Rikio Shinohara

The purification and properties of formamidase from rat liver

Abstract Formamidase (aryl-formylamine amidohydrolase, EC 3.5.1.9, formerly known as kynurenine formamidase) was purified by head treatment, (NH4)2SO4 fractionation, gel filtration by Sephadex G-200 and DEAE-cellulose column chromatography. The purified enzyme preparation appears as a single peak in ultracentrifuge and in acrylamide electrophoresis. The properties of the highly purified formamidase are as follows: 1. 1. Formamidase has a broad pH optimum in the range between pH 5.5 and 9.0. 2. 2. The K m values for formylkynurenine and formylanthranilic acid were 50 μM and 211 μM, respectively. 3. 3. The activity of formamidase was inhibited by Ag+, Hg−, Cu2+, NaAsO2, Na2S2O4, HaHSO3, and NaF but not affected by Ca2+, Mg2+, Zn2+, Fe2+, NH2OH, NaHAsO4, sodium formate and Na2S2O5. 4. 4. Anthranilic acid and o- aminobenzamide showed a mixed-type inhibition. Furthermore, o- aminophenol and o- aminotoluidine could activate formamidase in the presence of formylanthranilic acid as its substrate.

An enzyme immunosensor for IgG

An enzyme immunosensor has been developed for assaying human immunoglobulin G (IgG). The sensor is composed of an oxygen sensoring system and an antibody-binding membrane. The assay procedure involves the competitive immunochemical reaction of the membrane-bound antibody with nonlabeled and catalase-labeled IgG and the electrochemical determination of membrane-bound catalase activity. The analytical result is directly displayed by the output current of the sensor. The sensor exhibited an excellent performance in monitoring specifically human IgG.

An immunosensor for syphilis

An immuno responsive membrane was prepared by immobilizing Wasserman antigen to acetylcellulose. The antigen-binding membrane was used in developing an immunosensor for determining specifically corresponding antibody in serum. The senor is characterized by electrochemical determination of the antigen-antibody complex formed at the membranesoluti on interface. Experimental data on the applicability of the sensor to serology tests for syphilis are given as well as an outline of a possible mechanism for evaluating the electrical potential difference across the antigen-binding membrane.

New formamidase having substrate specificity for o-formylaminoacetophenone in pig liver

The supernatant (13 000 x g, 20 min) of pig liver homogenate was filtered with Sephadex G-200 and formamidase (aryl-formylamine amidohydrolase, EC 3.5.1.9)activity in each fraction was measured. When formylkynurenine was used as substrate, two peaks of formamidase activity were observed but, with formylaminoacetophenone as substrate, only one peak was observed. Formamidase in the lower molecular weight fraction is known as kynurenine formamidase (FA I), formamidase found here in the higher molecular weight fraction has not been previously reported. This form, designated FA II has been purified about 160-fold from pig liver. The formamidase obtained has substrate specificity for o-formylaminoacetophenone only and could not hydrolyze formylkynurenine. The optimal pH was 8.5 and the Km for o-formylaminoacetophenone was 1.66-10(-3) M. This formamidase was considered to be a new enzyme and was different from FA I in molecular weight and substrate specificity. This new formamidase was present in pig, rabbit and guinea pig liver and not present in rat or mouse liver.

Isolation and properties of protein component as activator for anthranilamide hydroxylation in rat liver

Abstract A protein component, which activates the hydroxylation of anthranilamide by rat liver microsomal fractions in the presence of NADPH and air, was isolated from rat liver by DEAE-Sephadex and Sephadex G-75 column chromatography after heat denaturation and (NH4)2SO4 fractionation of the soluble fraction. The component contained one atom of copper and one atom of zinc per molecule. The molecular weight was 25 700. One tyrosine, six phenylalanine and no tryptophan residues were found per molecule. The activation of anthranilamide hydroxylation by the protein component was removed, when metals were separated from the protein. The apoprotein regained the activity after incubation with 5 μM CuSO4 in 50 mM Tris-HC1 buffer at pH 8.2. 5-Hydroxyanthranilamide and 3-hydroxyanthranilamide were formed in the enzymatic reaction in about equal quantities.