Hiroshi Oneda

Substrate-dependent Activation of Thermolysin by Salt

Salt-activation of thermolysin was examined using a positively charged fluorescent substrate, (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH2 [MOCAc-PLGL(Dpa)AR]. Thermolysin activity increased in a biphasic exponential fashion and was 40 times higher in the presence of 4 M NaCl than in its absence. The degree of activation at x M NaCl was expressed as 4.7x when [NaCl]o<0.5 M and 2.3x when [NaCl]o>0.5 M respectively.

Anomalous pH-dependence of the activity of human matrilysin (matrix metalloproteinase-7) as revealed by nitration and amination of its tyrosine residues

Matrilysin activity exhibits a broad bell-shaped pH-dependence profile, with pK(a) values of 4.0 and 9.8. A maximum of five out of eight tyrosine residues in matrilysin were nitrated with tetranitromethane. On nitration of between one and five tyrosines, pK(a) at the alkaline side (pK(e2)) was shifted from 9.8 to 10.3-10.6, while that at the acidic side (pK(e1)) was not altered. The pK(e2) that was shifted by nitration to 10.3-10.6 was restored to 9.4-9.7 by subsequent amination, suggesting that the shift in pK(e2) is induced by a negative charge introduced on the most reactive tyrosine, Tyr-150. The Michaelis constant (K(m)) observed at pH 10 was decreased by nitration as a result of the increase in pK(e2), suggesting that the residue with pK(e2) may play a role in the recognition of substrate. When four or five tyrosines were nitrated, the activity at pH <7 decreased significantly, while that at pH 7-10 was unchanged, and thus the pH-dependence was not bell-shaped, but anomalous, with a third pK(a) (pK(e3)) of 6.2-6.4 in addition to pK(e1) and pK(e2). This suggests the possibility that a newly introduced nitrotyrosine residue has a strong influence on the activity as an ionizable group.

Dissociation/association properties of a dodecameric cyclomaltodextrinase : Effects of pH and salt concentration on the oligomeric state

As an effort to elucidate the quaternary structure of cyclomaltodextrinase I‐5 (CDase I‐5) as a function of pH and salt concentration, the dissociation/association processes of the enzyme were investigated under various pH and salt conditions. Previous crystallographic analysis of CDase I‐5 indicated that it existed exclusively as a dodecamer at pH 7.0, forming an assembly of six 3D domain‐swapped dimeric subunits. In the present study, analytical ultracentrifugation analysis suggested that CDase I‐5 was present as a dimer in the pH range of 5.0–6.0, while the dodecameric form was predominant at pH values above 6.5. No dissociation of the dodecamer was observed at pH 7.0 and the above. Gel filtration chromatography showed that CDase I‐5 dissociated into dimers at a rate of 8.58 × 10−2 h−1 at pH 6.0. A mutant enzyme with three histidine residues (H49, H89, and H539) substituted with valines dissociated into dimers faster than the wild‐type enzyme at both pH 6.0 and 7.0. The tertiary structure indicated that the effect of pH on dissociation of the oligomer was mainly due to the protonation of H539. Unlike the pH‐dependent process, the dissociation of wild‐type CDase I‐5 proceeded very fast at pH 7.0 in the presence of 0.2–1.0 m of KCl. Stopped‐flow spectrophotometric analysis at various concentrations of KCl showed that the rate constants of dissociation (kd) from dodecamers into dimers were 5.96 s−1 and 7.99 s−1 in the presence of 0.2 m and 1.0 m of KCl, respectively.

Inhibitory Effects of Alcohols on the Activity of Human Matrix Metalloproteinase 7 (Matrilysin)

Aliphatic alcohols inhibited the activity of human matrix metalloproteinase 7 (matrilysin) competitively with Ki of 6.1–19.4% (v/v) or 0.66–4.80 M. From the relationship between the structures of alcohols and their Ki values, alcohols are considered to bind the hydrophobic S1′ subsite most plausibly, and the size of the pocket was estimated to be large enough to accommodate the length of 1-butanol (4-carbon chain) and the bulk of tertiary alcohols. Alcohols might be suitable probes for exploring the active-site geometry of enzymes.

Detection of antigen-antibody reaction using a fluorescent probe and its application to homogeneous competitive-type immunoassay for insulin.

The interaction of bovine insulin with anti-human insulin antibody (mAb) was examined using a fluorescent probe. The fluorescence intensity of fluoresceinthiocarbamyl (FTC)-insulin was increased by adding mAb, and the increase was saturated at 53% at a molar ratio of FTC-insulin to mAb of 2.0. Based on the change in fluorescence intensity, a standard curve of the homogeneous competitive-type immunoassay was constructed, and the detection range of insulin was found to be 50–400 nM.

Determination of hydrophobicity of dry-heated wheat starch granules using sucrose fatty acid esters (SFAE)

Sucrose fatty acid esters (SFAE) were adsorbed onto dry-heated (120 °C for 10, 20, 40, 60, and 120 min) wheat starch granules and extracted with ethyl ether in a Soxhlet apparatus without gelatinization of the starch granules. The amount of sucrose in the extracted SFAE was determined by the phenol sulfate method. A gradual increase of the sucrose from 159 to 712 μg, in SFAE per gram of starch, occurred with increasing dry-heating time and demonstrated the increased hydrophobicity of the starch granules. Increase of the SFAE was highly correlated (r = 0.9816) to increase of the oil-binding capacity of the dry-heated wheat starch granules. Non-waxy rice, waxy rice, sweet potato, and potato starch granules also showed higher hydrophobicity after dry-heating by this method. Graphical Abstract Outline of determination of hydrophobicity in dry-heated starch granules by sucrose fatty acid esters.