Quantitative dissolution of the membrane and preparation of photoreceptor subunits from Rhodopseudomonas spheroides

Abstract

An effective and optimal combination of three reagents commonly used for modifying the structure of proteins has been developed for causing quantitative dissolution of membrane structure. Alkaline solution, 1.5 Triton X-100, and 6 M urea at 0" for about 2 hr causes quantitative conversion of chromatophores from wild-type Rhodopseudomoms spheroides into Triton-protein complexes having particle weights of less than 200,000. No trap activity is lost as evidenced by light-induced absorbance and electron paramagnetic resonance changes. Good rates for photoxida-

The Question of the Primary Electron Acceptor in Bacterial Photosynthesis

“…They found that it could be observed in their reaction-center preparations from the R-26 mutant of R. spheroides after addition of sodium dodecyl sulfate, and presented evidence for iron separation under these conditions. We want to underscore the fact that, of the methods used to purify reaction-center or phototrap systems, the AUT procedure is the only one that: (a) completely dissociates all wild-type, mutant, green-plant, and algae systems tested; (b) results in sufficient dissociation to allow separation of nearly all iron-containing material; (c) allows observation of a reduced radical that may be from the primary electronacceptor molecule; and (d) gives a low particle weight prel)aration in which the antenna bacteriochlorophyll and carotenoids are still a functioning part of the complex (1,2).…”

“…Details for dissolution of chromatophores by the alkaline-urea-Triton method have also been given (1, 2). The original conditions (3% Triton X-100-6 M urea, pH 11.5) were modified by the addition of 1 RESULTS When alkaline urea-Triton (AUT) particles were subjected to column electrophoresis, phototrap activity was found to correspond very closely with bacteriochlorophyll absorbance, as measured at 875 nm ( Fig. 1) (3).…”

“…The largest iron concentration is found in a fast-moving band at the bottom of the column, and there is no indication of a significant band of iron-containing material corresponding to the AUT-e bands. These data were obtained with a batch preparation (1); that is, sucrose density gradient centrifugation was omitted, and the AUT-treated chromatophores were placed directly onto 11.0 0.30 * Appropriate fractions from electrophoresis columns were dialyzed against 1 mM EDTA, followed by distilled deionized water or against distilled water only, pelleted by centrifugation, washed several times with distilled deionized water, and finally resuspended for analysis in 0.01 M Tris buffer (pH 7.5)-0.2% Triton X-100.…”

“…Recent results from this laboratory (1,2) have demonstrated that photosynthetic membrane fragments (chromatophores) of Rhodopseudomonas spheroides and Rhodospirillum rubrum can be quantitatively dissociated to yield active photoreceptor subunits. The procedure, in which chromatophores are treated with Triton X-100 and urea at high pH (AUT conditions), followed by sucrose density gradient centrifugation, causes a displacement of much of the phospholipid and a separation of about half of the protein originally present in the chromatophore membranes.…”

A Survey of Epr Investigations of Bacterial Photosynthesis

“…They are generally referred to as reaction center prep arations. Another pigment-protein complex which has been extensively studied and characterized (Loach et al, 1970a;1970b) is the so-called 'photoreceptor subunits' isolated from Rhodospirillum rubrum using alkaline, urea and Triton X-100. This unit retains a portion of the antenna pigments.…”