菠菜叶片中乙醇酸氧化酶3种同工酶的生化特性

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.

Biochemical Properties of Glycolate Oxidase Isozymes of Spinacia oleracea

By DEAE-cellulose and Sepharose-6B chromatography, the proteins containing glycolate oxidase isozymes GO Ⅱ and GO Ⅲ were extracted from spinach green leaves. The protein containing GO Ⅱ 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 GOⅡ was 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 GO Ⅲ was about 8.3 detected by IEF. The 40 + 2 kD might be the subunits of GO Ⅱ and GO Ⅲ . Antibodies of the protein containing GO Ⅱ and GO Ⅲ were prepared respectively. GO Ⅱ was very unstable and could change into GO Ⅲ artifact; GO Ⅲ was also unstable and could change into GO I artifact whose Mr ≈470 kD and pI ≈ 7.4 . This GO I (specific activity: 9.8 U·rng-1· min-1), showing one 40 + 2 kD band in SDS-PAGE, could be purified on another Sepharose-6B chromatography. The specific activity of GO Ⅱ 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 GO Ⅱ was extracted difficultly, and GO Ⅲ were easily confused with GO I and GO Ⅱ .