Purification And Properities Of Glycolate Oxidase Isozyme With High Isoelectric Point And High Activity From Plant Green Leaves

Glycolate oxidase(EC 1. 1.3.15 GO) was considered a homogeneous enzyme consisted of 40kD subunit and FMN before, but this view could not explain why GO had about ten kinds of isoelectric point(pI) varied from 7.1 to 10. 0; GO from spinach leaves was first reported as an isozyme by Xu Jie in our laboratory, which contained 40kD acidic subunit and 66kD basic subunit. Variation of pI was related to subunits dissociation. GO isozyme was unstable and its molecular weight(Mr), specific activity and pI decreased during purification. All pI of purified GO were smaller than 9. 6 but pI of GO in crude protein greater than 9. 6 had never been purified. This might be related to the unstability of GO during purification. It was necessary to find a simple and fast purification procedure to obtain the GO isozyme with high pI, by wich a physiological GO isozyme can be acquired.In this paper, by shorting the length of DEAE-Cellulose column and increasing the pH of elution solution, one kind of GO isozyme was purified rapidly from green leaves of spanictu Brassica parachinensis Bailey and Vigna unguiculateW. ssp. sesquipedalis(L). Verd in nine hours. GO isozyme displayed high specific activity varied from 90 to 197U/mg. min. and high pI, a value greater than 10.0 assayed by acetate cellulose membrane electrophoresis. 40kD main band appeared when this isozyme preparation was subjected to SDS-PAGE, indicating Mr of GO isozyme subunit from different plants was similar.GO isozyme from Brassica parachinensis Bailey was purified for many times. Its pI was greater than 10. 0 assayed in acetate cellulose membrane electrophoresis, but about ten kinds of pI varied from 4.5 to 10.0 were observed when the same GO isozyme was assayed in IEF. 40kD main band did not alter when GO isozyme was subjected to SDS-PAGE, implying thephenomenon of pI change was not related to 40kD subunit degradability.One 66kD band appeared except 44kD main band when GO isozyme above was subjected to SDS-PAGE and CE-SDS, indicating this GO isozyme was similar to that from spinach leaves which contained 40kD and 66kD simultaneously. Whether B-mercaptoethanol was added or not when GO isozyme was subjected to in SDS-PAGE and CE-SDS, 40kD main band and 66kD band still appeared, indicating two subunits were not linked by covalent disulfide. Amino acid analysis shew that the ratios of basic to acidic amino acid of GO isozyme and its 40kD acidic subunit were 0. 66 and 0. 54, respectively, implying 40kD subunit was a acidic protein, 66kD band was a basic protein.Similar to GO isozyme from spinach leaves, it is most likely that this GO isozyme comprised two noncovalently associated subunits: 66kD basic subunit and 40kD acidic subunit by comparing the differences of electrophoresis pattern in SDS-PAGE and CE-SDS, and by analyzing the amino acid component of GO isozyme and its 40kD subunit. Based on the results in acetate cellulose membrane electrophoresis and IEF, implying the pi of 66kD basic subunit was greater than 10. 0 and that of 40kD acidic subunit was smaller than 4.5. Under electric field, noncovalently associated subunits of GO isozyme dissocated easily which resulted in about ten kinds of pI observed in IEF.