Purification And Properties Of Glycolate Oxidase From Plant Green Leaves

Glycolate oxidase (EC 1. 1. 3. 15 GO) was purified by the improved method. Crude extract was adjusted to pH5. 3 with 10% acetic acid, the solution was centrifuged at 3000*g for 5 minutes, the supernatant obtained was taken to 0. 15 saturation of (NH4)2SO4. After 30 minutes, the suspension was centrifuged at 3000*g for 10 minutes. The supernatant was taken to 0.35 saturation of (NH4)2SO4. After 30 minutes, the precipitation was collected by centrifugation and dissolved in 5mM Tris-HCl(pH8. 3). Then the solution was applied to SephadexG-50 column equilibrate with 5mM Tris-HCl (pH8.3). GO was in the first fraction. The fraction FMN was added was applied to DEAE-Cellulose column. GO was obtained by collecting the yellow fraction. During the process, partly purified GO was also obtained. 40kD subunit of GO was obtained by SDS-PAGE. Mouse antiserum against 40kD subunit and GO was raised respectively.Different samples of purified GO were adjusted to SDS-PAGE separately and the activities of the samples were compared. The results shew that the molecular of weight of GO subunit could fluctuate in some certain range. But only subunit molecular weight of 40kD and most purified GO had the high activities. Subunits of 45kD, 43kD and 40kD could coexist, but activity of GO was much lower than that 40kD exist alone.Results of western blot analysis of GO from Brassica parachinensis Bailey at various purification steps, using the mouse antiserum raised against GO, shew the main immunity band of crude proteins was 49kD, but the bands of 75kD, 61kD were also clear. Many immunity bands of DEAE-Cellulose fraction were shown but different from the crude proteins. The main immunity band was 40kD. 40kD subunit was not the native subunit of GO was proved by these results.Western blot analysis of GO from Brassica parachinensis Bailey atvarious purification steps after separation by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), mouse antiserum raised against 40kD subunit was used, shew that result of purified GO was different from those of partly purified GO. In some case, there was no immunity band of crude proteins. But most of the time, results of crude proteins and SephadexG-50 fraction were almost the same. The main band was more than 40kD. Immunity bands of crude proteins accorded with the bands of SDS-PAGE. Immunity band of 31kD was obtained by western blot analysis of 35% (NH4)2SO4 precipitation. Two immunity bands were obtained by western blot analysis of purified GO. 40kD was the main band. The results also indicated that 40kD subunit probably was not the native subunit of GO. 40kD subunit was obtained by the degradation of native subunit whose molecular weight was much more than 40kD.Partly purified GO in various purification steps was adjusted to SDS-acetate cellulose membrane electrophoresis. Peptides combined with SDS were observed in cathode and anode. The proportion in cathode decreased along with the purification. Proteins combined with SDS were electrophoresed to cathode was an unusual phenomenon.