title: Phospholipid topography of the photosynthetic membrane of Rhodopseudomonas sphaeroides.
abstract: The topography of phospholipids in the photosynthetic membranes of Rhodopseudomonas sphaeroides was investigated by using purified chromatophores and spheroplast-derived vesicles (SDVs). Chromatophores are closed vesicles oriented inside out with respect to the cytoplasmic membrane (cytoplasmic side out) and obtained from French-pressed cell lysates. SDVs are oriented right side out (periplasmic side out) and are obtained after osmotic lysis of lysozyme-treated cells. Phosphatidylethanolamine (PE) comprised approximately 62% and phosphatidylglycerol (PG) comprised approximately 33% of the total phospholipid of both vesicle preparations. The relatively membrane impermeable reagent trinitrobenzenesulfonate (TNBS) at 3 mM concentration and 5 degrees C modified chromatophore and SDV PE with kinetics indicating the occurrence of fast- and slow-reacting pools of PE. The fast-reacting pools comprised 33% and 55% of the total PE of chromatophores and SDVs, respectively. The slow-reacting pools comprised 61% and 32% of the total PE of chromatophores and SDVs, respectively. Phospholipase A2 treatment of chromatophores (1 unit/mg of vesicle protein) for 1 h at 37 degrees C resulted in hydrolysis of 73% and 77% of the total PG and PE, respectively. Similar enzyme treatment of SDVs resulted in 14% and 60% hydrolysis of the total PG and PE, respectively. Phospholipase A2 treatment inhibited 60% of the succinate dehydrogenase activity of chromatophores but only 8% of the activity of SDVs, indicating the membrane impermeability of phospholipase A2. Incubation of chromatophores for 10 min with 3 mM TNBS at 5 degrees C and then treatment with phospholipase A2 for 10 min and 1 h resulted in the hydrolysis of 10% and 61%, respectively, of unmodified PE. The results indicate asymmetric distributions of PE polar head groups (32-33% cytoplasmic side, 55-61% periplasmic side) and PG (73% cytoplasmic side, 14% periplasmic side) across the membrane. Also, a rapid and unidirectional transbilayer movement of PE polar head groups from the periplasmic to cytoplasmic surfaces of the membrane appears to occur during phospholipase A2 hydrolysis on the chromatophore surfaces.
mesh_terms: D020130:Bacterial Chromatophores / Q000032:analysis; D001436:Bacteriorhodopsins / Q000032:analysis*; D002338:Carotenoids / Q000032:analysis*; D007700:Kinetics; D008051:Lipid Bilayers; D008563:Membrane Lipids / Q000032:analysis*; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D010743:Phospholipids / Q000032:analysis*; D010788:Photosynthesis; D012242:Rhodobacter sphaeroides / Q000032:analysis*; D013385:Succinate Dehydrogenase / Q000378:metabolism
chemical_list: D008051:Lipid Bilayers; D008563:Membrane Lipids; D010743:Phospholipids; D002338:Carotenoids; D001436:Bacteriorhodopsins; D013385:Succinate Dehydrogenase; D010741:Phospholipases A; D054467:Phospholipases A2