Characterization of bovine angiogenin-1 and lactogenin-like protein as glycyrrhizin-binding proteins and their in vitro phosphorylation by C-kinase.

Angiogenin-1 (p15, an angiogenesis inducer with RNase activity) and lactogenin-like protein (p17) isolated from partially purified bovine lactoferrin (bLF) preparations were characterized as glycyrrhizin (GL)-binding proteins (gbPs). As expected, bLF-affinity column chromatography confirmed these two gbPs to be bLF-binding proteins. These two purified gbPs exhibited RNase activities when incubated with poly(C) as a substrate. Both GL and glycyrrhetinic acid (GA) at 100 microM significantly inhibited RNase activities of these two gbPs, both of which functioned as phosphate acceptors of C-kinase in vitro. Phosphorylation of p15 and p17 by C-kinase was inhibited by GA in a dose-dependent manner with the 50% inhibition dose (ID50) of approx. 10 microM, whereas GL required a relatively high dose (300 microM) to inhibit significantly it. A GA derivative (oGA, ID50=approx. 0.3 microM) was found to be a potent inhibitor of the C-kinase-mediated phosphorylation of these two gbPs in vitro. In addition, a possible physiological significance of C-kinase on the physiological interaction between bLF and two bLF-binding proteins (p15 and p17) is noted.

Characterization of bovine and human lactoferrins as glycyrrhizin-binding proteins and their phosphorylation in vitro by casein kinase II.

The binding ability of bovine and human lactoferrins (bLF and hLF; LFs) to a glycyrrhizin (GL)-affinity column and their phosphorylation by casein kinase II (CK-II) in vitro were biochemically investigated. It was found that (i) both bLF and hLF are GL-binding proteins; (ii) purified both proteins function as phosphate acceptors of CK-II; and (iii) this phosphorylation is completely inhibited by two polyphenol-containing anti-oxidant compounds (quercetin and epigallocatechin gallate) at I microm, whereas a glycyrrhetinic acid derivative (oGA) inhibits it at one tenth the concentration of GL. The DNA-binding affinity of hLF was reduced by GL in a dose dependent manner. However, no significant effect of the CK-II-mediated hLF phosphorylation on its DNA-binding affinity was detected. These results suggest that the GL-induced inhibition of the DNA-binding affinity and the CK-II-mediated phosphorylation of hLF may be closely correlated with the anti-inflammatory effect of GL in the human body.

Structures of novel acidic galactooligosaccharides synthesized by Bacillus circulans beta-galactosidase.

The structures of acidic oligosaccharides synthesized by a transglycosylation reaction by Bacillus circulans beta-galactosidase, using lactose as the galactosyl donor, and N-acetylneuraminic acid (NeuAc) and glucuronic acid (GlcUA) as the acceptors were investigated. Acidic oligosaccharides thus synthesized were purified by anion exchange chromatography and charcoal chromatography. The MS and NMR studies indicated that the acidic oligosaccharides from NeuAc were Gal beta-(1-->8)-NeuAc, Gal beta-(1-->9)-NeuAc, and Gal beta-(1-->3)-Gal beta-(1-->8)-NeuAc, and those from GlcUA were Gal beta-(1-->3)-GlcUA and Gal beta-(1-->4)-Gal beta-(1-->3)-GlcUA. These are novel acidic galactooligosaccharides.

Effects of lactitol-oligosaccharides on calcium and magnesium absorption in rats.

Lactitol-oligosaccharide (LO) was prepared from lactitol by transglycosylation reaction with Aspergillus oryzae beta-galactosidase. LO is resistant to metabolism in the small intestine but not in the large intestine. The effects of LO, lactose (Lac), lactitol (Lacol) and galactooligosaccharide (GL) on calcium and magnesium absorption were determined by feeding 8-week-old Sprague-Dawley male rats diets containing 5% of the above carbohydrates for two weeks. The results obtained were as follows. 1) A significant increase of calcium absorption was observed in the LO diet. 2) A significant increase of magnesium absorption was observed in the LO, GL and Lacol diets. 3) The concentration of total volatile fatty acids (VFA) in the cecal contents increased significantly in the LO, GL and Lacol diets. The main constituent of VFA in the cecal contents was found to be acetic acid. 4) The correlations between calcium and magnesium absorption ratios and total VFA concentration in the cecum were found to be significantly related. These findings suggest that LO is metabolized to VFA, among which acetic acid concentration seems to have the most stimulatory effect on the absorption of calcium.

Formation of oligosaccharides from lactose by Bacillus circulans beta-galactosidase.

Eleven oligosaccharides formed by a transglycosylation reaction during lactose hydrolysis with Bacillus circulans beta-galactosidase were purified by gel permeation chromatography, charcoal chromatography, and HPLC. From the results of methylation analysis, and MS and NMR studies, it was concluded that these oligosaccharides were beta-D-Galp-(1-->3)-D-Glc, beta-D-Galp-(1-->6)-D-Glc, beta-D-Galp-(1-->2)-D-Glc, beta-D-Galp-(1-->4)-beta-D-Galp-(1-->4)-D-Glc, beta-D-Galp-(1-->6)-[beta-D-Galp-(1-->2)]-D-Glc, beta-D-Galp-(1-->6)-[beta-D-Galp-(1-->4)]-D-Glc, beta-D-Galp-(1-->4)-beta-D-Galp-(1-->3)-D-Glc, beta-D-Galp-(1-->4)-beta-D-Galp-(1-->2)-D-Glc, beta-D-Galp-(1-->4)-[beta-D-Galp-(1-->2)]-D-Glc, beta-D-Galp-(1-->4)-beta-D-Galp-(1-->6)-D-Glc, beta-D-Galp-(1-->6)[beta-D-Galp-(1-->3)]-D-Glc. The last five are newly observed oligosaccharides. The results of a use test (in vitro) by human intestinal bacteria showed that the oligosaccharides containing lactose units were predominantly used by human intestinal bifidobacteria.

Effects of lactitol-oligosaccharides on the intestinal microflora in rats.

Lactitol-oligosaccharide (LO) was prepared from lactitol by a transgalactosylation reaction catalyzed by Aspergillus oryzae beta-galactosidase. The utilization of LO by human intestinal bacteria, the digestion of LO by rat jejunum mucosal homogenates and the effects of LO on the intestinal microflora in rats were compared with those of lactitol. 1) LO was utilized in vitro by Bifidobacterium, but lactitol was not utilized. 2) Neither LO nor lactitol were digested by rat jejunum mucosal homogenates. 3) A significant increase in the fecal counts of Bifidobacterium was observed in the LO diets. 4) The concentration of organic acids in feces and cecal contents significantly increased in the LO diets. 5) The concentration of fecal putrefactive products significantly decreased in the LO and lactitol diets. These findings suggest that LO is effective for improving intestinal conditions.

Formation of oligosaccharides from lactitol by Aspergillus oryzae beta-D-galactosidase.

Six oligosaccharides were first formed from lactitol by a transgalactosylation reaction catalyzed by Aspergillus oryzae beta-D-galactosidase. From the results of methylation analysis, MS, and 1H- and 13C-NMR studies, it was concluded that these oligosaccharides are O-beta-D-galactopyranosyl-(1----4)-O-beta-D-galactopyranosyl-(1----4)-D- glucitol, O-beta-D-galactopyranosyl-(1----3)-O-beta-D-galactopyranosyl-(1----4)-D- glucitol, O-beta-D-galactopyranosyl-(1----4)-[O-beta-D-galactopyranosyl-(1----6)]- D- glucitol, O-beta-D-galactopyranosyl-(1----6)-O-beta-D-galactopyranosyl-(1---4)- glucitol, O-beta-D-galactopyranosyl-(1----4)-[O-beta-D-galactopyranosyl- (1----5)]-D-glucitol, and O-beta-D-galactopyranosyl-(1----4)-[O-beta-D-galactopyranosyl-(1----1)]- D-glucitol. The last three are newly observed oligosaccharides.