Koji Tadasa

Synthesis of N-kojic-amino acid and N-kojic-amino acid-kojiate and their tyrosinase inhibitory activity

Ten amino acid derivatives of kojic acid were synthesized to improve the tyrosinase inhibitory activity of kojic acid. Almost all derivatives showed stronger activities than kojic acid, and in general, N-kojic-amino acid-kojiate was found to have a higher inhibitory potency than N-kojic-amino acid. Among them, the N-kojic-L-phenylalanyl kojiate was the strongest inhibitor and its IC50 value was 1/380 that for kojic acid. The inhibition mechanism of these derivatives is considered to be noncompetitive which is similar to that of kojic acid.

Superoxide-scavenging and Tyrosinase-inhibitory Activities of the Extracts of Some Chinese Medicines

The superoxide-scavenging and the tyrosinase-inhibitory activities of 28 kinds of plants used as Chinese medicines were evaluated. Methanol/water extracts were used for the screening tests, and for those which represented high activities, other kinds of extracts were also studied. The extracts of Mallotus japonicus Muell. Arg. scavenged superoxide strongly; the half-inhibiting concentration (IC50) of its 50% methanol/water extract was 10.57 μg of dried material in 1 ml of reaction mixture. The extracts of Fntillaria thunbergii Miq., Carthamus tinctorius L., and Prunus persica (L.) Batsch had strong tyrosinase-inhibitory activities, and the extracts of Scutellariu baicalensis Georgi represented both kinds of activities. These facts suggested that Chinese medicines may be a treasure house of chemical compounds that have the superoxide-scavenging and the tyrosinase-inhibitory activities.

Kinetic behavior of activation of thermolysin by normal alcohols

SummaryThis paper reports an insight into the kinetic mechanism of activation and then inhibition in a thermolysin - catalyzed peptide synthesis of dipeptide N -(benzyloxycarbonyl)-L-phenylalanyl-L-phenylalanine methyl ester in aqueous - organic one phase system containing n-alcohols as activator. The jump in catalytic efficiency - as an effect of alcoholic solvents and the kinetic mode of activation are discussed.

Organic solvents cause spectral changes and pKa shifts of thermolysin in peptide synthesis in aqueous-organic one-phase reaction system

Thermolysin underwent spectral changes and pKa shifts upon mixing with alcoholic solvents. A hyperchromic spectra and shift occured upon activation of the enzyme by solvents used in dipeptide (Z-Phe-Phe-OMe) synthesis. This effect was decreased when enzyme was pre-treated with inhibitor. Model substrates did not undergo any significant change on mixing with the same solvents. pKa shift of upto 0.3 units was observed at solvent concentration showing maximum activity and the pKa shifts directly correlated with activity profiles of thermolysin.

Novel stabilization pattern of thermolysin due to the binding substrate induced by electrostatic change in enzyme active site caused by the temperature elevation

During the synthesis of the dipeptide, N-benzyloxycarbonyl-l-phenylalanyl-l-phenylalanine methyl ester, from N-benzyloxycarbonyl-l-phenylalanine and l-phenylalanine methyl ester by thermolysin, the enzyme was stabilized by 20 °C up to 110 °C. The stabilization was caused by the interaction of the enzyme with Phe-OMe, a counterpart of the substrate, which was bound at the enzyme active site due to the drop in pH and dielectric constant following the temperature elevation of the medium. The binding of the enzyme to Phe-OMe suggested the induction of the transition state formation at around 80 °C based on the UV spectra, resulting in the increase in the stability in the higher temperature region. The fluorescence second-order derivative spectra suggested that the binding Phe-OMe interacted with Trp 115 at the active site of the enzyme. The phenomenon was considered to be a novel stabilization pattern of the enzyme resulting from the conduction due to the chemical modification by the binding substrate.

Stabilization of Thermolysin Induced by a Decrease in the PH and Dielectric Constant of the Reaction Medium Resulting from a Temperature Increase in Z-Phe-Phe-OMe Synthesis

Thermolysin was stabilized by increasing the reaction temperature from 90°C to 110°C during peptide synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-phenylalanine methyl ester (Z-Phe-Phe-OMe). The stabilization energy was acquired from the drop in both pH and dielectric constant due to the temperature increase. The acquired stabilization energy was as high as ca. 42 kJ/mol (corresponding to 20°C). This acquired stabilization energy did not result from a single event such as a change in electrical charges. It was evaluated as the overall stabilization energy at and around the active site area of the enzyme using an electrostatic potential equation.

Kinetics of inhibition of thermolysin-catalyzed peptide synthesis by alcohols in aqueous organic one-phase system

The kinetic patterns and parameters of 12 alcoholic organic solvents of different classes inhibiting thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-phenylalanine methyl ester (Z-Phe-Phe-OMe) in aqueous organic one-phase reaction system have been determined. All alcohols showed a linear mixed type inhibition. A kinetic model of inhibition is suggested. It was presumed that alcohols interact with substrate in the active site of thermolysin.

Method of Culture of Human Cancer Cells with a Thermoresponsive Polymer and Dextran Sulfate.

A method for cell culture with a thermoresponsive polymer and dextran sulfate was developed. In the culture of human cancer cell lines, the polymer had no cytotoxicity and the cells attached, spread, and grew well on a substrate conjugated with type I collagen and the polymer. Use of different collagen : polymer ratios in the primary culture of cells from human cancer which had serially been transplanted into nude mice showed that the ratio of 2 : 1 gave satisfactory attachment and detachment of the cells. At each ratio tested, cell attachment was greater with type IV collagen than with type I collagen. When human cancer cells obtained from surgical specimens were cultured on type IV collagen-polymer substrate with a medium containing dextran sulfate (10μg/ml), the growth of contaminating fibroblasts was suppressed and the cancer cells proliferated. During subculture, the cancer cells detached from the primary culture on the type IV collagen-polymer substrate by treatment with ethyleneglycol bis (2-aminoethylether) tetraacetic acid and low-temperature treatment (at about 25°C) without trypsin, and the cells continued to proliferate. These observations suggest that culture with a type IV collagen-polymer substrate and a culture medium containing dextran sulfate is useful for establishment of human cancer cell lines.

Selective Method for Primary Culture of Hnman Cancer Cells with Dextran Sulfate.

ヒト癌細胞の初代培養系に混入した線維芽細胞の増殖を選択的に抑制する方法を開発するために,われわれはデキストラン硫酸を添加した培地を用いて,癌細胞と線維芽細胞に対する増殖抑制効果を調べた. (1) 分子量の異なる4種類のデキストラン硫酸(8k, 15k, 50k, 500k)を用いて,ヒト線維芽細胞株(MRC-5)に対する増殖抑制効果を調べた結果,分子量500kのデキストラン硫酸がもっとも強い増殖抑制効果を示した. (2) 分子量500kのデキストラン硫酸は, 7種類のヒト線維芽細胞株の増殖を濃度依存的に抑制した.しかし, 9種類のヒト癌細胞株に対する増殖抑制効果は非常に弱かった.さらに,ヒト線維芽細胞株の細胞質の形態はデキストラン硫酸によって濃度依存的に肥大化した. (3) 外科手術から得たヒト癌細胞を10μg/mlのデキストラン硫酸を含む培地で培養したとき,癌間質から混入したヒト線維芽細胞の増殖は抑制された.そして,ヒト癌細胞が優先的に増殖した. 以上の結果から,デキストラン硫酸を添加した培地は,ヒト癌細胞の選択的培養方法として有用であることが示唆される.