Separation of sulphur-containing substances, amino acids and nucleotides during gel filtration on trisacryl GF05 by specific gel interaction

Krauss, Friedrich; Schmidt, Ahlert

doi:10.1016/s0021-9673(01)95010-1

Cited by 7 publications

(1 citation statement)

References 15 publications

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“…Formation of cysteine was analyzed by incubation ofcysteine synthase and O-acetyl-L-serine with bound sulfide isolated from Chlorella (S°) in the presence and absence of thiols. Cysteine was determined as the red ninhydrin complex (9) modified as described (20). Cysteine synthases from Chlorella and spinach were purified as described earlier (28,29).…”

Section: Methodsmentioning

confidence: 99%

Formation of Elemental Sulfur by Chlorella fusca during Growth on l-Cysteine Ethylester

Krauss

Schäfer²,

Schmidt³

1984

Plant Physiol.

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During growth on L-cysteine ethylester, Chlorella fisca (211-8b) accumulated a substance which contained bound sulfide, which could be liberated by reduction with dithioerythritol (DTE) as inorganic sulfide.This substance was extracted with hot methanol and purified by thin layer chromatography. This substance liberated free sulide when incubated with mono-and dithiols, and thiocyanate was formed after heating with KCN. The isolated substance cochromatographed with authentic sulfur flower using different solvent systems for thin layer chromatography, high pressure liquid chromatography, and the identical spectrum with a relative Xmax at 263 nm was found. The chemical structre was confirmed by mass spectrometry showing a molecular weight of 256 m/ e for the S% configuration. No labeled elemental sulfur was detected when the cells were grown on [Sjsulfate and L-cysteine ethylester indicating the origin of elemental sulfur from L-cysteine ethylester. C. fusca seems to have enzymes for the metabolism of elemental sulfur, since it disappeared after prolonged growth into the stationary phase. Cysteine was formed from O-acetyl-L-serine and elemental sulfur in the presence of thiol groups and purified cysteine synthase from spinach or Chlorella.Phototrophic organisms form elemental sulfur (S°) under certain growth conditions. This was observed for Rhodospirillaceaelike Rhodopseudomonas and Rhodospirillum (13), Chromatiaceae-like Chromatium (12), and Chlorobiaceae-like Chlorobium (12). In blue green algae such as Oscillatoria (23) in 10-L bottles at light intensities of 7000 lux. For growth on radio-labeled sulfate carrier-free sulfate was added to the growth medium to reach a specific activity of 1725 cpm/nmol sulfate. Growth on [35S]sulfate was performed in 400-ml glass bottles connected to a Zn-acetate trap for labeled volatile sulfur compounds.Isolation of Bound Sulfide. Bound sulfide was isolated from cultures growing in 10-L bottles on sulfate and L-cysteine ethylester when the culture reached an optical density of 0.6 at 680 nm. Cells were harvested by centrifugation and washed once with distilled H20. Bound sulfide was extracted with hot methanol. After incubation in methanol at 80°C for 10 min, the cells were centrifuged for 10 min at 5000g and the pellet was extracted again with hot methanol. The combined supernatants were evaporated to drynes and resuspended in a small volume of petrolether. This crude extract was spotted on thin layer plates (Silica GF254, 0.5 mm) and developed with petrolether/diethylether (99.5/0.5) (solvent system 1). Bound sulfide with an RF of 0.84 was scraped off the plates, eluted with hot methanol, and spotted on a second plate which was developed with the solvent system 2 (acetone/water, 1/9) where bound sulfide stayed at the origin. The bound sulfide was eluted from the gel and spotted again on a TLC plate which was developed with ethylacetate (solvent system 3; RF = 0.86). The last plate was prerun with acetone and ethylacetate to remove impurities. This material was used ...

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Section: Methodsmentioning

confidence: 99%

Formation of Elemental Sulfur by Chlorella fusca during Growth on l-Cysteine Ethylester

Krauss

Schäfer²,

Schmidt³

1984

Plant Physiol.

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Oxidation of cysteine to cystine by membrane fractions of Chlorella fusca

Krämer

Schmidt

1984

Planta

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Isolated membrane fractions of Chlorella fusca 211-8b obtained by french-press treatment and sonication catalyzed the oxidation of L-cysteine to L-cystine. The pH-optimum of this reaction was determined to be around 8-8.5 and a stoichiometry of 4 SH-groups oxidized for one O2 consumed was obtained. This thiol-oxidation system was specific for D-and L-cysteine; DL-homocysteine and cysteamine were oxidized at about half the rate whereas all other thiols tested including glutathione, mercaptoethanol, mercaptopropionic acid and dithioerythritol were not oxidized by these membrane fractions. The apparent Km for L-cysteine was determined as 3.3 mmol l(-1). Rates of 200 μmol cysteine oxidized mg(-1) chlorophyll h(-1) were normally obtained. Extremely high rates of oxygen uptake were measured using L-cysteine methyl ester and L-cysteine ethyl ester. This thioloxidation system was not inhibited by mitochondrial electron-transport inhibitors such as rotenone or antimycin A, nor by the chloroplast electron-transport inhibitors 2,5-dibromothymochinone and 2,4-dinitrophenylether of iodonitrothymol. The cysteine oxidation catalyzed by C. fusca membranes was inhibited, however, by salicylhydroxamic acid, o-phenanthrolin, N,N'-disalicyliden-1,3-diaminopropane 5,5'-disulfonic acid, ethylenediaminetetraacetic acid, high KCN levels and by the buffers, N-[2-hydroxyl-1,1-bis(hydroxymethyl) ethyl] glycine and phosphate. This cysteine-oxidation system seems to function as a counterpart of thioredoxin-mediated light activation of enzymes, allowing reduced thiol groups to be oxidized again by O2 (dark inactivation).

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Mononucleotides

Frank¹

2013

Chemistry of Plant Phosphorus Compounds

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Separation of sulphur-containing substances, amino acids and nucleotides during gel filtration on trisacryl GF05 by specific gel interaction

Cited by 7 publications

References 15 publications

Formation of Elemental Sulfur by Chlorella fusca during Growth on l-Cysteine Ethylester

Formation of Elemental Sulfur by Chlorella fusca during Growth on l-Cysteine Ethylester

Oxidation of cysteine to cystine by membrane fractions of Chlorella fusca

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