The proteins with glycolate oxidase activity from B.parachinensis Bailey and rice( Oryza sativa )green leaves were prepared respectively.From the second protein peak on DEAE\|Cellulose column,two glycolate oxidases,expressed as B.parachinensis Bailey GO Ⅲ(specific activity natove 13 2 U·mg -1 ·min -1 )and rice GOⅢ(specific activity 8 8 U·mg -1 ·min -1 ),could not migrate anywhere in 4%～20% native\|PAGE under a pH8.3 buffer system.GOⅢ’s p I was about pH8.3. The protein containing B.parachinensis Bailey GOⅢ showed 67±2,43±2,and 38±2 kD in SDS PAGE,band 43±2 kD was the subunit of B.parachinensis Bailey GOⅢ.From the two proteins above,another group of glycolate oxidases,expressed as B.parachinensis Beiley GOⅠ(specific activity 5 U·mg -1 ·min -1 )and rice GOⅠ(specific activity 1 2 U·mg -1 ·min -1 ),showed only one 43±2 kD band in SDS\|PAGE,and was purified on the Sepharose\|6B column which migrated towards anode in the same native\|PAGE showing the M r about 420 kD,or 460 kD and 260 kD respectively.GOⅠ’s p I was smaller than pH8.3.Antibody against B.parachinensis Bailey GOⅠ was prepared and its efficacy was about 1/1600 in ELISA.By native\|PAGE,Western blot and rocket immunoelectrophoresis,the third group of glycolate oxidases,expressed as B.parachinensis Bailey GOⅡ and rice G0Ⅱ,were confirmed in crude protein of green leaves and migrated towards cathode under the same native\|PAGE,so GOⅡ’s p I was higher than pH8.3.The M r of B.parachinensis Bailey GOⅡ was about 669 kD determined by native\|PAGE Western blot.Rice GOⅠ,rice GOⅢ and rice GOⅡ showed different quantitation under different physiological conditions.Rice GOⅡ could be induced by glycolate.
Protein components of which p I <8.3 and molecular weights 35±2 kD and 25±2 kD may be degradation products of GO isozyme 40±2 kD subunit.But protein band of which p I >8 3 and molecular weight 67±2 kD from the proteins containing GOⅢ and GOⅡ was stable.There was a cross immunoreaction between specific GOⅠ antibody and only 40±2 kD protein band from crude protein of spinach green leaves assayed by SDS PAGE Western blot.Specific antigen from spinach crude protein immunoadsorbed by specific GOⅠ antibody showed four bands with the molecular weights 67±2 kD,40±2 kD,38±2 kD,and 25±2 kD assayed by SDS PAGE, Western blot using the antibody against the protein containing GOⅡ.So 67±2 kD protein could be immunoadsorbed by specific GOⅠ antibody.By native PAGE and SDS PAGE,Western blot using the antibody against the protein containing GOⅡ,rice GOⅡ and a rice 67 kD protein of which p I >8 3 was confirmed to be induced by glycolate substrate.On the results above,a new theory was put forwards that spinach GOⅡ consisted of 67±2 kD and 40±2 kD subunits.
Glycolate oxidase (GO) isozyme with high specific activity (75.0～279.0 U/mg) is purified quickly on DEAE- Cellulose column from Brassica parachinensis Bailey. Its pI is greater than 10.0 assayed by acetate cellulose membrane electrophoresis for 1 hour. In view of about ten kinds of pI varied from 4.5 to 10.0 are observed when the same GO isozyme is assayed in IEF for 14 hours, it is obvious that its pI decreases in IEF. Its pI also decreases when this GO isozyme is assayed in PAGE for 14 hours. Based on the results in SDS-PAGE, CGE-SDS, and IEF, it is most likely that this GO isozyme comprises two noncovalently associated 66 kD basic subunit and 40 kD acidic subunit, the phenomenon of pI change is related to subunit dissociation. The basic/acidic amino acid residues ratios in GO isozyme and its 40 kD acidic subunit are detected to be 0.66 and 0.54, respectively, a value much lower than that (0.96) in 40 kD peptide encoded by GO cDNA reported previously, indicating neither M r nor charge characteristic of this 40 kD peptide is similar to that of GO isozyme subunits, two subunits of GO isozyme may be the modified products of the same GO gene after post-translation.
By DEAE cellulose and Sepharose 6B chromatography, the proteins containing glycolate oxidase isozymes GOⅡ and GOⅢ were extracted from spinach green leaves. The protein containing GOⅡ showed two bands of 67±2 kD and 40±2 kD in SDS PAGE whose specific activity of glycolate oxidase was 33.4 U·mg -1 ·min -1 .It migrated towards cathode in Native PAGE in pH 8.3 buffer system. pI of GOⅡwas about 9.4 detected by IEF. The protein containing GOⅢ showed three bands of 67±2 kD, 40±2 kD and 38±2 kD in SDS PAGE whose specific activity of glycolate oxidase 14.4 U·mg -1 ·min -1 and could not migrate anywhere in the same Native PAGE. pI of GOⅢ was about 8.3 detected by IEF. The 40±2 kD might be the subunits of GOⅡ and GOⅢ. Antibodies of the protein containing GOⅡ and GOⅢ were prepared respectively. GOⅡ was very unstable and could change into GOⅢ artifact; GOⅢ was also unstable and could change into GOⅠartifact whose Mr ≈470 kD and pI ≈7.4 . This GOⅠ(specific activity: 9.8 U·mg -1 ·min -1 ), showing one 40±2 kD band in SDS PAGE, could be purified on another Sepharose 6B chromatography. The specific activity of GOⅡ decreased rapidly to about half of its original value and then was relatively stable when stored in 50% glycerol at -20℃. The results above explained why GOⅡ was extracted difficultly, and GOⅢ were easily confused with GOⅠ and GOⅡ.
