PURIFICATION AND PROPERTIES OF GLYCOLATE OXIDASE ISOZYME GO I FROM SPINACH LEAVES

Abstract:

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 (M_r) of 40 000± 2 000 in SDS-PAGE. The specific activity of purified GO I was 8.4 U min^-1mg^-1 protein. GO I showed its M_r 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-difusion reaction between the antibody raised against GO I and crude protein from spinach green leaves．The specitic G0 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 polvmers of 470 000，280 000，and 80000 were found when crude protein were conduced to SDS-PAGE Ⅵ stern blot or ND-PAGE Western blot using this speciftc GO I antibody.