PURIFICATION AND IMMUNOHISTOCHEMISTRY OF FETAL ACID α-GLUCOSIDASE

概要

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Fetal rat liver acid α-glucosidase was purified more than 20,000 fold from the fetal rat liver 2 days before delivery. The disc gel electrophoresis of this preparation showed a single band of protein. The Michaelis constants of this enzyme were 3.3 mM with maltose and 3.1 mM with 4-methylumbelliferyl α-glucoside as substrates. Molecular weight measured by SDS electrophoresis and TOYOPEARL gel filtration was about 100,000. The pH optima was 4.0 with 4-methylumbelliferyl α-glucoside as substrate and 3.7 with maltose as substrate. Antibody was obtained by injection of this enzyme into rabbits with Freund's complete adjuvant. The anti-fetal acid α-glucosidase IgG showed the cross-reaction to the adult rat liver acid α-glucosidase. F(ab')_2 was separated by the filtration of Sephacryl S-200 column chromatography after digestion of anti acid α-glucosidase IgG pepsin and then F(ab')_2 was treate d with 2-mercaptoethanol. Coupling of Feb', obtained by use of Sephacryl S-200 column chromatography after treatment of F(ab')_2 with 2-mercaptoethanol, to horseradish peroxidase was performed according to the method of Wilson and Nakane (14). Light microscopical observation of the immunohistochemical localization of this enzyme in fetal rat and newborn rat liver showed small granular deposits of DAB reaction products in the cytoplasm.
日本組織細胞化学会の論文