Comparative immunological and chemical analysis of lipids and carotenoids of the D1-peptide and of the light-harvesting-complex of photosystem II of Nicotiana tabacum

The light-harvesting-complex (LHCP) was isolated from photosystem II of Nic otiana tabacum var. John William's Broadleaf by means of the detergent acet yl-beta-D-glucopyranoside and fractionating centrifugation. The D1-peptide of photosystem II was isolated as a dimer with the molecular mass of 66 kDa from the chlorophyll-deficient tobacco mutant N. tabacum Su/su by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis. Both preparatio ns were characterized by means of the Western blot procedure using monospec ific antisera to the proteins of photosystem II and monospecific antisera t o lipids with which the lipids bound to peptides were determined. In parall el to this, lipids bound to the isolated LHCP-complex and to the isolated D 1-peptide were determined by lipido-chemical methods. The extraction of the isolated core peptide D1 with a mixture of boiling me thanol and chloroform and subsequent HPLC-chromatography showed that in the D1-peptide isolated via SDS-polyacrylamide gel electrophoresis, monogalact olipids, phosphatidylglycerol and sulfolipid molecules are bound in the mol ar ratio 1:3:17. By means of the immunological procedure of Western blottin g we were able to show that the 66 kDa band of the isolated dimeric D1 reac ts positively only with the antisera to monogalactolipid, sulfolipid, beta- carotene and violaxanthin. With the antiserum to digalactolipid and that to phosphatidylglycerol a positive reaction is only observed if the preparati on used in the Western blot is not the isolated D1-peptide but a "total" ph otosystem II-preparation. The lipid extraction of the LHCP-complex and the subsequent analysis by thi n-layer chromatography led to the result that the isolated LHCP-complex con tained in bound form 3 molecules monogalactolipid, 1 molecule of digalactol ipid, 1 molecule of phosphatidylglycerol and 1 molecule of lutein. Less tha n 1 molecule of sulfolipid, beta-carotene, neoxanthin and violaxanthin are found. In the Western blot analysis only the antiserum to monogalactolipid and phosphatidylglycerol and among the carotenoid antisera only the antiser a to beta-carotene, violaxanthin and to neoxanthin reacted. With the antise ra to the digalactolipid, to the sulfolipid and the antisera to the xanthop hylls, namely to lutein and neoxanthin, a positive reaction occurred only i f the material used in the Western Blot was the "total" photosystem II-prep aration. By gas chromatography of the fatty acids of the isolated peptide fractions it was shown that, compared to the lipids of photosystem II and of the thyl akoid membrane, in lipids of the isolated D1-peptide and of the LHCP-comple x the saturation degree of fatty acids is strongly increased. Whereas palmi tic acid in chloroplast lipids makes up for only 11% of the fatty acids, th is saturated fatty acid increases in the lipids of the LHCP to 20% and make s up for 74% of total fatty acids in the lipids of the D1-peptide. Linoleic and linolenic acids are completely absent and oleic acid makes up for 14% of total fatty acids. In contrast to the lipids of the thylakoid membrane, the lipids bound to proteins/peptides are characterized by a strongly satur ated character.