Augusto F. García

Comparative studies on membranes isolated from Rhodopseudomonas viridis grown in the presence and in the absence of yeast extract

Rhodopseudomonas viridis was grown in the presence and in the absence of yeast extract. The cells grown under this latter condition present a ten fold diminished bacteriochlorophyll (bchl) content. This decrease was paralleled by a similar increase in the ratio lipid phosphorous/bchl, whereas the ornithine lipid/bchl ratio remains constant. Some quantitative differences in the fatty acid composition are also reported.The protein composition of both membranes was also studied, only indicating quantitative differences.An active reaction center preparation was obtained from both types of cells. When isolated from cells grown in the presence of yeast extract, this reaction center preparation shows the presence of proteins a, b, c and d. Further treatment of this active reaction center results, in cells grown under either condition, in the isolation of green (oxidized behl) and brown (inactive reaction center) bchl containing fractions.The protein composition and absorption spectrum of the inactive reaction centers obtained from both types of cells were identical (proteins a, c and d).On the other hand the green complexes differ in their protein composition as well as in their absorption spectrum.

Bioenergetic factors controlling in vitro phosphorylation of LHIα (B870) polypeptides in membranes isolated from Rhodobacter capsulatus

Abstract Membranes of Rhodobacter capsulatus strain U43 (pTX35) showed qualitatively very similar phosphorylation patterns under in vitro and in vivo conditions. In vitro, it was irrelevant whether the phosphate source was orthophosphate or ATP. Inhibitors of electron transport did not inhibit light-harvesting complex I (LHIα) (B870) polypeptide phosphorylation, except for o-phenanthroline, which was strongly inhibitory. Redox conditions regulated the amount of protein phosphorylated; external redox potentials between +200 and +300 mV promoted the reaction. Phosphorylation was inhibited by uncouplers such as carbonyl cyanide m-chlorophenyl hydrazone and nigericin plus valinomycin plus potassium ions. Inhibitors of the H+-ATPase were also inhibitory when the phosphate source was [32P]Pi or [γ-32P]ATP. From these results, it was concluded that an operative reaction center, a coupled membrane, and external redox potentials higher than +200 mV are required for optimum LHIα phosphorylation. We also demonstrated that phosphorylation of LHIα polypeptide occurs before insertion into the membrane and that phosphate is preferentially incorporated into specific domains within the cytoplasmic membrane. Intracytoplasmic membranes, identified here as light membranes, were found to contain a dephosphorylated LHIα polypeptide.

Isolation of a “basic membrane” fraction enriched in an ornithine-containing lipid, from a blue-green mutant of Rhodospirillum rubrum

An ornithine-containing lipid (OCL) that lacks phosphorus has been observed in some photosynthetic bacteria [l-5] . It has been found among the phospholipids of different subcellular fractions of Rhodospirillum rubrum, namely the chromatophores, and also in non-pigmented, aerobically grown organisms. Gorchein [ 1 ] suggests that this lipid could be a membranestructural component, at least in anaerobically grown organisms. On the other hand, De Pinto [2] observed that unlabelled omithine added to a culture containing (14Q) ornithine yields no dilution of the isotope in the lipid fraction. This lack of turnover of the OCL may suggest a structural role for this compound. The intracellular distribution of this lipid [3, S] and its physical and chemical properties, are consistent with a possible structural role within the cytoplasmic membrane system of the non-sulphur purple bacteria. We attempted to prove this hypothesis by looking for the distribution of the OCL in different subchromatophore fractions, from a non-carotenoid-containing mutant ofRhodospirrilum rubrum (a blue-green, BG 1). The cell membrane repeatedly treated with Triton X-100 yields an insoluble lipoprotein fraction. This fraction presents some properties characteristic of the so called “structural proteins” and was enriched in OCL, the only lipid detectable by thin-layer chromatography (TLC).

Isolation of a membrane-bound protein having coupling factor capacity as well as ADP—Pi exchange and ADPase activities from Rhodopseudomonas viridis

It was earlier observed by Garcia et al., that a crude preparation of coupling factor isolated from Rhode pseudomonas capsulata showed a high dark phosphate esterification activity [l] . It was then postulated that this incorporation could be the result of an as yet unknown ADP-Pi exchange reaction, and no relationship was established between the coupling activity and the dark esterification activity. We report here on the purification of a protein from Rhod@seudomonas viridis having ADP-Pi exchange activity, Mg’+ activated ADPase activity and the ability of restoring a light-induced phosphate esteriiication capacity when rebound to uncoupled membranes.

Tryptophan metabolism in experimental porphyria

Abstract Although there seems to be no qualitative difference in the excretion of certain urinary metabolites of l -tryptophan between normal and porphyric rats, quantitative determination showed that there is a decrease in the level of excretion of l -kynurenine and 3-OH-kynurenine. At the same time, tryptophan pyrrolase activity is diminished in porphyric animals. This is interpreted as being the result, at least in part, of a competitive inhibition between the porphyrins accumulated in the liver during the state of porphyria and the tryptophan pyrrolase heme coenzyme, for the tryptophan pyrrolase apoenzyme.

Some environmental factors influencing the state of the membranes isolated by gradient centrifugation from cell-free extracts of Rps. viridis☆

Most of the studies on the purification of bacterial intracytoplasmic-photosynthetic systems have been carried out in bacteria containing an internal vesicular system. No similar studies have been carried out in photosynthetic microorganisms containing extensive internal lamellar structure. Chromatophores have been isolated in, a variety of states of purity by sucrose density centrifugation of cell-free extracts of R. rubrum [l-3] and Rps. spheraides [4-81. As part of our investigation on the morphogenesis of the photosynthetic apparatus in bacteria containing internal lamellar structures, we have studied the effect of different environmental conditions on the state of the membranes isolated by sucrose gradient centrifugation of cell-free extracts of Rps. viridis.

Protein composition of intact and fractionated memberanes isolated from dark and light grown cells of a blue green mutant of Rhodospirillum Rubrum (BG1)

Abstract 1. By means of different fractionation procedures we have separated two major protein fractions present in photosynthetically grown cells of the blue-green mutant of Rhodospirillum rubrum (BG 1 ). These proteins are found in differing amounts in the two subchromatophore fragments produced by the detergent Triton X-100. A more extensive breakdown caused by digestion of intact photosynthetic membrane with Triton X-100 and trypsin produced four main protein fractions which were separated by means of gel electrophoresis. Some of these bands originated from proteins that were intimately associated with bacteriochlorophyll in the intact membrane. The other protein fractions seem to derive from a group of proteins whose function could be a supporting one. These protein fractions seem to be a somewhat degraded form of the basic membrane core that cannot be further attacked by the combined action of the detergent and the trypsin. 2. Electron micrographs of the fractions obtained by the digestion with Triton X-100 and trypsin are presented, showing a very different structure for the degraded basic membrane and a unique structure for the aggregated units of the bacteriochlorophyll-containing fraction.

[31] Subchromatophore fragments: Chromatium, Rhodospirillum rubrum, and Rhodopseudomonas palustris

Publisher Summary The photosynthetic membrane systems of some purple bacteria are readily cleaved by both nonionic and ionic detergents to yield distinct subchromatophore fragments, which are characterized by the unique forms of bacteriochlorophyll contained in the particles. The types of subchromatophore fragments obtained depend largely upon the nature of the detergent, the presence or absence of carotenoids in the membrane, and the conditions used for growing the bacteria. In the bacteria examined to date, at least two fragments are produced—one containing the reaction center bacteriochlorophyll (BChl) and varying amounts of other associated BChl, while the other one containing only light-harvesting BChl. In the ease of Chromatium , fragmentation with sodium dodecyl sulfate (SDS) produces three fragments, but two of the three are quite similar in their general properties. In vivo , the BChl α (the form of BChl contained in the bacteria under consideration) exhibits an absorption band in the orange and a series of bands in the near infrared region. Although the exact locations of the absorption maxima of the three BChl a forms differ slightly in different bacteria, they are termed “B800,” “B850,” and “B890.” It is generally held that the different absorption properties of the BChl molecules in the near infrared region reflect different complexes of BChl with lipoproteins.