Immunochemistry, 1977, Vol. 14, pp. 129-137.

Pergamon Press.

Printed in Great Britain

IMMUNOCHEMICAL STUDIES ON ACETYLCHOLINE RECEPTOR FROM TORPEDO CALIFORNICA A H A R O N A H A R O N O V , 1 REBECA T A R R A B - H A Z D A I ) ISRAEL S I L M A N 2 and SARA F U C H S 1 1Department of Chemical Immunology, and 2Department of Neurobiology, The Weizmann Institute of Science, Rehovot, Israel (First received 15 July 1976; in revised fi~rm 1 October 1976) Abstract--Acetylcholine receptor (AChR) was purified from electric organ tissue of Torpedo californica by solubilization of membrane fragments with 1% Triton X-100 followed by affinity chromatography on a Naja naja siamensis neurotoxin-Sepharose resin. The molecular and pharmacological properties of the purified receptor were determined, and it was shown to have a specific activity of 8-12 nmoles toxin bound per mg protein, corresponding to 80,00(~125,000 daltons per toxin-binding site. Experimental autoimmune myasthenia gravis was consistently induced in rabbits following a single injection of purified AChR. The cellular and humoral immune responses to the purified AChR were analyzed in the injected animals. By lymphocyte transformation in vitro it was shown that such animals responded not only to the homologous immunogen Torpedo AChR--but also to extracts of rabbit skeletal muscle or thymus. Humoral rabbit anti-AChR antibodies gave one precipitation line with AChR in immunodiffusion and immunoelectrophoresis. The antigenic determinants against which antibodies were produced are different from the toxin-binding sites of the AChR. However, anti-AChR antibodies inhibit toxin-binding, probably due to steric hindrance. A possible molecular model is presented for explaining the relationship between the physiological active site, the ~myasthenic determinant" and other antigenic determinants on the AChR molecule.

MATERIALS AND METHODS

INTRODUCTION The nicotinic acetylcholine receptor (AChR) is an integral protein of the postsynaptic membrane. This protein has been extracted and isolated mainly from electric organs of electric fish such as Electrophorus and Torpedo (see for reviews, Meunier et al., 1974; Karlin, 1974; Heilbronn, 1975; Vandlen et al., 1976) and more recently from mammalian muscle (Dolly & Barnard, 1975; Brockes & Hall, 1975). Availability of purified AChR protein permits many new approaches to the study of synaptic function in general, and of the involvement of the receptor as an autoantigen in the neuromuscular disease, myasthenia gravis (MG), in particular. Experimental autoimmune myasthenia gravis (EAMG) was first observed by Patrick & Lindstrom (1973) following injection of purified electric eel AChR into rabbits. Subsequently, E A M G was produced in a variety of experimental animals (Heilbronn et al., 1975; Green et al., 1975; Lennon et al., 1975; Tarrab-Hazdai et al., 1975a; Granato et al., 1976; Fuchs et al., 1976). Direct confirmation that AChR is an autoantigen in the human disease was provided by the observation of cellular and humoral immune responses against AChR in patients with M G (Abramsky et al., 1975a,b; Aharonov et al., 1975a; Appel et al., 1975; Bender et al., 1975). Much of the most recent work on this topic was presented in a symposium devoted to myasthenia gravis (Grob, 1976). In this report we present an immunological analysis of a highly purified preparation of AChR from electric organ tissue of Torpedo californica. IMM 14/2

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Materials Torpedo californica electric organ tissue frozen in liquid nitrogen was purchased from Pacific Bio-Marine, Venice, CA, and stored at -70°C until used. Naja naja siamensis neurotoxin was prepared from venom of the Thailand cobra, Naja naja siamensis (Miami Serpentarium, Miami, FL) according to Karlsson et al. (1971). :~-Bungarotoxin (~-Bgt) was prepared according to Clark et al. (1972). Carrier-free 12sI was obtained from Amersham Searle Co., Sepharose 2B from Pharmacia, complete Freund's adjuvant (CFA) from Difco and Triton X-100 from Packard. Phenylmethylsulfonylfluoride (PMSF) was purchased from Sigma. Buffers: Buffer A, 0.01 M Tris HCI pH 7.5 containing 0.1M NaC1, 10-3M EDTA, 10-SM PSMF and 5 × 10-4M NAN3. Buffer B, 0.01 M Tris HCI, pH7.5 containing 0.1 M NaCI, 10 -3 M EDTA, 10 -5 M PMSF, 5 x 10-4M NaN 3 and 0.1~o Triton X-100. METHODS Assay for the nicotinic receptor site. 125I-:t-Bungarotoxin (1251

Immunochemical studies on acetylcholine receptor from Torpedo californica.

Immunochemistry, 1977, Vol. 14, pp. 129-137. Pergamon Press. Printed in Great Britain IMMUNOCHEMICAL STUDIES ON ACETYLCHOLINE RECEPTOR FROM TORPEDO...
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