Phospholipid topography of the photosynthetic membrane of Rhodopseudomonas sphaeroides.

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.