Shigeyoshi Osaki

Crystal structures of isomaltase from Saccharomyces cerevisiae and in complex with its competitive inhibitor maltose

The structures of isomaltase from Saccharomyces cerevisiae and in complex with maltose were determined at resolutions of 1.30 and 1.60 Å, respectively. Isomaltase contains three domains, namely, A, B, and C. Domain A consists of the (β/α)8‐barrel common to glycoside hydrolase family 13. However, the folding of domain C is rarely seen in other glycoside hydrolase family 13 enzymes. An electron density corresponding to a nonreducing end glucose residue was observed in the active site of isomaltase in complex with maltose; however, only incomplete density was observed for the reducing end. The active site pocket contains two water chains. One water chain is a water path from the bottom of the pocket to the surface of the protein, and may act as a water drain during substrate binding. The other water chain, which consists of six water molecules, is located near the catalytic residues Glu277 and Asp352. These water molecules may act as a reservoir that provides water for subsequent hydrolytic events. The best substrate for oligo‐1,6‐glucosidase is isomaltotriose; other, longer‐chain, oligosaccharides are also good substrates. However, isomaltase shows the highest activity towards isomaltose and very little activity towards longer oligosaccharides. This is because the entrance to the active site pocket of isomaltose is severely narrowed by Tyr158, His280, and loop 310–315, and because the isomaltase pocket is shallower than that of other oligo‐1,6‐glucosidases. These features of the isomaltase active site pocket prevent isomalto‐oligosaccharides from binding to the active site effectively.

Steric hindrance by 2 amino acid residues determines the substrate specificity of isomaltase from Saccharomyces cerevisiae

The structures of the E277A isomaltase mutant from Saccharomyces cerevisiae in complex with isomaltose or maltose were determined at resolutions of 1.80 and 1.40Å, respectively. The root mean square deviations between the corresponding main-chain atoms of free isomaltase and the E277Α-isomaltose complex structures and those of free isomaltase and the E277A-maltose complex structures were found to be 0.131Å and 0.083Å, respectively. Thus, the amino acid substitution and ligand binding do not affect the overall structure of isomaltase. In the E277A-isomaltose structure, the bound isomaltose was readily identified by electron densities in the active site pocket; however, the reducing end of maltose was not observed in the E277A-maltose structure. The superposition of maltose onto the E277A-maltose structure revealed that the reducing end of maltose cannot bind to the subsite +1 due to the steric hindrance from Val216 and Gln279. The amino acid sequence comparisons with α-glucosidases showed that a bulky hydrophobic amino acid residue is conserved at the position of Val216 in α-1,6-glucosidic linkage hydrolyzing enzymes. Similarly, a bulky amino acid residue is conserved at the position of Gln279 in α-1,6-glucosidic linkage-only hydrolyzing α-glucosidases. Ala, Gly, or Asn residues were located at the position of α-1,4-glucosidic linkage hydrolyzing α-glucosidases. Two isomaltase mutant enzymes - V216T and Q279A - hydrolyzed maltose. Thus, the amino acid residues at these positions may be largely responsible for determining the substrate specificity of α-glucosidases.

Electrical properties of form III poly(vinylidene fluoride)

Abstract Form III poly(vinylidene fluoride) crystals were easily obtained by an isothermal crystallization between 165 and 175°C. The dielectric relaxation for the form III crystal was observed at high temperatures, where values of dielectric constant e′ and loss factor e″ were very large. The e′ underwent almost a tenfold increase when the temperature was raised from 150°C to the melting point (198°C) of the sample. The sharp drop in e′ beyond the melting point was even more striking. Also, at 100 Hz, the e″ reached a very sharp maximum at about 180°C. The relaxation strength was about 2.8 × 104. According to the results of mechanical and DSC measurements, it may be concluded that this high temperature relaxation is due to the form III crystallites. There are two possibilities for the mechanism responsible for the relaxation: (I) the orientational motion of crystallites accompanied by the applied ac electric field; (2) the motion of the domain boundaries of the form III polar crystallites which bring abo...

Is the mechanical strength of spider’s drag-lines reasonable as lifeline?

Drag-lines play a role as a lifeline for a spider to move and fall from trees. The mechanical strength of the drag-lines may be related to the spiders weight since spiders hang from them. The safety coefficient of drag-lines as the lifeline should be considered for the mechanical strength of the drag-lines consisting of double filaments. It was found that the elastic limit strength and breaking strength increase linearly with spiders weight, about twice the spiders weight corresponds to the elastic limit strength, and about six times the spiders weight corresponds to the breaking strength of drag-lines. In other words, the spiders weight corresponds to the elastic limit strength for the single filament. This means that a spider can act safely by one filament even though another filament is broken down. It should be an outcome from spiders long history of 400-million-years evolution. These findings may give a maximal efficiency for the mechanical strength of spiders drag-lines.

DISTRIBUTION MAP OF COLLAGEN FIBER ORIENTATION IN A WHOLE CALF SKIN

It is important to obtain information about the collagen fiber orientation in biological fibrous tissues such as human and animal skins because the collagen fiber orientation in the skins may closely relate to the motional functions of the body. However, no reports are yet available on the distribution of collagen fiber orientation in a whole skin.

Determination of the orientation of collagen fibers in human bone

A human calcaneus bone, consisting of hydroxyapatite and collagen fibers, was successively sliced into samples in a direction perpendicular to the long axis of the bone and parallel to the long axis of the human lower limb. The transmitted microwave intensities of 12 GHz, reflecting the dielectric property, were measured for the slice samples using Osakis microwave method (Tappi J., 1987 ; 70:105–108). The complex dielectric constant of 12 GHz of the collagen fiber film was much greater than that of hydroxyapatite disc, which demonstrated that the dielectric anisotropy observed for the sliced bone was mainly affected by the collagen fibers. The angular dependence of the transmitted microwave intensity gives the orientation angle reflecting the direction of the collagen‐fiber orientation, and the degree of orientation reflecting the anisotropic property of collagen fibers. The orientation angle and the degree of orientation for the slice samples changed with changing position along the long axis of the calcaneus bone. The direction of orientation deviated to the lateral side at the heel part of the left calcaneus, and to the medial side at the middle part. The degree of orientation is relatively high at the heel part and low at the middle. Such results give a two‐dimensional distribution of collagen‐fiber orientation in the left calcaneus, and suggest that the direction and degree of orientation are closely related to the direction and magnitude of the stress applied to the bone, respectively. Anat Rec 266:103–107, 2002.

Explanation of orientation patterns determined for sheet materials by means of microwaves

Unstretched, biaxially stretched, and uniaxially stretched poly(ethylene terephthalate) (PET) films showed different types of three‐dimensional pattern, i.e., the frequency and angular dependencies of transmitted microwave intensity. The resonance frequency and the intensity at the resonance frequency for the biaxially and uniaxially stretched PET films changed with rotation angle, while those for the unstretched PET films stayed unchanged. Thus these properties reflected the dielectric anisotropy of the polymer films. The angular dependence of transmitted microwave intensity at a fixed frequency, i.e., the orientation pattern, gave an orientation angle and a maximum‐to‐minimum ratio of transmitted microwave intensity. The orientation pattern can be obtained by cutting the three‐dimensional pattern at the fixed frequency. This frequency should be selected above the resonance frequency, where the transmitted microwave intensity is one‐half that at the resonance frequency in the resonance curve which is loc...

Dielectric anisotropy of stretched poly(ethylene terephthalate) at microwave frequencies

A new method for determining quickly and nondestructively the complex dielectric constant at microwave frequencies was applied to poly(ethylene terephthalate) (PET) films. The dielectric loss e‘ at 4.0 GHz decreased with increasing degree of crystallinity, indicating that it affects molecular motions in the amorphous region. From the angular dependence of e‘ for the uniaxially stretched PET film dielectric anisotropy was detected, showing e‘ to be larger in the transverse direction (TD) than in the machine direction (MD) or draw direction. The ratio of e‘ in the TD to MD increased with increasing draw ratio. The PET molecules in the amorphous region are considered to be oriented mainly in the MD. This anisotropy was considered to result from a change in dipole moment due to torsional motions of the ethylene glycol unit around the main chain in the amorphous region. Values of e‘max/e‘min were found to be useful as a measure of the degree of orientation, where e‘max and e‘min denote the maximum and minimum ...

A new microwave cavity resonator for determining molecular orientation and dielectric anisotropy of sheet materials

A new type of microwave instrument is developed for quick determination of the molecular orientation and dielectric anisotropy in sheet materials. Its cavity resonator differs from the conventional one in that the waveguide is separated by a narrow gap. Thus, it consists of a pair of rectangular waveguides, each partitioned by an iris plate. A given sheet is inserted into the gap, and can be rotated around the axis normal to its plane. The iris diameter and gap width are determined so as to meet the requirement that the transmitted microwave power and the Q value may not drop markedly after insertion of thick films into the gap. Polarized microwaves are irradiated perpendicularly to the sheet, and the transmitted wave intensity is measured at every 1° of rotation angle as the sheet is rotated at a speed of 6.0 s per turn. This operation quickly gives information related to the molecular orientation in the given sample. Dielectric measurements can be made at any rotation angles, allowing the angular depend...

Determination of the refractive index and birefringence for biaxially stretched poly(ethylene terephthalate) at microwave frequencies

Abstract Refractive index and birefringence were determined without contact for biaxially stretched poly(ethylene terephthalate) (PET) films at a microwave frequency. The following relationship was obtained between the birefringences ΔnMW at a frequency of 4.0 GHz and ΔnOPT at a visible wavelength of 589 nm for many kinds of biaxially stretched PET films: ΔnMW = 1.3933 ΔnOPT. The correlation coefficient between ΔnMW and ΔnOPT was 0.969, indicating a very good correlation. On the other hand, the observed relation between the refractive indices nMW at 4.0 GHz and nOPT at 589 nm could be expressed as nMW = −0.5567 + 1.4243 nOPT. The nMW was larger than the nOPT in all the directions of the sheet plane. This difference may arise from difference in contribution of e′ associated with the local motion (β-relaxation) of PET molecules.