Vol. 107, No. 1, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS July 16, 1982 Pages 272-278 THE AMINO ACID SEQUENCE OF TOXIN V FROM ANEMONIA SULCATA Jean-Jacques Scheffler, Akira Tsugita, Guy Linden", Hugues Schweitzi and Michel Lazdunskit, European Molecular Biology Laboratory, Postfach 10.2209, D-6900 Heidelberg; ?Service de Biochimie Appliquee de Nancy I, B.P. 239, F-54506 Vandoeuvre Les Nancy Cedex, and 'fcentre de Biochimle du CNRS de 1'UniversitP de Nice, Part Valrose, F-06034 Nice Cedex. Received May 19, 1982 INTRODUCTION : Sea apemone toxins have become very useful tools to study the voltage-dependent Na channel in nerve, cardiac and muscle cells (l-4). Four different neurotoxins - AS ASI AS and AS - have been isola- ted in the pure form from Anemonla dicata (5,6f!IThese txxins are polypepti- des consisting of 46, 47, 27 and 46 amino acid residues respectively. Although they are all specific for the voltage-dependent Na channel, they display a marked difference in theLr toxicity for different animal species. Some are more active on crustaceans, others on mammals (6). Three of the four toxins - ASI, ASII and AS1 I 1 - have already been sequenced (7-10). A vered more recent y 'r has been disco- (6); it is the most toxic of the four po ypeptides Pen injected to mice and the most useful one in studies dealing with the Na chan- nel. The present paper reports the primary structure of ASV. MATERIALS AND METHODS : Toxin V was purified according to (6). S-carboxymethy- lation was made according to (11) with minor modifications as follows : 1.3 mg toxin V was reduced with 1.5 ~1 &mercaptoethanol in 250 ill of a 0.36 M Tris- HCl buffer at pH 8.6 containing 8 M urea and 0.2% efbylenediamine-tetraacetate at 37°C under N 3 atmosphere. After 4 hr 22 pmol of C iodoacetic acid (1.14 uCi, New Englan Nuclear Co. and Serva Feinbiochemica) were added and the mix- ture was incubated further for 2 hr at 37'C. The CM-toxin was recovered by gel filtration on a Rio-Gel P2 column (1 x 70 cm) equilibrated with 70% formic acid. Trypsin digestion - 200 nmol of CM-toxin V was digested with trypsin (50/l : w/w, Worthington Biochemicals) in a 0.1 M pyridine-collidine acetate buffer at pH 8.2 at 37°C for 12 hr. Peptide separation was carried out by gel filtration on a Sephadex G25 column (1 x 100 cm) equilibrated in a 0.1 M pyri- dine-acetate buffer at pH 7.0. Peptide elution was monitored by radioactivity and by amino acid analysis after hydrolysis. Amino acid analysis - The toxin or peptides were hydrolysed in a mixture of CF3C02H/HC1 (1:2) at 166°C between 25 to 50 min (12). For cystine analysis, the toxin was oxidized with performic acid (13) and hydrolysed. For tryptopha- ne analysis, hydrolysis was made with 3 N mercaptoethanesulfonic acid according to (14) using a microscale modification (Maeda, Scheffler & Tsugita, unpubli- shed data). Amino acid analysis was performed on a Durrum D500 analyser, set to a sensitivity of 2.5 nmol of amino acids. Abbreviations : Tris : tris(hydroxymethyl)aminomethane; CM : carboxymethyl; AS : toxin I from the sea anemone se; anemone Anemonia sulcata; ASIII Anemonla sulcata; AS : toxin II from the : toxin III from th$'sea anemone Anemonia sulcata; ASV : toxin V from the sea anemone Anemonia sulcata; AX1 : toxin I from the sea anemone Anthopleura xanthogrammica. Enzymes : carboxypeptidase A (EC.3.4.17.1), carboxypeptidase B (EC.3.4.17.2), carboxypeptidase P (EC.3.4.16. l), trypsin from bovine pancreas (EC.3.4.21.4). 0006-291X/82/130272-07$01.00/0 Copyrighi 0 1982 by Academic Press, Inc. All righfs of reproducrion in any form reserved. 272