, A. and Cohn, V.H., Jr.: column chromatography (1.1 x 8 cm). The A method for the fluorometric assay of histamine most active fraction which was eluted by in tissues. J. Pharmacol. exp. Ther. 127, 182 400 mM NaCI in 10 mM Tris-HCI buffer 186 (1959) 7) Alender, C.B., Feigen, G.A. and Tomita, J.T.: (pH 7.0) was obtained with a purity about Isolation and characterization of sea urchin 15 times that in the crude extract. We are toxin. Toxicon 3, 9-17 (1965) presently studying this fraction. 8) Joh, T.H. and Goldstein, M.: Isolation and characterization of multiple forms of phenyl Acknowledgement: We wish to thank Dr. ethanolamine N-methyltransferase. Mol. Phar Y. Ishimura (Department of Pharmacology, macol. 9, 117-123 (1973) Tokushima University School of Medicine)