Immunochemical analysis of inner and outer membranes of Escherichia coli by crossed immunoelectrophoresis

Abstract

Isolated membrane fractions of Escherichia coli K-12 yielded complex immunoprecipitate patterns when Triton X-100 and sodium dodecyl sulfate extracts were examined by crossed immunoelectrophoresis with antienvelope immunoglobulins. Twelve of the 46 antigens in the immunoprecipitate patterns of inner (plasma) membranes were identified by zymograms and/or by the use of specific antisera. The following enzyme activities were detected in immunoprecipitates: 6-phosphogluconate dehydrogenase (EC 1.1.1.43); adenosine triphosphatase (EC 3.6.1.3); glutamate dehydrogenase (EC 1.4.1.4), two separate components; malate dehydrogenase (EC 1.1.1.37); dihydroorotate dehydrogenase (EC 1.3.3.1); succinate dehydrogenase (EC 1.3.99.1); lactate dehydrogeanse (EC 1.1.1.27); reduced nicotinamide adenine dinucleotide dehydrogenase (EC 1.6.99.3); protease (EC 3.4.21.1); and glycerol 3-phosphate dehydrogenase (EC 1.1.99.5). The corresponding immunoprecipitate pattern for isolated outer membranes consisted of at least 25 discrete antigens and differed strikingly from that obtained with inner membranes. Two major immunogens were identified as lipopolysaccharide and Braun lipoprotein. A protease-active immunoprecipitate was also detected in this fraction, but attempts to identify the Rosenbusch matrix protein in the crossed immunoelectrophoretic profile were unsuccessful.