Localization of the tri- and digalactosyl diglyceride in the thylakoid membrane with serological methods

Trigalactosyl diglyceride was isolated from leaves of Urtica dioica and characterized by thin layer chromatography, infrared spectroscopy and by its fatty acid composition. An antiserum to the trigalactolipid was obtained by immunization of rabbits. By means of inhibition experiments with oligosaccharides and mono- and digalactosyl glycerol it was demonstrated that the antibodies are directed towards the alpha-galactosyl-(1 leads to 6)-alpha-galactosyl-(1 leads to 6)-beta-galactosyl-(1 leads to 1)-glycerol configuration of the trigalactosyl diglyceride. Monogalactosyl diglyceride and sulfoquinovosyl diglyceride do not react with this antiserum. However, a cross reaction was observed with digalactosyl diglyceride. The presence of antibodies to tri- and digalactosyl diglyceride was demonstrated in antisera to different chloroplast preparations of Antirrhinum majus and Spinacia oleracea. The antiserum to the trigalactolipid agglutinates stroma-freed chloroplasts. Membrane fragments obtained by the ultra sonication were precipitated. The antiserum is exhausted by trigalactosyl diglyceride but not by digalactosyl diglyceride or digalactosyl glycerol. The antiserum treated with digalactosyl glycerol and digalactosyl diglyceride also agglutinated stroma-freed chloroplasts. 1 g stroma-freed chloroplasts binds 0.17 g antibodies to trigalactolipid. Membrane fragments bind more antibodies to trigalactolipids than stroma-freed chloroplasts. From the agglutination tests it follows that the antigenic determinants of the trigalactolipid and the digalactolipid are localized in the outer surface as well as in the surface directed towards the inside of the thylakoid membrane.