Binding of Antibodies onto the Thylakoid Membrane IV. Phosphatides and Xanthophylls in the Outer Surface of the Thylakoid Membrane

Abstract

Antisera to the phosphatides phosphatidyl inositol and phosphatidyl choline as well as to the xanthophylls violaxanthin and zeaxanthin were obtained by immunization of rabbits. The phosphatidyl choline antiserum did not give cross reactions with thylakoid membrane glycolipids, nor with phosphatidyl glycerol and phosphatidyl inositol. The antiserum to phosphatidyl inositol also did not react with the glycolipids and phosphatidyl choline. However, a cross reaction with phosphatidyl glycerol was observed. Exhaustion tests revealed, that the phosphatidyl inositol antiserum was fully neutralized with the homologous phosphatide, but not with phosphatidyl glycerol. Antibodies to the phosphatides phosphatidyl inositol and phosphatidyl choline react with stroma- freed, not swellable chloroplasts of Antirrhinum majus only in a monovalent manner as demon­strated by means of the anti-y-globuline consumption test. Sonicated and spun down chloroplasts, however, are agglutinated. Also an antiserum to phosphatidyl inositol treated with exhausting quantities of phosphatidyl glycerol agglutinates this ultrasonic sediment. This type of chloroplast preparation binds approximately double the amount of antibodies to phosphatides. Small membrane fragments of the ultrasonic supernatant in which the outer as well as the inner surface of the thylakoid membrane is accessible to antibodies, are not precipitated. They react with phosphatide antisera just like untreated chloroplasts. Chloroplasts of Antirrhinum majus and of Nicotiana tabacum, which were isolated in Tris buffer and whose thylakoids are swellable and exhibit high Hill reaction rates, were agglutinated by the phosphatide antisera. The antisera to violaxanthin and zeaxanthin gave with the described chloroplast preparations the same reactions as the phosphatide antisera. From this it is concluded that antigenic determinants of the phosphatides and xanthophylls are located in the surface of the thylakoid membrane directed towards the outside. Quantitative investigations on the maximal binding of antibodies showed that 1g stromafreed not swellable chloroplasts of Antirrhinum majus bind maximally 0.06 g antibodies to phosphatidyl choline and approximately four times the amount of antibodies to phosphatidyl inositol. Antibodies to the xanthophylls were bound in an amount of 0.04 to 0.09 g. The order of magnitude of these values fits the values of the maximal binding of antibodies to the glycolipids and also to proteins, which participate in photosynthetic electron transport. Onto the outer surface of the thylakoid membrane, accessible to antibodies, 1 g of antibodies can be maximally bound by 1 g chloroplasts.