Evaluation of enamel crystallites in subsurface lesion by microbeam X-ray diffraction.

Early caries lesion is a demineralization process that takes place in the top 0.1 mm layer of tooth enamel. In this study, X-ray microbeam diffraction was used to evaluate the hydroxyapatite crystallites in the subsurface lesion of a bovine enamel section and the results are compared with those obtained by transversal microradiography, a method commonly used for evaluation of tooth mineral. Synchrotron radiation from SPring-8 was used to obtain a microbeam with a diameter of 6 microm. Wide-angle X-ray diffraction reports the amount of hydroxyapatite crystals, and small-angle X-ray scattering reports that of voids in crystallites. All three methods showed a marked decrease in the enamel density in the subsurface region after demineralization. As these diffraction methods provide structural information in the nanometre range, they are useful for investigating the mechanism of the mineral loss in early caries lesion at a nanometre level.

Substrate selectivity in Aspergillus niger KU-8 acid phosphatase II using phosphoryl oligosaccharides.

The intracellular acid phosphatase II (ACPase II) produced by Aspergillus niger KU-8 preferentially dephosphorylates C-6 phosphate groups rather than C-3 phosphate groups of phosphoryl oligosaccharides. In this study, the kinetic parameters of ACPase II were measured. 3(2)-phosphoryl maltotriose and 6(2)-phosphoryl maltotriose, which differ only in the binding position of the phosphate group, were prepared and used as the substrates. The Km for both substrates were similar. However, the k(cat) value for the 6(2)-phosphoryl maltotriose was about three-fold of that for the 3(2)-phosphoryl maltotriose.

Rice bifunctional alpha-amylase/subtilisin inhibitor: characterization, localization, and changes in developing and germinating seeds.

A bifunctional alpha-amylase/subtilisin inhibitor (RASI) was purified to electrophoretic homogeneity from rice (Oryza sativa L.) bran. Its molecular mass was 21 kDa by SDS-PAGE and its isoelectric point was 9.05. Purified RASI inhibited subtilisin Carlsberg strongly and inhibited alpha-amylase from germinating rice seeds weakly. It inhibited rice alpha-amylase more than barley alpha-amylase, and the inhibition of rice alpha-amylase was greater at higher pHs. RASI did not inhibit trypsin, chymotrypsin, cucumisin, or mammalian alpha-amylase. The RASI was in the outermost part of the rice grain and its subcellular site seemed to be aleurone particles in aleurone cells. SDS-PAGE and western blotting showed that RASI was synthesized in the late milky stage in developing seeds, and it remained fairly constant during the first 7 days of germination.

A novel acid phosphatase from Aspergillus niger KU-8 that specifically hydrolyzes C-6 phosphate groups of phosphoryl oligosaccharides.

We had analyzed the detailed structures of the phosphoryl oligosaccharide-1 (PO-1) fraction that was the main component of phosphoryl oligosaccharides (POs) prepared from a potato starch hydrolysate. PO-1 fraction was made up of 3-phosphoryl oligosaccharides and 6-phosphoryl oligosaccharides. Aspergillus niger strain KU-8 produced two types of intracellular acid phosphatase (EC 3.1.3.2, ACPase); ACPase I and II. ACPase II preferentially dephosphorylated 6-phosphoryl oligosaccharides rather than 3-phosphoryl oligosaccharides. The molecular weight of the enzyme was estimated as 66 kDa by SDS-polyacrylamide gel electrophoresis and about 260 kDa by gel filtration, implying the active form to be a tetramer. The optimum pH and temperature of the enzyme were 2.0-2.5 and 60 degrees C, respectively. ACPase II was stable below 50 degrees C for 30 min and pH 2.0-10.0 for 60 min. In spite of the strict specificity toward 6-phosphoryl oligosaccharides in the PO-1 fraction, ACPase II was able to hydrolyze Fru-1,6-di-P, ATP, pyrophosphate, and polyphosphate as well as pNPP and Glc-6-P, a broad substrate specificity.

Inhibitory effect of phosphorylated oligosaccharides prepared from potato starch on the formation of calcium phosphate.

The inhibitory effect of phosphorylated oligosaccharides, which were prepared from potato starch, on the formation of calcium phosphate in vitro were investigated. Phosphorylated oligosaccharides from potato were fractionated by ion-exchange chromatography into two fractions, PO-1 and PO-2. Fraction PO-1 was composed of maltotriose, maltotetraose, and maltopentaose to which one phosphate group was attached. Fraction PO-2 was predominantly composed of maltopentaose and maltohexaose to which at least two phosphate groups were attached. The average degree of polymerization of dephosphorylated PO-1 and PO-2 was evaluated to be 4.02 and 5.82, respectively. Fraction PO-2 was the main component having an inhibitory effect on calcium phosphate formation. In addition, among the phosphorylated monosaccharides, glucose-1,6-diphosphate and fructose-1,6-diphosphate were more effective inhibitors of the formation of calcium phosphate than glucose-6-phosphate and fructose-6-phosphate. These results suggest that the strength of the inhibitory effect might depend on the number of phosphate groups attached to each sugar molecule.