Immunological Characterization of the Cytochromes of Escherichia Coli (Hybridonas, Antibodies, Membranes)

170 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1984.Investigations were carried out on the cytochrome components of the E. coli aerobic electron transport system. This system involves two parallel electron transport chains: (UNFORMATTED TABLE FOLLOWS)(high aeration)cyt b(,556) (--->) cyt oDehydrogenases (--->) quinones O(,2)cytochrome b(,558) a(,1) d(low aeration)(TABLE ENDS)Major studies concerned the development of antisera, structural analysis and the biosynthesis of each cytochrome component. In addition, mutants isolated in our lab were analyzed with various immunological reagents. Using these various antisera as probes, the following conclusions could be made about the 2 subunit cytochrome b(,558) a(,1) d oxidase complex: (1) The 55 K dalton subunit possesses b(,558) heme (indicated by the analysis of the cyt d mutants), (2) The UQ-1 binding site is present on the 55 K dalton subunit. Monoclonal antibodies A14-5 and A16-1 bind at this site. (3) Some of the 55 K dalton subunit appears to be present in a dissociated, free form, at least in detergent extracts. (4) The 28 K dalton subunit is present as a dimer in the native cytochrome b(,558) a(,1) d oxidase. (5) The TMPD site is located on the 28 K dalton subunit. (6) The 28 K dalton subunit is largely inaccessible to antibodies and proteases in the native cytochrome b(,558) a(,1) d oxidase.The cytochrome o terminal oxidase from E. coli was immunochemically purified and monospecific antiserum towards cytochrome o was obtained. This antiserum is able to precipitate 100% of the ubiquinol-1 oxidase activity in solubilized membranes from an E. coli strain in which cytochrome o is the only terminal oxidase. Cytochrome o was analyzed and quantitated using CIE, RIE, and SDS-PAGE. Analysis of SDS-PAGE shows that cytochrome o is composed of four subunits of approximate equimolar stoichiometry with molecular weights of 51,000, 28,500, 18,000 and 12,700. The low temperature (77(DEGREES)K) reduced-minus-oxidized spectrum of the immunoprecipitate shows two peaks at 555 nm and 562 nm, indicating b-type cytochromes. Both appear to bind CO. Preliminary results indicate that the biosynthesis of cytochrome o is repressed when cytochrome d is induced by lowering the dissolved oxygen concentration during cell growth. It was shown that cytochrome b(,556) and cytochrome o are not present in a constant ratio in the membrane.Finally, a rapid, sensitive, and quantitative radioimmunoassay for specific translation products was developed using E. coli cells grown in 96-well microtiter plates.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD