Receptor destroying enzyme (RDE) を作用させる条件について

Abstract

In a previous paper, some purification studies on the receptor destroying enzyme (RDE) produced in a particular medium containing hog stomac mucin, which allowed the highest production of RDE by V. cholerae, have been reported.For the application of this purified preparation on serological studies of influenza infections and also for detecting the viral hemagglutinins in the organ of animals infected with influenza virus, some systematic researches on the optimum conditions for the action of RDE, seemed advisable to investigate. The results of basic studies obtained along this line are the subject of this communication.1) Using chicken red cells and ovomucin as the substrates, quantitative studies of RDE activity have been performed. The higher the concentration of the substrate, the higher concentration of enzyme was necessary. Linear relationship was obtained between the log concentration of the substrate and of enzyme in order to destroy the activity of the substrate completely after fixed period of incubation.2) Optimum temperature and pH for the action of RDE was 37°C and between 6.5 and 7.0, respectively.3) Ca ion concentration was an important factor for the RDE action and the optimum concentrations laied between M/100 and M/200. Deionization with EDTA, sod. citrate and sod. oxalate worked out to inhibit the RDE action and the effectiveness of these substances was in this order.4) The provisional working standard of determining the titer of both α-inhibitor and RDE has been proposed. Following to this standard, x/4 unit of RDE was necessary to destroy the x unit of α-inhibitor under standard condition of incubation, i.e. at 37°C for 1hr.5) Methods to remove the residual activity of RDE after the completion of the reaction have been examined. Heating was favourable for practical use particularly at the time of serodiagnosis. However, there was the difference of heat stability between purified RDE and just cholerae filtrate. The assay with 2.5% sod. citrate saline, which have been customly used as the routine procedure to remove the residual RDE activity was not so satisfactory when high concentration of RDE was used. 1% aqueous solution of EDTA was preferrable with regard to this respect.The conditions determined above will give us an idea that how much unit of RDE should be used in order to remove the known amount of α-inhibitor contained in the specimens to be tested, and also how to remove the residual activity of used RDE. However, the existence of the other kind of α-inhibitors among the biological specimens which is not impaired by the above procedure was not ruled out.