Impr�gnierung chromatographischer D�nnschichten mit Polyestern

Knappe, E.; Yekundi, K. G.

doi:10.1007/bf01105918

Cited by 10 publications

(2 citation statements)

References 8 publications

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“…Labeled GSH (15S, 19 mCi/mmol, obtained from Schwartz/Mann) was used as the second label for the product. Hydroxamate derivatives were prepared from synthetic monomethyl ester of citric acid (prepared by the diazomethane procedure, with 0.9 mol of diazomethane per mol of citrate in methanol) and from the enzymatically synthesized product by reacting it directly (after freeze-drying) with a methanolic solution of NH2OH (13) for 1 hr at 400 after 16 hr at room temperature. Hydroxylaminolysis was 60% under these conditions.…”

Section: Methodsmentioning

confidence: 99%

Enzymatic formation of glutathione-citryl thioester by a mitochondrial system and its inhibition by (-)erythrofluorocitrate

Kun

Kirsten

Sharma

1977

Proc. Natl. Acad. Sci. U.S.A.

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A soluble extract of the mitochondrial compartment composed of the inner membrane and matrix catalyzes the enzymatic synthesis and hydrolysis of the 1:1 adduct of citric acid and glutathione. (9) was also explained by the stringent requirement of preincubation of mitochondria with fluorocitrate in the absence of millimolar concentrations of citrate (cf. 7, 8), allowing for the formation of two protein-fluorocitrate thioesters (8).The present report is concerned with the identification of a mitochondrial enzyme system that catalyzes the formation of the 1:1 adduct of glutathione and citric acid. The adduct was identified as the thioester of glutathione, and kinetic evidence was also obtained that the mitochondrial extract contained, in addition to the thioester-forming enzyme system, a hydrolase capable of degrading the thioester. The citryl-glutathioneforming enzyme system proved to be the most sensitive catalytic component of mitochondria towards the irreversible inhibitory action of (-)erythrofluorocitrate, suggesting that the molecular site of action of this highly toxic agent has been identified. METHODSLysosome-free mitochondria and mitoplasts (defined as the matrix surrounded by the inner membrane) were isolated from rat livers as described (12). An extract of mitoplasts was prepared by stirring (at 00) of a suspension of mitoplasts (30 mg of protein per ml) in 0.25 M sucrose/mannitol medium (12) containing 1 mg of Brij 56 (Sigma Chemicals) per 10 mg of mitoplast protein for 15 min. The resulting lysate was diluted 1:3 with the medium; after removal of large granules (unbroken mitoplasts) by centrifugation at 10,000 X g (10 min at 40), the supernatant (containing soluble extract of the inner membrane and soluble proteins of the matrix and inner membrane vesicles) was subjected to ultracentrifugation (145,000 X g for 1 hr at 20). The clear supernatant was concentrated by centrifugation (at 20) in Amicon CF-25 membrane cones at 100 X g to one-fourth of the original volume. Approximately 250 mg of protein extract was obtained by this technique from 500 mg (protein) of mitoplast starting material. The concentrated extract was used directly in most experiments, unless specified, whereas the sediment containing the inner membrane vesicles was rehomogenized in the suspending medium. Both fractions were stored at -15°until used. The enzymatic activity of the soluble extract decayed slowly at -150 (about half of the activity was lost in 2 weeks) but much more rapidly after repeated thawing and refreezing. The activity was completely recovered when the stored extract was preincubated for 5-10 min with 5 mM reduced glutathione (GSH), demonstrating the essential role Abbreviations: GSH, reduced glutathione; GSSG, oxidized glutathione; Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; citryl-SG and malyl-SG, glutathione thioesters of citric and malic acids, respectively. 4942

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Section: Methodsmentioning

confidence: 99%

Enzymatic formation of glutathione-citryl thioester by a mitochondrial system and its inhibition by (-)erythrofluorocitrate

Kun

Kirsten

Sharma

1977

Proc. Natl. Acad. Sci. U.S.A.

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“…In TLC, impregnation with silver nitrate or with stationary phases of partition systems (138,1022,1897) is also used frequently; derivatives, such as the p-phenylazophenacyl esters (2168) and the Hg adducts of polyene fatty acids (2202), are not often prepared. TLC methods are capable of resolving mixtures of geometric isomers of monounsaturated acids (138, 160, 259, 1359, 1483, 2187), position isomers of hydroxy acids (1359,2002), stereoisomers (1359), acids differing in degree of unsaturation (1696), and other critical pairs (733).…”

Section: Tetranitrobenzylpolystyrenementioning

confidence: 99%

Chromatography

Heftmanń¹

1966

Anal. Chem.

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Fortschritte auf dem Gebiet der Lipoid‐Analyse V: Dünnschicht‐Chromatographie, Teil 2

1970

Fette, Seifen, Anstrichm.

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Die Margarine-Probe 3 hatte eine ausgezeichnete Strukturhaltbarkeit. Durch gas-chromatographische Analyse d e r Methylester d e r Fettsauren konnte festgestellt werden, dai3 d a s Margarinefett wahrscheinlich aus e t w a 30 O i u Kokosol, 15 O/o zum T e i l gehartetem Palmol, 15 '10 Rub01 und 40 Oio gehartetem Fischol zusamniengestellt war. Mit Hilfe d e r DTA konnte in d e r isolierten festen Phase keine polymorphe Zustandsanderung nachgewiesen werden.

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Impr�gnierung chromatographischer D�nnschichten mit Polyestern

Cited by 10 publications

References 8 publications

Enzymatic formation of glutathione-citryl thioester by a mitochondrial system and its inhibition by (-)erythrofluorocitrate

Enzymatic formation of glutathione-citryl thioester by a mitochondrial system and its inhibition by (-)erythrofluorocitrate

Chromatography

Fortschritte auf dem Gebiet der Lipoid‐Analyse V: Dünnschicht‐Chromatographie, Teil 2

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