Purification of cytoplasmic membranes and outer membranes from Proteus mirabilis

Summary A crude cell envelope suspension has been prepared from Proteus mirabilis after osmotic shock of penicillin-induced spheroplasts. Employing discontinuous sucrose gradients this cell envelope suspension can be fractionated into four fractions. Besides a pellet of remaining spheroplasts and an intermediate fraction with mixed composition a highly purified cytoplasmic membrane fraction and an outer membrane fraction have been obtained. The cytoplasmic membrane fraction is not contaminated with mucopeptide or outer membrane material. It has a buoyant density of 1.13 g/ml and a protein content of 38%. The specific activities of formate dehydrogenase and nitrate reductase and the content of cytochrome b₁ have increased sixfold in comparison with the crude cell envelope suspension. The outer membrane fraction contains only few contaminations with cytoplasmic membrane components and with mucopeptide.The gradient fractions have been characterized by electron microscopy and by polyacrylamide gel electrophoresis.