Spectrophotometric and ESR evidence for vanadium(IV) deferoxamine complexes.

Complexes of vanadium(IV), vanadyl, are reported to be formed with the trihydroxamic acid deferoxamine (H3DF+). One complex exhibits a reddish-violet color, with a major absorbance peak at 386 nm and a smaller peak at 520 nm. This complex is potentially useful for the microdetermination of vanadyl. The apparent molar absorptivity is 3.91 mM-1 cm-1, and the complex obeys Beer's law in the concentration range of 0.6-63 ppm. Electron spin resonance studies indicate the formation of two vanadyl complexes that are 1:1 in vanadyl and deferoxamine, but have two or three bound hydroxamate groups. ESR and spectrophotometric evidence indicate that the red, low pH form, involves an octahedral vanadium (4+) ion coordinated by three hydroxamate ligands. One of these hydroxamates is displaced by an oxygen at pH greater than 2.8 according to the following equilibria: VO2+ + H3DF+ in equilibrium with VIV(DF)2+ + H3O+, VIV(DF)2+ + H2O in equilibrium with VO(HDF)+ + H+, where pk2 = 2.8.     

Mechanism of intestinal formation of deoxycholic acid from cholic acid in humans: evidence for a 3-oxo-delta 4-steroid intermediate

12 alpha-Hydroxy-3-oxo-4-cholenoic acid coupled to an adenosine nucleotide has been shown to be a metabolite of cholic acid in the intestinal anaerobic bacteria, Eubacterium species VPI 12708 (1987. J. Biol. Chem. 262: 4701-4707) and it has been suggested that this may be an intermediate in the conversion of cholic acid into deoxycholic acid. The possibility that the intestinal conversion of cholic acid into deoxycholic acid involves a 3-oxo-delta 4-steroid as an intermediate has been studied in the present work by use of [3 beta-3H]- and [5-3H]-labeled cholic acid. Whole cells as well as cell extracts of Eubacterium sp. VPI 12708 catalyzed conversion of [3 beta-3H] + [24-14C]cholic acid into deoxycholic acid with loss of about 50% of 3H label. When unlabeled chenodeoxycholic acid (20 microM) was added along with [3 beta-3] + [24-14C]cholic acid, then approximately 85% of the [3 beta-3H]-labeled was lost from deoxycholic acid. After administration of the same mixture to two healthy volunteers, deoxycholic acid could be isolated that had lost 81 and 84%, respectively, of the 3H label. Conversion of a mixture of [5-3H]- and [24-14C]labeled cholic acid by the above intestinal bacteria or cell extracts led to loss of 79-94 of the [5-3H] label.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thiamine pyrophosphate requirement for o-succinylbenzoic acid synthesis in Escherichia coli and evidence for an intermediate.

Cell-free extracts of various strains of Escherichia coli synthesize the menaquinone biosynthetic intermediate o-succinylbenzoic acid (OSB) when supplied with chorismic acid, 2-ketoglutaric acid, and thiamine pyrophosphate (TPP). To assay for OSB synthesis, 2-[U-14C]ketoglutaric acid was used as substrate, and the synthesized OSB was examined by radiogas chromatography (as the dimethyl ester). [U-14C]Shikimic acid also gave rise to radioactive OSB if the cofactors necessary for enzymatic conversion to chorismic acid were added. Use of 2-[1-14C]ketoglutaric acid does not give rise to labeled OSB. In the absence of TPP during the incubations, OSB synthesis was much reduced; these observations are consistent with the proposed role for the succinic semialdehyde-TPP anion as the reagent adding to chorismic acid. Extracts of cells from menC and menD mutants did not form OSB separately, but did so in combination. There was evidence for formation of a product, X, by extracts of a menC mutant incubated with chorismic acid, TPP, and 2-ketoglutaric acid; X was converted to OSB by extracts of a menD mutant. It appears that the intermediate, X, is formed by one gene product and converted to OSB by the second gene product.     

The acid-catalyzed decompostion of phenacylcobalamin: evidence for the formation of an enol-Co(III) pi-complex intermediate.

Phenacylcobalamin has been synthesized and characterized by thin-layer chromatography and uv-visible spectroscopy, as well as identification of the cobalt-containing and organic products of its cleavage in acid and base and by aerobic photolysis. The major organic product from all three cleavage reactions is acetophenone and the cobalt-containing product is aquacobalamin (or hydroxocobalamin, its conjugate base). In aqueous acidic solution (pH 0 to 7.3, ionic strength 1.0 M, and 25.0 degrees C), the kinetics of the formation of aquacobalamin are biphasic representing the linear sum of two exponential terms. The pH dependence of the first-order rate constant of both phases shows a first-order dependence on proton concentration but with an inflection point ot pH 3.55 for the faster phase and at pH 4.03 for the slower phase. This behavior is interpreted in terms of the specific acid catalyzed formation of an intermediate from both "base on" and "base off" phenacylcobalamin with different second-order rate constants for each form, followed by an intermediate decompotion step with a similar formal mechanism. The nature of the intermediate is discussed and it is concluded to be a pi-complex between cob(III)alamin and the enol of acetophenone.     

Anthranilic acid metabolism by microorganisms. Formation of 5-hydroxyanthranilate as an intermediate in anthranilate metabolism byNocardia opaca

5-Hydroxyanthranilic acid was isolated and characterized as an intermediate in the metabolism of anthranilate byNocardia opaca. This compound appeared during the log phase of growth and led to the subsequent induction of high levels of enzymes for its subsequent degradation. Gentisate was also found as a product of 5-hydroxyanthranilate metabolism. Both 5-hydroxyanthranilate and gentisate were oxidized to pyruvate by extracts of anthranilate-grown cells, the ring cleavage of the diphenol being effected by an oxygenase with a typical Fe²⁺ requirement. Features of the metabolism of 5-hydroxyanthranilate and gentisate byN. opaca, which can also oxidize anthranilate through catechol, suggest that the gentisate pathway is a secondary route.     

5,6-Anhydro-l-ascorbic acid. A reactive intermediate for the formation of 6-substituted derivatives of l-ascorbic acid

Cell-wall material was isolated from the alcohol-insoluble residue of carrot by treatment with Pronase, phenol—acetic acid—water, and aqueous 90% methyl sulphoxide. Some pectic material was solubilised, but the major component was a highly esterified, acidic arabinogalactan. The purified cell-wall material, which contained ～1% of protein, was sequentially extracted with water at 80°, ammonium oxalate at 80°, and m and 4m KOH at 20°, to leave a residue of α-cellulose, which contained some pectic material. From the hot-water-soluble fraction, a major pectic polymer was isolated by anion-exchange chromatography. Methylation analysis showed that it was a rhamnogalacturonan, probably having highly branched arabinan and slightly branched galactan side-chains linked to O-4 of rhamnopyranosyl residues. An unusual feature of this pectic polymer is that it contained a small but significant proportion of 1,4-linked xylopyranosyl residues. From the alkali-soluble fractions, a range of pectic polymers associated with various amounts of xylans and possibly xyloglucans was isolated. The main linkages present in these complexes were 1,4-linked galactopyranosyluronic acid, 1,4-linked galactopyranosyl, and 1,5-linked arabinofuranosyl residues, terminal arabinofuranosyl and galactopyranosyl groups, and, in some fractions, 1,4-linked xylopyranosyl residues. The possible association of some of these polymers with proteins and phenolics is discussed.