Purification of membrane fragments derived from the non excitable surface of the eel electroplax

Volume 8, number 3 FEBS LETTERS June 1970 PURIFICATION OF MEMBRANE FRAGMENTS DERIVED FROM THE NON EXCITABLE SURFACE OF THE EEL ELECTROPLAX Annie BAUMAN, Jean-Pierre CHANGEUX and Philippe BENDA* with the technical collaboration of Monique HUCHET D~partement de Biologic Moldculaire, lnstitut Pasteur, Paris Received 17 March 1970 1. Introduction The monocellular electroplax, the elementary unit of the electric organ of various fishes, presents, in the Gymnotid: Electrophorus electricus, two different types of plasma membranes [1]. One, the innervated membrane, receives nerve terminals and responds to both chemical and electrical stimuli. The other, the non-innervated membrane, is not excitable but spe- cialized in active transport. A purification procedure of membrane fragments derived from the innervated surface and based upon their particularly high con- tent in acetyl-cholinesterase (AcChE) has been pre- viously reported [2]. In this letter, we describe the preparation of another type of membrane fragments, whfch do not contain appreciable amounts of AcChE, but are extremely rich in another enzyme: the ouabain sensitive, Na ÷ K ÷ activated, adenosine tri- phosphatase (ATPase). Evidence is given that these fragments are derived from the non-innervated mem- brane of the electroplax. 2. Material and methods Enzyme assays: The ouabain sensitive ATPase was assayed following exactly the procedure of Bonting, Simon and Hawkins [3]. The incubations were carried * We thank Dr. L. Benedetti for the electron micrograph pub- lished in this letter. Research was aided by grants from the "Coll~ge de France", the "Centre National de la Recherche Scientifique", the "D616gation G6n~rale A la Recherche Scien- tifique et Technique", the "National Institutes of Health" and the "Commissariat ~t l'Energie Atomique'. at 37 ° in samples of a total volume of 1 ml and the three reagent blanks and the two sets of inorganic phosphate standards suggested by Bonting et al. were included in each experiment. AcChE was measured by the method of Ellman'~4] in a medium containing: 5 X 10 -4 M acetylthiocholine, 5 X 10-4 M sodium -5,5'-dithiobis-2-nitrobenzoate and 5 X 10 "2 M sodium phosphate pH 7.0. Chemicals: Acetylthiocholine chloride, sodium-5,5'- dithiobis-2-nitrobenzoate, were from Sigma Chemicals Co.; Ouabain was from Serlabo. 3. Results Membrane fragments rich in ATPase were routine- ly purified by the following method: 10 g of fresh electric tissue, cut with scissors into fragments of about 1 cm, are suspended in 50 ml of 0.2 M sucrose in distilled water and homogenized in a Virtiss apparatus for 1 min 30 sec at 75% of its maximal speed. The homogenate (H) is first centri- fuged at 5000 g (20 min in a refrigerated ServaU cen- trifuge rotor SS34 at 6,500rpm). This slow centrifu- gation eliminates unbroken cell fragments, nuclei and most of the connective tissue. The supernatant S is then centrifuged at high speed (105,000 g) in a SW 25 rotor of a Beckman preparative centrifuge. The best separations were obtained with discontinuous gradients established immediately before centrifuga- tion in the following manner. From the bottom to the top are carefully layered, in a 25 ml lusteroid tube: 8 ml of 1.4 M sucrose, 12 ml of 1.0 M sucrose and 5 ml of S. The gradients are centrifuged at 4 ° North-Holland Publishing Company - Amsterdam 145