Protein Composition of the Outer Membrane of Rhodopseudomonas Sphaeroides: Isolation of the Major Outer Membrane Protein

162 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1982.The outer membrane protein profile of Rhodopseudomonas sphaeroides was characterized on SDS-polyacrylamide gels. Solubilization of the outer membrane at 75(DEGREES)C as opposed to room temperature solubilization resulted in the dissociation of 75, 72, and 68 kilodalton polypeptide aggregates into 29, 26.5, and 21.5 filodalton polypeptides, and a common subunit, the major outer membrane protein.Solubilization and isolation of the major outer membrane protein has been achieved by both nondetergent and detergent based methods. The protein was differentially solubilized from other outer membrane proteins in 5 M guanidine thiocyanate which was exchanged by dialysis for 7 M urea. The urea-solubile protein was purified to homogeneity by a combination of DEAE-Sephadex chromatography and preparative electrophoretic techniques. The protein was also purified by differential temperature extraction of the outer membrane in the presence of SDS, followed by preparative SDS-polyacrylamide gel electrophoresis. Immunochemical analysis established the immunochemical identity and homogeneity of each preparation. Immunoblots of SDS-polyacrylamide gels revealed that antibody directed against the major outer membrane protein reacted with the three high molecular weight aggregates present in the outer membrane.Molecular weight determined by electrophoretic mobility in SDS and urea polyacrylamide gels was in agreement with that obtained by amino acid compositional studies. Gas chromatography of the hydrolyzed protein confirmed the presence of covalently associated fatty acid. Peptide mapping of tryptic and chymotryptic digests localized the fatty acid in one peptide in each digest. Isolation of the fatty acid containing peptide resulted in the identification of the fatty acid amide-linked to an amino-terminal alanine.Immunoblots of SDS-polyacrylamide gels of outer membranes from various strains of Rp. sphaeroides revealed proteins which crossreacted with antibody directed against the Rp. sphaeroides 2.4.1. major outer membrane protein, though the number of high molecular weight aggregates varied with the strain examined. Quantitation of the major outer membrane protein demonstrated equivalent amounts in cells grown chemoheterotrophically and photoheterotrophically. Rs. rubrum outer embranes contained a crossreactive protein, while Rp. capsulata and P. denitrificans did not.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD