Separation of inner and outer membranes of Rickettsia prowazeki and characterization of their polypeptide compositions.
Rickettsia prowazeki were disrupted in a French pressure cell and fractionated into soluble (cytoplasm) and envelope fractions. The envelope contained 25% of the cell protein, with the cytoplasm containing 75%. Upon density gradient centrifugation, the envelope fraction separated into a heavy band (1.23 g/cm3) and a lighter band (1.19 g/cm3). The heavy band had a high content of 2-keto-3-deoxyoctulosonic acid, a marker for bacterial lipopolysaccharide, but had no succinic dehydrogenase, a marker for cytoplasmic membrane activity, and therefore represented outer membrane. The lighter band exhibited a high succinate dehydrogenase activity, and thus contained inner (cytoplasmic) membrane. Outer membrane purified by this method was less than 5% contaiminated by cytoplasmic membrane; however, inner membrane from the gradient was as much as 30% contaminated by outer membrane. The protein composition of each cellular fraction was characterized by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. The outer membrane contained four major proteins, which were also major proteins of the whole cell. The cytoplasmic membrane and soluble cytoplasm exhibited a more complex pattern on gels.
D001426:Bacterial Proteins / Q000032:analysis*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000648:ultrastructure; D002473:Cell Wall / Q000648:ultrastructure; D007661:Ketoses / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis*; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis*; D012283:Rickettsia prowazekii / Q000648:ultrastructure*; D013385:Succinate Dehydrogenase / Q000032:analysis; D013400:Sugar Acids / Q000032:analysis
D001426:Bacterial Proteins; D007661:Ketoses; D008565:Membrane Proteins; D010455:Peptides; D013400:Sugar Acids; C002532:2-keto-3-deoxyoctonate; D013385:Succinate Dehydrogenase