Membrane fragments were prepared from electric organs of the electric ray, Torpedo maromorata and shown to contain exposed acetylcholine receptor (AChR) as evidenced by their binding of radio-labelled alpha-bungarotoxin and Naja naja toxin. When the membranes were labelled with the affinity ligand 3H-MBTA, specific for the acetylcholine binding site of AChR, and subjected to SDS-PAGE analysis, 12 major protein components were detected, only one of which (M.W. 40,000) contained the radiolabel. The protein components of the purified membranes were examined by using a number of approaches. Membranes solubilized in Triton X-100 showed a single precipitin line when analyzed by double diffusion using rabbit anti-(Torpedo membrane) antisera and the presence of four to six antigenic components when analyzed by rocket and crossed immun-electrophoresis using the same antisera. Treatment of purified Torpedo membrane fragments with pronase released soluble glycopeptides which in double diffusion experiments against rabbit anti-(Torpedo membrane) antisera, gave two precipitin lines, one of which showed a reaction of identity with the single line given by solubilized whole membrane fragments. The soluble glycopeptides were fractionated on Sephadex G-50 when three hexose-containing protein peaks A, B and C were separated. Peak A, showed antigenic cross reactivity with rabbit anti-(Torpedo membrane) antiserum in immunodiffusion and electrophoresis assays and inhibited the precipitation of 125I-labelled purified Torpedo acetylcholine receptor by rabbit anti-AChR IgG. Glycopeptide peaks A, B and C when analysed by gas chromatographic and colourimetric techniques all showed the presence of fucose, mannose and galactose, but not sialic acid. Peaks A and B contained glucose while peak C showed additionally glucosamine and galactosamine. These sugar compositions were compared with that obtained by similar analysis of affinity purified AChR from Torpedo marmorata.
|Date of Award||1982|
The isolation and characterization of antigenic glycopeptides from electroplax membranes of Torpedo marmorata.
Roast, C. R. (Author). 1982
Student thesis: Doctoral Thesis › PhD