639
574th MEETING, BATH
Antigenic Glycopeptides from Electroplax Membranes of Torpedo marmorata ROGER HARRISON, GEORGE G . L U N T and CHRISTOPHER R. ROAST Department of Biochemistry, University of Bath, Claverton Down, Bath BA2 7 A Y, U.K.
The successful isolation and characterization of the nicotinic acetylcholine receptor has been made possible by the availability of an unusual tissue, the electric organ of several species of electric fish. The innervated face of the electroplax cells from these organs is composed of densely packed arrays of receptor molecules and provides a unique opportunity for the study of the receptor in its membrane environment. The human disease myasthenia gravis involves an autoimmune response to antigenic determinants associated with the acetylcholine receptor at the subsynaptic membrane of the neuromuscular junction. It is accordingly important to learn the structural basis of the antigenicity of acetylcholine-receptor-rich membrane preparations, and that from the electric ray Torpedo marmorata constitutes a particularly rich source of material for such studies. Membranes rich in acetylcholine receptor were prepared from the electric organs of Torpedo marmorata as described by Sobel et al. (1977), and samples (1 ml containing 2.5mg of protein) emulsified in complete Freund’s adjuvant were injected into rabbits at weekly intervals for 4 weeks and then at 5-weekly intervals. Samples (50ml) of antisera were collected every 6 weeks starting 10 weeks after the first injection, and individual serum samples were pooled. Crossed-immunoelectrophoresis experiments were done in which the acetylcholine-receptor-rich membrane preparation (25 mg of protein) was solubilized in Triton X-100 (25nil), and samples (1Opl) were electrophoresed in the w/v) containing Triton X-100 ( 1 %, w/v) and second dimension into an agarose gel ( I rabbit antiserum (2%, v/v). The resulting pattern of precipitin lines indicated the presence of at least six antigenic structures on the Triton-soluble membrane preparation. A soluble glycopeptide fraction that contained antigenic determinants also present on receptor-rich membranes was prepared from electroplax membranes as follows. Frozen electric organ (500s) of Torpedo marmoratu was homogenized in IOmM-potassium phosphate buffer, pH 7.4, (500ml) containing I mM-EDTA, 0.1 mM-phenylw/v). The homogenate was centrimethanesulphonyl fluoride and sodium azide (0.2 fuged (7000g, IOmin) and the supernatant was recentrifuged (20000g, I h) giving a pellet that was washed in l0mM-potassium phosphate buffer and centrifuged again (20000g, 1 h). A sample (IOmg of protein) of the washed pellet was suspended in 5Om~-Tris/ HCI buffer, pH 7.8 ( 1 5 ml), containing 4 m ~ - C a C l ,and Pronase ( I mg) and incubated at 37°C for 30min. The incubation mixture was centrifuged (80000g, I h), and the supernatant retained. This supernatant gave two precipitin lines when examined in doublediffusion experiments with antibodies raised against acetylcholine-receptor-rich membranes. One of the precipitin lines showed a reaction of identity with the single line given by Triton-solubilized receptor-rich membrane fragments against the same antibodies. The above supernatant was concentrated and applied to a column of Sephadex (3-50. Elution with double-distilled water gave three major peaks with mol.wts. higher than 30000, 16000 and 12000 respectively, all of which contained both protein and hexose. Material corresponding to the highest-molecular-weight peak gave two precipitin lines in double diffusion experiments against antibodies raised to receptor-rich membranes. Material corresponding to the other two hexose-protein peaks gave no precipitin lines in similar double-diffusion experiments. Crossed immunoelectrophoresis of the highestmolecular-weight gfycopeptide fraction against anti-membrane antibodies gave a complex pattern of peaks, three of which corresponded t o peaks given under similar conditions by Triton-solubilized receptor-rich membrane fragments.
x,
x,
We thank the Science Research Council for a research studentship (to C. R. R). Sobel, A., Weber, M. & Changeux, J.-P. (1977) Eur. J. Biochem. 80,215-224
Vol. 6
574th MEETING, BATH
Antigenic Glycopeptides from Electroplax Membranes of Torpedo marmorata ROGER HARRISON, GEORGE G . L U N T and CHRISTOPHER R. ROAST Department of Biochemistry, University of Bath, Claverton Down, Bath BA2 7 A Y, U.K.
The successful isolation and characterization of the nicotinic acetylcholine receptor has been made possible by the availability of an unusual tissue, the electric organ of several species of electric fish. The innervated face of the electroplax cells from these organs is composed of densely packed arrays of receptor molecules and provides a unique opportunity for the study of the receptor in its membrane environment. The human disease myasthenia gravis involves an autoimmune response to antigenic determinants associated with the acetylcholine receptor at the subsynaptic membrane of the neuromuscular junction. It is accordingly important to learn the structural basis of the antigenicity of acetylcholine-receptor-rich membrane preparations, and that from the electric ray Torpedo marmorata constitutes a particularly rich source of material for such studies. Membranes rich in acetylcholine receptor were prepared from the electric organs of Torpedo marmorata as described by Sobel et al. (1977), and samples (1 ml containing 2.5mg of protein) emulsified in complete Freund’s adjuvant were injected into rabbits at weekly intervals for 4 weeks and then at 5-weekly intervals. Samples (50ml) of antisera were collected every 6 weeks starting 10 weeks after the first injection, and individual serum samples were pooled. Crossed-immunoelectrophoresis experiments were done in which the acetylcholine-receptor-rich membrane preparation (25 mg of protein) was solubilized in Triton X-100 (25nil), and samples (1Opl) were electrophoresed in the w/v) containing Triton X-100 ( 1 %, w/v) and second dimension into an agarose gel ( I rabbit antiserum (2%, v/v). The resulting pattern of precipitin lines indicated the presence of at least six antigenic structures on the Triton-soluble membrane preparation. A soluble glycopeptide fraction that contained antigenic determinants also present on receptor-rich membranes was prepared from electroplax membranes as follows. Frozen electric organ (500s) of Torpedo marmoratu was homogenized in IOmM-potassium phosphate buffer, pH 7.4, (500ml) containing I mM-EDTA, 0.1 mM-phenylw/v). The homogenate was centrimethanesulphonyl fluoride and sodium azide (0.2 fuged (7000g, IOmin) and the supernatant was recentrifuged (20000g, I h) giving a pellet that was washed in l0mM-potassium phosphate buffer and centrifuged again (20000g, 1 h). A sample (IOmg of protein) of the washed pellet was suspended in 5Om~-Tris/ HCI buffer, pH 7.8 ( 1 5 ml), containing 4 m ~ - C a C l ,and Pronase ( I mg) and incubated at 37°C for 30min. The incubation mixture was centrifuged (80000g, I h), and the supernatant retained. This supernatant gave two precipitin lines when examined in doublediffusion experiments with antibodies raised against acetylcholine-receptor-rich membranes. One of the precipitin lines showed a reaction of identity with the single line given by Triton-solubilized receptor-rich membrane fragments against the same antibodies. The above supernatant was concentrated and applied to a column of Sephadex (3-50. Elution with double-distilled water gave three major peaks with mol.wts. higher than 30000, 16000 and 12000 respectively, all of which contained both protein and hexose. Material corresponding to the highest-molecular-weight peak gave two precipitin lines in double diffusion experiments against antibodies raised to receptor-rich membranes. Material corresponding to the other two hexose-protein peaks gave no precipitin lines in similar double-diffusion experiments. Crossed immunoelectrophoresis of the highestmolecular-weight gfycopeptide fraction against anti-membrane antibodies gave a complex pattern of peaks, three of which corresponded t o peaks given under similar conditions by Triton-solubilized receptor-rich membrane fragments.
x,
x,
We thank the Science Research Council for a research studentship (to C. R. R). Sobel, A., Weber, M. & Changeux, J.-P. (1977) Eur. J. Biochem. 80,215-224
Vol. 6
