Masatake Ohnishi

Structure of β-Glucan Oligomer from Laminarin and Its Effect on Human Monocytes to Inhibit the Proliferation of U937 Cells

We analyzed the human monocyte-stimulating ability of laminarin from Eisenia bicyclis, lichenan from Cetraria islandica, and their oligomers depolymerized with endo-1,3-β-glucanase from Arthrobacter sp. The respective β-glucan oligomers with different degrees of polymerization (DP) were fractionated from hydrolytic products of laminarin and lichenan using gel-filtration chromatography. The monocyte-conditioned medium pre-cultured in the presence of a fraction of β-glucan oligomer (DP≥8) from laminarin exhibited inhibitory activity against the proliferation of human myeloid leukemia U937 cells, while those pre-cultured with other β-glucan oligomers and the original laminarin and lichenan showed little or no activity. NMR analysis indicated that the β-glucan oligomer (DP≥8) has an average DP value of 13, and its ratio of β-1,3- to β-1,6-linkages in glucopyranose units was estimated to be 1.3:1. These results indicate that the β-1,3-glucan oligomer with a higher content of β-1,6-linkage stimulates monocytes to inhibit the proliferation of U937 cells.

Elucidation of the subsite structure of bacterial saccharifying alpha-amylase and its mode of degradation of maltose

The subsite structure of bacterial saccharifying alpha-amylase (BSAm) was elucidated by two methods using a series of maltooligosaccharides labeled with [14C]D-glucose at the reducing end. The rate parameter k0/Km and the cleavage frequency were obtained using the labeled substrates at sufficiently low concentrations to eliminate transglycosylation and condensation. This evaluation showed that the active center is composed of five subsites, with the catalytic site located between the 3rd and the 4th subsites from the nonreducing end. The evaluated affinity values of a subsite varied with the set of data used, which suggests some stimulation factor resulting from the chain length effect. The appearance of a time lag during the digestion of the poor substrate, maltose, was studied using radioactively labeled maltose (81.6 mM). Radioactive oligosaccharides larger than maltose were found at a significant level of more than 2% of the initial substrate in the digests, including a product peculiar to condensation, G-G*-G, as 8-10% of the maltotriose in the digests. This indicates that transglycosylation is a main side reaction (ca. 90%). A degradation pathway for maltose via maltosyl transfer was proposed, in which G3 behaves as a kind of catalyst.

Gymnemic acids inhibit rabbit glyceraldehyde-3-phosphate dehydrogenase and induce a smearing of its electrophoretic band and dephosphorylation

Gymnemic acids (GA) inhibited rabbit muscle glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) activity. Binding of GA to GAPDH was observed by surface plasmon resonance measurement. Incubation of GAPDH with GA induced a smearing of the GAPDH band in SDS–PAGE. The GA‐induced smearing was diminished by prior incubation of GA with γ‐cyclodextrin or by GA treatment with NAD. GA treatment did not affect the electrophoretic mobility of glucose‐6‐phosphate isomerase and dehydrogenase. GA treatment diminished the GAPDH band detected by an antibody to phosphoserine, but did not affect the phosphoserine bands of glucose‐6‐phosphate isomerase and dehydrogenase. These results indicated that GA specifically induced dephosphorylation of GAPDH.

Crystallization and preliminary crystallographic analysis of endo‐1,3‐β‐glucanase from Arthrobacter sp.

Endo-1,3-beta-glucanases hydrolyze internal 1,3-beta-glucosyl linkages. The endo-1,3-beta-glucanase from Arthrobacter sp. was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to space group P4(1), with unit-cell parameters a = 71.31, c = 60.07 A, and contained one molecule per asymmetric unit. The Matthews coefficient (VM) and the solvent content were 2.35 A3 Da(-1) and 47.63%, respectively. Diffraction data were collected to a resolution of 1.66 A at SPring-8 using a MAR CCD area detector and gave a data set with an overall Rmerge of 5.4% and a completeness of 99.4%.

Effects of Alkalinization and ATPase Inhibition on Stromal Free Mg2+ Concentration in Spinach Chloroplasts

Our earlier studies indicate that stromal alkalinization is essential for light-induced increase in free Mg2+ concentration ([Mg2+]) in chloroplast. Stromal [Mg2+] was increased by dark incubation of chloroplasts in the K+-gluconate medium (pH 8.0), or by NH4Cl. These results indicate that stromal alkalinization can induce an increase in stromal [Mg2+] without illumination. Some inhibitors of envelope proton-translocating ATPase activity involved in H+ efflux inhibited the alkalinization-induced increase in [Mg2+].