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The asymmetric structure of the Rhodopseudomonas sphaeroides chromatophore membrane was examined in detail by crossed immunoelectrophoresis techniques. Because these methods are quantitative and allow increased resolu

Triton extracts of intracytoplasmic photosynthetic membranes (chromatophores) purified from Rhodopseudomonas sphaeroides were subjected to crossed immunoelectrophoresis with antiserum raised in rabbits to purified chromatophores. A total of 31 immunoprecipitates was visualized; 2 of the immunoprecipitates were identified as reduced nicotinamide adenine dinucleotide (EC 1.6.99.3) and L-lactate dehydrogenases by enzyme staining techniques. Reaction with a monospecific antiserum identified the photochemical reaction center. Photopigments were associated with a major precipitate in the pattern which was identified on the basis of immunological identity as light-harvesting bacteriochlorophyll aprotein complex. These results provide the basis for a detailed structural and functional analysis of the chromatophore membrane by crossed immunoelectrophoresis.Growth of the facultative photoheterotrophic bacterium Rhodopseudomonas sphaeroides under low oxygen tension results in the development of intracytoplasmic (chromatophore) membranes in which the photosynthetic apparatus is localized. Formation of the chromatophore membrane is repressed under high aeration and derepressed upon reduction of oxygen tension. Fractionation and analysis of functional components of the R. sphaeroides chromatophore membrane has been restricted mainly to the photochemical reaction center (RC) (2,13), the light-harvesting (LH) bacteriochlorophyll a (BCHL) complex (1,5, 16), and the L-lactate dehydrogenase (LDH) (10). Additional chromatophore polypeptide components have been observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (6,15, 19), but the function of these components has remained largely unknown because this procedure results in the loss of any biological activity. Recently, crossed immunoelectrophoresis (CIE) has been applied to the study of bacterial membranes (7,14,17,18). Since nondenaturing detergents are used for membrane disruption before CIE, both enzymatic and antigenic activities are usually retained, thus permitting a functional analysis of membrane components. In addition, a high order of resolution is achieved through CIE by virtue of the two-dimensional distribution of antigenic components. This communication describes a CIE analysis of chromatophores isolated from R. sphaeroides.(This work was presented in part at the 79th Annual Meeting of the American Society for Microbiology, Los Angeles, Calif., 4-8 May 1979.) Triton X-100 was selected as the detergent for the solubilization of chromatophores for CIE because a recent study demonstrated that it was superior to other detergents for the maximal recovery of Micrococcus lysodeikticus membrane components (4). Chromatophores were purified from phototrophically grown (15) R. sphaeroides NCIB8253 by differential and sucrose density gradient centrifugation (12). Chromatophore preparations for immunization were purified further by a second cycle of differential and rate-zone sedimentation. Previous reconstitution studies have shown that such chromato...

1. Dark equilibrium potentiometric titrations were conducted on membranes purified from Rhodospirillum rubrum in an effort to identify b-type cytochrome components reported in other Rhodospirillaceae. In preparations from aerobically grown cells virtually devoid of bacteriochlorophyll a, three components were observed at 560-540 nm. Their oxidation-reduction midpoint potentials assigned by computer-assisted analysis were +195, +50 and -110 mV at pH 7.0; each of these fitted closely to theoretical single-electron equivalent curves. 2. In chromatophores from phototrophically grown carotenoidless mutant G-9, three components were also observed with E0' +190, +50 and -90mV. 3. The alpha-band of the +50mV component exhibited an absorption maximum near 560nm in difference spectra obtained at fixed oxidation-reduction potentials. 4. This component could be demonstrated most readily in purified membrane preparations and may have been obscured in previous studies by residual cytochrome c'. 5. This is the first definitive report of cytochrome b+50 in membranes from Rs. rubrum and aligns this bacterium with other Rhodospirillaceae in which this component functions in light-driven cyclic electron flow.