Impact of Surface Active Compounds on Iron Catalyzed Oxidation of Methyl Linolenate in AOT–Water–Hexadecane Systems

Edible oils contain minor surface active components that form micro-heterogeneous environments, such as reverse micelles, which can alter the rate and direction of chemical reactions. However, little is known about the role of these micro-heterogeneous environments on lipid oxidation of bulk oil. Our objective was to evaluate the ability of water, cumene hydroperoxide, oleic acid, and phosphatidylcholine to influence the structure of reverse micelles in a model oil system: sodium bis(2-ethylhexyl) sulfosuccinate (aerosol-OT; AOT) in n-hexadecane. The influence of reverse micelle structure on iron catalyzed lipid oxidation was determined using methyl linolenate as an oxidizable substrate. The size and shape of the reverse micelle were investigated by small-angle x-ray scattering, and water contents was determined by Karl Fischer titrations. Lipid hydroperoxides and thiobarbituric acid reactive substances were used to follow lipid oxidation. Our results showed that AOT formed spherical reverse micelles in hexadecane. The size of the reverse micelles increased with increased water or phosphatidylcholine concentration, but decreased upon addition of cumene hydroperoxide or oleic acid. Iron catalyzed oxidation of methyl linolenate in the reverse micelle system decreased with increasing water concentration. Addition of phosphatidylcholine into the reverse micelle systems decreased methyl linolenate oxidation compared to control and reverse micelles with added oleic acid. These results indicate that water, cumene hydroperoxide, oleic acid, and phosphatidylcholine can alter reverse micelle size and lipid oxidation rates. Understanding how these compounds influence reverse micelle structure and lipid oxidation rates could provide information on how to modify bulk oil systems to increase oxidative stability.     

Methyl ketone formation during degradation of phenoxybutyric acid by Penicillium canescens SBUG-M 1139

Penicillium canescens SBUG-M 1139 was shown to be able to grow using phenoxybutyric acid as the sole carbon source. The rapid conversion of the phenoxyalkanoic acid resulted in the formation of phenol, which was metabolized completely. These reactions were accompanied by an accumulation of the methyl ketone phenoxypropan-2-one. Furthermore, during the metabolism of phenoxybutyric acid, 4-phenoxy-2,3-dehydrobutyric acid, 4-phenoxy-3-hydroxybutyric acid, phenoxyacetic acid, and phenoxypropan-2-ol accumulated in minor amounts. Clearly, fungi can metabolize phenoxyalkanoic acids to produce methyl ketones in a manner analogous to that used for the conversion of short- or medium-chain fatty acids by fungi. Received: 7 May 1999 / Accepted: 23 August 1999     

Expression, purification, and characterization of a novel methyl parathion hydrolase

The mpd gene coding for a novel methyl parathion hydrolase (MPH) was previously reported and its putative open reading frame was also identified. To further confirm its coding region, the intact region encoding MPH was obtained by PCR and expressed in Escherichia coli as a hexa-His C-terminal fusion protein. The fusion protein was purified to homogeneity by metal-affinity chromatography. The enzyme activity and zymogram assay showed that the fusion protein was functional in degrading methyl parathion. The amino terminal sequencing of the purified recombinant MPH indicated that a signal peptide of the first 35 amino acids was cleaved from its precursor to form active MPH. A rat polyclonal antiserum was raised against the purified mature fusion protein. The results of Western blot and zymogram demonstrated that mature MPH in native Plesiomonas sp. strain M6 was also processed from its precursor by cleavage of a putative signal peptide at the amino terminus. The production of active MPH in E. coli was greatly improved after the coding region for the signal peptide was deleted. HPLC gel filtration of the purified mature recombinant MPH revealed that the MPH was a monomer.     

Purification and partial characterization of azoreductase from Enterobacter agglomerans

Azoreductase, an enzyme catalyzing the reductive cleavage of the azo bond of methyl red (MR) and related dyes, was purified to electrophoretic homogeneity from Enterobacter agglomerans. This bacterial strain, isolated from dye-contaminated sludge, has a higher ability to grow, under aerobic conditions, on culture medium containing 100mg/L of MR. The enzyme was purified approximately 90-fold with 20% yield by ammonium sulfate precipitation, followed by three steps of column chromatography (gel-filtration, anion-exchange, and dye-affinity). The purified enzyme is a monomer with a molecular weight of 28,000 Da. The maximal azoreductase activity was observed at pH 7.0 and at 35 degrees C. This activity was NADH dependent. The K(m) values for both NADH and MR were 58.9 and 29.4 microM, respectively. The maximal velocity (V(max)) was 9.2 micromol of NADH min(-1)mg(-1). The purified enzyme is inhibited by several metal ions including Fe(2+) and Cd(2+).     

X-ray diffraction and high-resolution NMR spectroscopy of methyl 3-azido-2,3-dideoxy-alpha-D-lyxo-hexopyranoside

The single-crystal X-ray diffraction and high-resolution 1H and 13C NMR spectral data for the title compound are reported. The influence of the ring oxygen atom on the J(1,2e) and J(4,5) coupling constants for 2-deoxy-D-lyxo- and -D-xylo-hexopyranosides is discussed.     

Synthesis and intramolecular transesterifications of pivaloylated methyl alpha-D-galactopyranosides

Selective pivaloylations of methyl alpha-D-galactopyranoside have been studied under various reaction conditions. Partially pivaloylated products were submitted to additional acetylations. The structures were established by 1H and 13C NMR spectroscopies. Both, 2,6- and 3,6-dipivalates underwent intramolecular cyclization in neutral conditions (phosphate buffered saline, pH 7.2) to give a stable 2,3-orthoacid with a parallel 6-->4 migration of the pivaloyl group.     

Acid-catalyzed isomerization of methyl 2-deoxy-D-arabino-hexosides: equilibria, kinetics and mechanism

Four isomers of methyl 2-deoxy-D-arabino-hexosides were isolated by HPLC as chromatographically homogeneous compounds. The rates of pyranoside isomerization (alpha(p) and beta(p)) at 40 degrees C and of furanoside isomerization (alpha(f) and beta(f)) at 26 degrees C were determined. A mechanism has been suggested for transformations taking place during isomerization of methyl 2-deoxy-D-arabino-hexosides in methanolic solution catalyzed with hydrogen chloride.     

In Vitro Evaluation of 11C-Labeled (S)-Nicotine, (S)-3-Methyl-5-(1-Methyl-2-Pyrrolidinyl)isoxazole, and (R,S)-1-Methyl-2-(3-Pyridyl)azetidine as Nicotinic Receptor Ligands for Positron Emission Tomography Studies

Abstract: The binding characteristics of the novel ¹¹C-labeled nicotinic ligands (R,S)-1-methyl-2-(3-pyridyl) azetidine (MPA) and (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418) were investigated in comparison with those of (S)-[¹¹C]nicotine in vitro in the rat brain to be able to predict the binding properties of the new ligands for positron emission tomography studies in vivo. The data from time-resolved experiments for all ligands indicated fast binding kinetics, with the exception of a slower dissociation of [¹¹C]MPA in comparison with (S)-[¹¹C]nicotine and [¹¹C]ABT-418. Saturation experiments revealed for all ligands two nicotinic receptor binding sites with affinity constants (K_D values) of 2.4 and 560 nM and binding site densities (B_max values) of 65.5 and 223 fmol/mg of protein for (S)-[¹¹C]nicotine, K_D values of 0.011 and 2.2 nM and B_max values of 4.4 and 70.7 fmol/mg of protein for [¹¹C]MPA, and K_D values of 1.3 and 33.4 nM and B_max values of 8.8 and 69.2 fmol/mg of protein for [¹¹C]ABT-418. In competing with the ¹¹C-ligands, epibatidine was most potent, followed by cytisine. A different rank order of potencies was found for (?)-nicotine, (+)-nicotine, MPA, and ABT-418 displacing each of the ¹¹C-ligands. Autoradiograms displayed a similar pattern of receptor binding for all ligands, whereby [¹¹C]MPA showed the most distinct binding pattern and the lowest nonspecific binding. We conclude that the three ¹¹C-labeled nicotinic ligands were suitable for characterizing nicotinic receptors in vitro. The very high affinity of [¹¹C]MPA to nicotinic acetylcholine receptors, its low nonspecific binding, and especially the slower dissociation kinetics of the [¹¹C]MPA from the putative high-affinity nicotinic acetylcholine receptor binding site compared with (S)-[¹¹C]nicotine and [¹¹C]ABT-418 raise the level of interest in [¹¹C]MPA for application in positron emission tomography.     

4-Hydroxybenzoate 3-geranyltransferase from Lithospermum erythrorhizon: purification of a plant membrane-bound prenyltransferase

Geranyldiphosphate:4-hydroxybenzoate 3-geranyltransferase is a regulatory enzyme in the biosynthesis of shikonin, a phytoalexin and pharmaceutical produced by cell cultures of Lithospermum erythrorhizon Sieb. et Zucc.. In Linsmaier-Skoog medium, the activity of this enzyme could be enhanced more than 200-fold by addition of methyl jasmonate, and this culture material was used for the solubilization and purification of the enzyme. Of various detergents examined, digitonin was the most suitable for the solubilization of the enzyme. The solubilized enzyme was purified 800-fold by chromatography over diethylaminoethyl (DEAE)-Sephacel, Heparin-Sepharose, Reactive Green 19-Agarose, and Cholic Acid-Agarose. The purified enzyme required magnesium ions as cofactor and was highly specific for geranyldiphosphate (GPP) and 4-hydroxybenzoate (4HB) as substrates. The K _m values for 4HB and GPP were calculated by the method of Lineweaver and Burk as 18.4 μM and 13.8 μM, respectively. Received: 2 July 1997 / Accepted: 14 October 1997