The isolation and characterization of antigenic glycopeptides from electroplax membranes of Torpedo marmorata.

  • Christopher Richard Roast

Student thesis: Doctoral ThesisPhD

Abstract

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 Award1982
LanguageEnglish
Awarding Institution
  • University of Bath

Cite this

The isolation and characterization of antigenic glycopeptides from electroplax membranes of Torpedo marmorata.
Roast, C. R. (Author). 1982

Student thesis: Doctoral ThesisPhD