20 48 4 826 830 * S - 2 **. 030006 2. 03080 GSH-agarose Oxya chinensis Thunberg 5 S - glutathione S-transferases GSTs 60% ~ 80% GSTs 90% 0. 3046 μmol / min / mg protein. 82 GSH-agarose 8. 585 μmol / min / mg protein 50. 88 SDS- PAGE 25. 4 ku S - Affinity chromatography for the purification of glutathione S-transferase from Oxya chinensis GUO Yan-Qiong 2 WU Hai-Hua XUAN Tao ZHANG Jian-Zhen GUO Ya-Ping MA En-Bo **. Institute of Applied BiologyShanxi UniversityTaiyuan 030006China 2. College of AgricultureShanxi Agriculture UniversityTaigu 03080China Abstract Glutathione S-transferase GST in the fifth-instar nymphs of Oxya chinensis Thunberg was purified by ammonium sulfate and GSH-agrose affinity chromatography. Higher specific activity of GSTs could be detected at 60% - 80% purityand the highest specific activity was found at 90%. The specific activity was 0. 3046 μmol / min / mg protein and the purification factor was. 82-fold. Further GSH-agrose affinity chromatography indicated that purification reached 50. 88 with specific activity of 8. 585 μmol / min / mg protein. SDS-PAGE analysis indicated that the purified GSTs preparation had a single band with a relative molecular mass of 25. 4 ku. These results provide the basis for further study of the enzymatic propertiesstructural characteristics and functions of the GSTs of O. chinensis. Key words glutathione S-transferases GSTs Oxya chinensispurificationaffinity chromatography S- glutathione S-transferases GSTs 2003 2006 Qin et al. 20 Oxya chinensis Thunberg Insecta Orthoptera Acridoidea Oedipodidae Oxya Serville 993 2003 2006 S - * 308003907 30870302 20003656 2009045359 ** E-mail maenbo2003@ sxu. edu. cn 20-05-0 20-05-06
4 S - 827 20% 30 min 2007 4 0 min 4 2 000 g 30 2007 S - min GSTs 30% ~ 00% 4 0 min 4 2 000g 30 min S - 0. mol / L ph7. 5 0. 3% Triton X- 00 GSTs GSTs 4 GSH-agrose 5. 2. 3 GSTs GSH-agrose GSTs 60% ~ 90% GSTs 2 h NH + 4. GSH-agrose.. Oxya GSH-agrose chinensis 0 0. mol / L ph7. 5. 5 mol / L NaCl ml / 5 2 ~ 3 d min 30-80 0 mmol / L ph7. 4 0. % Triton X - 00.. 2 GSH-agrose Sigma 50mmol / L ph9. 0 Tris-HCl 8 mmol / L GSH GSH BBI GSTs - 2 4 - CDNB GSTs BSABio-Rad 4. 2. 4 GSTs Zhu He.. 3 DY89 - Ⅱ 2000 CDNB GSH 0 μl 90 μl Sigma DYZC-24DN 200 mmol / L GSH 0. 35 mmol / L CDNB SpectraMAX 90 GSH CDNB = 88 2 340 nm Molecular Device 0 s min OD. 2 3 0 μl. 2. GSTs - 80 CDNB 9. 6 m mol L - - 5 0. cm - mol / L ph7. 5 0. 3% Triton X-00 0 W / V 5 Bio-Rad Protein 500 rpm 5 min Assay 2008 4 5 000 g 20 min. 2. 6 GSTs GSTs SDS-. 2. 5 Smith 985 PAGE 5%. 2. 2 GSTs 4% R - 250
828 48 2. 2 GSTs 20 2 2. GSTs 5 GSTs 60% ~ 90% GSTs 2 GSTs 3 90% 0. 3046 μmol / 5 6 8 GSTs min / mg 0. 674 μmol / min / mg. 82 GSTs GSTs 60% ~ 90% GSH-agrose GSH-agrose Fig. Fig. 2 GSH-agrose affinity chromatography of the GSTs from Oxya chinensis GSTs Specific activity of GSTs in different NH 4 2 SO 4 saturation 2 GSH-agrose GSTs Table GSTs GSH-agrose Purification results of the GSTs from Oxya chinensis with ammonium sulfate and GSH-agrose affinity chromatography Purification step GSTs GSTs Total enzyme GSTs specific Total protein Purification activity activity Yield % mg fold μmol / min μmol / min / mg Crude extract 446. 695 74. 773 0. 674 00 60% ~ 90% Ammonium sulfate with saturation 60% 90% GSH-agrose 58. 683 7. 720 0. 3046. 82 23. 70 GSH-agrose affinity chromatography 0. 7497 6. 3866 8. 585 50. 88 8. 54
4 S - 829 GSTs GSTs 0. 674 μmol / min / mg 60% ~ 90% Qin GSTs 0. 3046 μmol / et al. 20 GSTs min / mg. 82 23. 70% GSH-agrose GSTs 8. 585 μmol / min / mg 50. 88 2009 8. 54% 2. 3 GSTs S - GSH-agrose 3 SDS - 2007 GSTs 3 Chien Dauterman 99 Helicoverpa zea GSTs 4. 3 Yawetz 3 GSTs SDS-PAGE Fig. 3 SDS-PAGE electrophoretogram of tube 3 with GSH-agrose affinity chromatograph M Protein markers 2 Crude GSTs 3 Dialysate 4 Purified GSTs. GSTs Koren 984 Ceratitis capitata GSTs 6. 3 GSTs GSH-agrose GSTs 3 GSH-agrose 3 GSTs SDS-PAGE 20. 0 ~ 26. 0 ku GSTs 200 Kunanz SDS-PAGE Nanen 2004 GSH-agrose Tetranychus urticae 3 Rauch Nanen 2004 Bemisia tabaci 8 20. 0 ~ 26. 0 ku 2009 25. 4 ku 3 GSH-agrose Locusta migratoria manilensis 3 S - S - S - 2 S -
830 48 2003. S -. GSH-agrose 2 GSTs 40 3 204-207 228. Qin GHJia MLiu TXuan TZhu KYGuo YPMa EB 6. 25 50. 88 Zhang JZ20. Identification and characterization of ten glutathione S-transferase genes from oriental migratory locustlocusta migratoria manilensis Meyen. Pest GSTs Manag. Sci. wileyonlinelibrary. com DOI 0. 002 / ps. GSTs 85% Rauch NNauen R2004. Characterization and molecular GSTs cloning of a glutathione S-transferase from the whitefly 90% Bemisia tabaci Hemiptera Aleyrodidae. Insect Biochem. GSTs 24 ku Mol. Biol. 34 32 329. GSTs 25. 4 ku Smith PKKrohn RIHermanson GTMallia AKGartner GSTs 24. 9 ± 3. ku FHProvenzano MDFujimoto EKGoeke NMOlson BJ Klenk DC985. Measurement of protein using 2009 bicinchoninic acid. Anal. Biochem. 50 76 85. 2007. S - GSH-agrose GSTs. 44 3 80 84. 20. S -. 7 35 37. 2007.... S - 2007.. 26 79 83. References 200.. 2006. 7 3 276 280. Chien CDauterman WC99. Studies on glutathione S-. 25 9 24. transferase in Helicoverpa = Heliothis zea. Insect Yawetz AKoren B984. Purification and properties of the Biochemistry2 8 857 864. Mediterranean fruit fly Ceratitis capitata W. glutathione S- 2009. S - transferase. Insect Biochemistry4 6 663 670.... 2006. Kunanz SNauen R2004. Purification and partial. 22 3 24 28. characterization of a glutathione S-transferase from the twospotted spider mitetetranychus urticae. Pestic. Biochem. Phys. 79 2 49 57. 2009. S -. 46 3 480 484. 993... 76 80. Zhu KYHe FQ2000. Elevated esterases exhibiting 2008. arylesterase-like characteristics in an organophosphateresistant. 24 4 662 clone of the greenbugschizaphis graminum. 666. 2003. Pestic. Biochem. Phys. 67 3 55 67. GST. 23 2 70 74.