Identification and localization of enzymes of the fumarate reductase and nitrate respiration systems of escherichia coli by crossed immunoelectrophoresis

Abstract

Crossed immunoelectrophoresis was used to analyze the components of membrane vesicles of anaerobically grown Escherichia coli. The number of precipitation lines in the crossed immunoelectrophoresis patterns of membrane vesicles isolated from E. coli grown anaerobically on glucose plus nitrate and on glycerol plus fumarate were 83 and 70, respectively. Zymogram staining techniques were used to identify immunoprecipitates corresponding to nitrate reductase, formate dehydrogenase, fumarate reductase, and glycerol-3-phosphate dehydrogenase in crossed immunoelectrophoresis reference patterns. The identification of fumarate reductase by its succinate oxidizing activity was confirmed with purified enzyme and with mutants lacking or overproducing this enzyme. In addition, precipitation lines were found for hydrogenase, cytochrome oxidase, the membrane-bound ATPase, and the dehydrogenases for succinate, malate, dihydroorotate, D-lactate, 6-phosphogluconate, and NADH. Adsorption experiments with intact and solubilized membrane vesicles showed that fumarate reductase, hydrogenase, glycerol-3-phosphate dehydrogenase, nitrate reductase, and ATPase are located at the inner surface of the cytoplasmic membrane; on the other hand, the results suggest that formate dehydrogenase is a transmembrane protein.

Nickel-containing hydrogenase isoenzymes from anaerobically grown Escherichia coli K-12

“…We have previously identified an antigen possessing hydrogenase activity by crossed immunoelectrophoretic analysis of the Triton X-100-dispersed membranes employing polyspecific antiserum obtained by using membrane vesicles as the immunogen (11). Van der Plas et al (24), describing a similar analysis, reported a major and a minor precipitin arc, both of which exhibited hydrogenase activity. When we subjected our immunoplates to prolonged activity staining in a container enclosing a hydrogen atmosphere, we observed two hydrogenasecontaining arcs ( Fig.…”

Cell Wall-Associated Proteases of Streptococcus cremoris Wg2

“…Specific antibodies against proteins A and B were obtained by excision of the appropriate immunoprecipitates from CIE gels. Ten of these gel slices were homogenized in 2.5 ml of 0.9% NaCl and injected into rabbits as described previously (7,29). The antibodies were isolated from the rabbit serum as described previously (7,29).…”

Energetics of Leucyl-Leucine Hydrolysis in Streptococcus cremoris Wg 2

“…Isolation of antibodies. Antibodies against cell-free extract of S. cremoris Wg2 were raised and isolated as described elsewhere (26). The protein concentration of the injected solution was 6 mg/ml, and the concentration of the isolated antibodies was 30 mg/ml.…”

“…The protein concentration of the injected solution was 6 mg/ml, and the concentration of the isolated antibodies was 30 mg/ml. CIE. Crossed-immunoelectrophoresis (CIE) was carried out as described previously (26). The gels were run at 2.5 V/cm for 90 min in the first dimension and at 1.5 V/cm in the second dimension.…”