Localization of Phospholipids in the Membrane of Bacillus megaterium

Phospholipase C treatment of Bacillus megaterium protoplasts at 37°C removed up to 63% of the total phospholipid without disrupting the membrane. More than 95% of the total phosphatidylethanolamine and 3′‐glucosaminylphosphatidylglycerol, 80% of the phosphatidylglycerol and about 20% of the cardiolipin and 2′‐glucosaminylphosphatidylglycerol were hydrolyzed. Similar results were obtained with isolated membranes and dispersions of lipids extracted from the membranes, demonstrating that these data do reflect, at least in part, the substrate specificity of phospholipase C and not necessarily the distribution of phospholipids over the two membrane layers. When protoplasts and isolated membranes were incubated with excess phospholipase C at temperatures in between 5°C and 25°C a different hydrolysis pattern was obtained and it was concluded that 50% of the phosphatidylethanolamine and 70% of the 3′‐glucosaminylphosphatidylglycerol are located in the outer membrane layer, the residual being in the cytoplasmic layer. At least 40% of the phosphatidylglycerol was found to be present also in the outer layer. The data do not allow conclusions about the localization of cardiolipin and 2′‐glucosaminylphosphatidylglycero1. Trinitroben‐zenesulfonic acid reacted with 50% of the phosphatidylethanolamine in protoplasts and with nearly 100% of this lipid in isolated membranes at various temperatures and concentrations, thus confirming the symmetric localization of this lipid. The data furthermore indicate that a temperature‐dependent transbilayer movement of phospholipids is induced by phospholipase C treatment of the Bacillus megaterium protoplasts.