Pharmacological prospection and structural characterization of two purified sulfated and pyruvylated homogalactans from green algae Codium isthmocladum.

Two sulfated polysaccharides (SPs), F2 and F3, isolated from Codium isthmocladum were found to contain galactose, sulfate, and pyruvate. The apparent molecular weights of F2 and F3 were determined to be 62 and 61 kDa, respectively. NMR spectroscopy combined with chemical analysis showed that F2 and F3 have the same structural features. However, F3 showed higher sulfate/sugar ratio (1/2.6) than F2 (1/4). F2 and F3 are essentially (1 → 3)-ß-D-galactans with some branching at C6. Pyruvylation occurs at O3 and O4, forming 3,4-O-(1-carboxyethylidene)-ß-D-Galp residues; some of these pyruvylated residues contain sulfate groups at C6. Some non-branching residues contain sulfate at C4. None of the SPs exhibited antioxidant activity. MTT results indicated that 1 mg/mL of both SPs about 40% of PANC-1 cell viability. At 10 µg/mL, F2 and F3 had 1.7-fold longer clotting times compared to that of Clexane® at the same concentration. The higher sulfate content of F3 is not a determining factor for pharmacological activities of galactans, considering that both F2 and F3 exerted the effects.

Quantification of Photorespiratory Intermediates by Mass Spectrometry-Based Approaches.

Photorespiration is an essential metabolic process in plants occurring via the oxygenase reaction of RuBisCO. In order to understand this process, it is essential to determine the amounts of intermediates involved. For this purpose we combined mass spectrometry-based approaches and the use of authentic standards for the quantification of photorespiratory intermediates. Here we describe protocols for the extraction and quantification of 2-phosphoglycolate (2PG) by LC-MS/MS and serine, glycine, glycolate, hydroxypyruvate, glyoxylate, and glycerate by GC-MS.

Production of hydroxypyruvate from glycerate by a novel biotechnological route.

In this work, bio-oxidation of glycerate was introduced for the green production of hydroxypyruvate. Whole cells of Pseudomonas sp. XP-LM were confirmed to have a good ability to produce hydroxypyruvate from glycerate. Under the optimal conditions, Hydroxypyruvate of 98.6 mM was produced from glycerate of 200 mM. Glycerate is now potentially producible from glycerol, a by-product during biodiesel fuel production, through a biotechnological process. Thus, the bioconversion system introduced in this work provided not only a green hydroxypyruvate production process but also a potential pathway of surplus glycerol utilization.

High-performance liquid chromatographic enantiomeric separation of an enzyme inhibitor which possesses both a chiral center and tautomeric moieties.

The enantiomeric separation using high-performance liquid chromatography (HPLC) on chiral stationary phases (CSPs) of a chiral compound which exists in solution in several tautomeric forms is described. 2,4-Dioxo-5-acetamido-6-phenylhexanoic acid is the most potent inhibitor known for peptidylamidoglycolate lyase (PGL, EC 4.3.2.5), an enzyme which plays an essential role in carboxyl-terminal amidation of many biological peptides. Synthesis of this inhibitor entails an alkaline hydrolysis step, under which condition the compound is racemized; thus, HPLC with a CSP was employed to obtain the individual enantiomers of this inhibitor. Since 2,4-dioxo-5-acetamido-6-phenylhexanoic acid exists in solution in several tautomeric forms, the strategy of first converting this compound from its multiple enol forms into a single diketo tautomer, which was then applied to various CSPs, was employed. Successful preparative scale enantiomeric separation of this compound was achieved using a Chiralpak AD CSP. Enantiomeric separation was also accomplished on a D-penicillamine column, but this CSP was found to be less satisfactory for preparative purposes.

Studies of enzyme polymorphisms in the Kamuela population of Drosophila mercatorum. II. Evaluation of glycolytic intermediates.

A simple and effective cryogenic procedure for the extraction of glycolytic intermediates from whole Drosophila has been developed. This procedure gives consistent results when a measure (microM/liter/OD260) is adopted which corrects for differences in extraction efficiency. Using this measure and a homozygous strain of D. mercatorum, there are no significant differences among extracts for the levels of any of the 15 glycolytic intermediate or energy molecules considered. The profile of means is consistent across experimental designs and instrument types. Coefficients of variation are well below 50% for most variables. The methodology presented has the statistical power to detect a mean change of 10 to 50% using an experimental design which requires as few as 32 observations. The estimated energy charge for resting Drosophila from these studies is the expected value of 0.86.

Application of high-performance liquid chromatography to the determination of glyoxylate synthesis in chick embryo liver.

The isolation and identification of three major alpha-keto end products (glyoxylate, pyruvate, alpha-ketoglutarate) of the isocitrate lyase reaction in 18-day chick embryo liver have been described. This was accomplished by the separation of these alpha-keto acids as their 2,4-dinitrophenylhydrazones (DNPHs) by high-performance liquid chromatography (HPLC). The DNPHs of alpha-keto acids were eluted with an isocratic solvent system of methanol-water-acetic acid (60:38.5:1.5) containing 5 mM tetrabutylammonium phosphate from a reversed-phase ultrasphere C18 (IP) and from a radial compression C18 column. The separation can be completed on the radial compression column within 15-20 min as compared to 30-40 min with a conventional reversed-phase column. Retention times and peak areas were integrated for both the assay samples and reference compounds. A relative measure of alpha-keto acid in the peak was calculated by comparison with the standard. The identification of each peak was done on the basis of retention time matching, co-chromatography with authentic compounds, and stopped flow UV-VIS scanning between 240 and 440 nm. Glyoxylate represented 5% of the total product of the isocitrate lyase reaction. Day 18 parallels the peak period of embryonic hepatic glycogenesis which occurs at a time when the original egg glucose reserve has been depleted.

Purification and properties of two 2-oxoacid:ferredoxin oxidoreductases from Halobacterium halobium.

Pyruvate:ferredoxin oxidoreductase and 2-oxoglutarate:ferredoxin oxidoreductase were obtained from cell-free extracts of Halobacterium halobium as homogeneous proteins after ammonium sulfate precipitation, salting-out chromatography with ammonium sulfate on unsubstituted agarose, gel filtration and chromatography on hydroxyapatite. The respective molecular weights are 256000 and 248000. Both enzymes consist of two sets of non-identical subunits of Mr 86000 and 42000 in the case of the pyruvate-degrading enzyme and of 88000 and 36000 in the case of the 20 -oxogluatarate-degrading enzyme. Analyses indicate that an intact enzyme molecule contains two [4 Fe-4S]2 + (2 + , 1+) clusters and two molecules of thiamin diphosphate. Flavin nucleotides, lipoic acid and pantetheine are absent. Thus the enzymes are very similar to the 2-oxoacid:ferredoxin oxidoreductases from fermentative and photosynthetic anaerobes described previously, but are clearly different from the 2-oxoacid dehydrogenase multienzyme complexes which commonly occur in anaerobic organisms.

A novel method for purification of prephenic acid.

Prephenic acid accumulated in culture filtrates of Neuro-spora crassa has been purified in 66% yield utilizing adsorption chromatography on Sephadex G-10 in the major purification steps.

Isolation of organic anions by extraction with liquid anion exchangers and its application to micromethods for acetylcholinesterase and 4-aminobutyrate aminotransferase.

Organic anions of particular importance to biochemistry such as Krebs cycle intermediates, glycolysis intermediates, simple fatty acids, adenine nucleotides and CoA derivatives can be quantitatively extracted from a buffered solution by high-molecular-weight ammonium salts in an organic solvent. Phosphate salts of tertiary amines in chloroform were the most efficient extractants. The isolation procedure was found to be an example of amine neutralization. The effect of pH, different inorganic anions, volume ratios between the two phases, concentration of the isolated anions and concentration of the ammonium salts have been investigated. The extraction technique has been applied to rapid and sensitive radiochemical methods for the determination of acetylcholinesterase and 4-aminobutyrate aminotransferase activities.

Carboxymethyl-selenopyruvic acid as the product of the oxidative deamination of carboxymethyl-selenocysteine.

CMSeC labeled with 14C in the carboxymethyl moiety was incubated with snake venom L-aminoacid oxidase. As the product of the reaction only one ketoacid was detected, which retained all the radioactivity of the oxidized substrate. This clearly shows that no breakdown of the C-Se bond of CMSeC occurs during its oxidation, and confirms previously reported data indicating that the ketoacid arising from CMSeC is CMSeP.

Molar absorptivities of beta-NADH and beta-NADPH.

Re-investigating the accuracy of the commonly used values for molar absorptivities (epsilon) of beta-NADH and beta-NADPH at Hg 334, Hg 365, or 340 nm, we obtained the following results: The maximum of absorbance of NADH is shifted from about 340 nm at 0 degrees C to about 338.5 nm at 38 degrees C; the corresponding maxima of NADPH are located at about 0.5-nm longer wavelengths. In addition, the absorption curves of both coenzymes broaden with increasing temperature. For these reasons, the epsilon-values of NADH and NADPH are generally different from each other, and are temperature-dependent. Only at 334 nm are they almost identical and nearly independent of temperature. Therefore this wavelength is recommended for precise measurements. The epsilon-values of these coenzymes are influenced by ionic strength and pH. To determine the absolute values of the molar absorptivities, we performed the glutamate dehydrogenase or lactate dehydrogenase assay with carefully purified 2-oxoglutaric acid or pyruvic acid in the presence of excess coenzyme. The purity of the substrates was checked through differential scanning calorimetry, moisture analysis, gas-liquid chromatography, gas chromatography in combination with mass spectrometry, and nuclear magnetic resonance spectroscopy. The epsilon-values observed under the various conditions are about 1-7% higher than those currently used.