Activity of Debaryomyces hansenii UFV-1 α-galactosidases against α-D-galactopyranoside derivatives

α-D-Galactopyranosides were synthesized and their inhibitory activities toward the Debaryomyces hansenii UFV-1 extracellular and intracellular α-galactosidases were evaluated. Methyl α-D-galactopyranoside was the most potent inhibitor compared to the others tested, with K(i)(') values of 0.82 and 1.12 mmolL(-1), for extracellular and intracellular enzymes, respectively. These results indicate that the presence of a hydroxyl group in the C-6 position of α-D-galactopyranoside derivatives is important for the recognition by D. hansenii UFV-1 α-galactosidases.     

A synthesis of methyl α-maltoside

A mixture of methanol, bromine, and silver carbonate readily converted ethyl 1-thio-β-maltoside into a mixture containing methyl α,β-maltoside and traces of maltose. The α-anomer, which initially formed 85% of the maltoside fraction, was enriched by selective oxidation of the β-anomer with chromic acid. Chromatographic purification gave a 65% yield of methyl α-maltoside of 97% anomeric purity. N.m.r. spectral data for methyl α-maltoside heptaacetate were obtained.     

Selective esterification of methyl α-d-glucopyranoside

6-Monoesters of methyl α-D-glucopyranoside have been prepared by transesterification with methyl laurate, methyl palmitate, methyl stearate, and methyl benzoate, catalyzed by sodium methoxide in the absence of a solvent. The benzoylation of methyl 4,6-O-benzylidene-α-D-glucopyranoside was separately performed with methyl benzoate and with benzoyl chloride, and the product distributions with the two reagents were compared; methyl 2-O-benzoyl-α-D-glucopyranoside and methyl 3-O-benzoyl-α-D-glucopyranoside were obtained by hydrolysis of the corresponding monobenzoates of methyl 4,6-O-benzylidene-α-D-glucopyranoside. Methyl 4-O- benzoyl-α-D-glucopyranoside was prepared by ring-opening of methyl 4,6-O-benzylidene-α-D-glucopyranoside with N-bromosuccinimide in the presence of water.     

Migration of thiolthiocarbonyl groups of methyl α-d-glucopyranoside xanthates

If similar reactant ratios are used, the xanthation of methyl α-d-glucopyranoside parallels the xanthation of starch. Degrees of substitution in the range of 0.3–0.4 were achieved, and changes in distribution from secondary to primary positions were observed. Synthesis of the individual, isomeric, xanthate salts of methyl α-d-glucopyranoside, and treatment of each with 18% sodium hydroxide, showed that the 2-, 3-, and 6-xanthate salts rearranged to mixtures in each of which the 6-isomer preponderated. Evidence is presented suggesting that the 2-isomer migrates intramolecularly to the 6-isomer by way of the 3-isomer. However, observation of the presence of minor proportions of polyxanthates in these mixtures suggests that intermolecular migration of thiolthiocarbonyl groups also occurs.     

Expression of enzymatically active,recombinant barley α-glucosidase in yeast and immunological detection of α-glucosidase from seed tissue

An -glucosidase cDNA clone derived from barley aleurone tissue was expressed in Pichia pastoris and Escherichia coli. The gene was fused with the N-terminal region of the Saccharomyces cerevisiae -factor secretory peptide and placed under control of the Pichia AOX1 promoter in the vector pPIC9. Enzymatically active, recombinant -glucosidase was synthesized and secreted from the yeast upon induction with methanol. The enzyme hydrolyzed maltose > trehalose > nigerose > isomaltose. Maltase activity occurred over the pH range 3.5–6.3 with an optimum at pH 4.3, classifying the enzyme as an acid -glucosidase. The enzyme had a K_m of 1.88 mM and V_max of 0.054 µmol/min on maltose. The recombinant -glucosidase expressed in E. coli was used to generate polyclonal antibodies. The antibodies detected 101 and 95 kDa forms of barley -glucosidase early in seed germination. Their levels declined sharply later in germination, as an 81 kDa -glucosidase became prominent. Synthesis of these proteins also occurred in isolated aleurones after treatment with gibberellin, and this was accompanied by a 14-fold increase in -glucosidase enzyme activity.Abbreviations: AGL, barley seed -glucosidase; rAGL, recombinant barley seed -glucosidase; BMGY, buffered glycerol-complex medium; BMMY, buffered methanol-complex medium; GA, gibberellic acid; UTR, untranslated region.     

Fluorescent detection of α-aminoadipic and γ-glutamic semialdehydes in oxidized proteins

The oxidative modification of proteins is believed to play a critical role in the etiology and/or progression of several diseases. α-Aminoadipic semialdehyde (AAS) and γ-glutamic semialdehyde (GGS) residues represent major oxidized amino acids generated in oxidized proteins. This paper describes a novel procedure for the specific and sensitive determination of AAS and GGS after their reductive amination with sodium cyanoborohydride and p-aminobenzoic acid, a fluorescence reagent, to their corresponding derivatives, followed by a high-performance liquid chromatography (HPLC) analysis. This fluorescent labeling of protein-associated aldehyde moieties is a simple and accurate technique that may be widely used to reveal increased levels of oxidatively modified proteins with reactive oxygen species during aging and disease.