PURIFICATION AND PROPERTIES OF GLYCOLATE OXIDASE ISOZYME GO I FROM SPINACH LEAVES
菠菜乙醇酸氧化酶同工酶GO Ⅰ的纯化和特性

By using ammonium sulfate fractionation, Sephadex G-25, DEAE-Cellulose, and Sepharose-6B chromatographies, glycolate oxidase isozyme GO Ⅰ was purified from spinach green leaves which showed only one band with molecular weight (Mr) of 40 000 + 2 000 in SDS-PAGE. The specific activity of purified GO Ⅰ was 8.4 U min-1mg-1 protein. GO Ⅰshowed its Mr of 470 000 in 4%-20% ND-PAGE and could catalyze both oxidations of glycolate and glyoxylate simultaneously. The isoelectric point (PI) of GO Ⅰ was about pH 7.4. There was a immunity precipitate line determined by immunity double-diffusion reaction between the antibody raised against GO Ⅰ and crude protein from spinach green leaves. The specific GO Ⅰ antibody was purified by protein A-Sepharose CL-4B column and specific antigen affinity elution. Only the 40 000 + 2 000 band could immunity cross-react with GO Ⅰ antibody, and different GO Ⅰ polymers of 470 000, 280 000, and 80 000 were found when crude protein were conduced to SDS-PAGE Western blot or ND-PAGE Western blot using this specific GO Ⅰ antibody.