Abstract
A crude preparation of ascorbate oxidase was prepared from Japanese pumpkin by treating extract successively with barium acetate, ammonium sulfate, magnesium sulfate and alumina. On the other hand, a purified preparation was obtained by barium acetate treatment, ammonium sulfate fractionation, acetone precipitation, twice chromatography on DEAE cellulose column and gel filtration with sephadex G 100. Both preparations contained antitumoric potentiality against sarcoma-180, even though ascorbate oxidase activity had been decreased after long storage in frozen state or had been completely lost by carboxymethylation of ascorbate oxidase preparation. In addition, antitumoric potentiality was maintained more or less when ascorbate oxidase had been inactivated by heating or splitting off with trypsin. Thus, it was indicated that the antitumoric potentiality of ascorbate oxidase preparation was attributed to the protein itself, but has no intimate relationship with the enzymatic activity. On the other hand, a crude preparation of poIypheno1 oxidase, a similar copper enzyme to ascorbate oxidase, was prepared from shiitake mushroom by treatments with acetone, calcium acetate and ammonium sulfate. Its antitumoric potentiality was also confirmed for sarcoma-180 and MH-134.
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