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Chelon auratus (Risso, 1810)’un Karaciğerinden Glukoz-6-Fosfat Dehidrojenaz’ın Saflaştırılması ve Kinetik Karakterizasyonu

Year 2021, Volume: 11 Issue: 2, 138 - 144, 15.12.2021

Salih Görgün Emre Koç

Abstract

Bu çalışmada, önemli bir kefal balığı türü olan Chelon auratus (Risso, 1810)’un karaciğerinden glukoz-6-fosfat dehidrojenaz (EC
1.1.1.49), 2′5′ ADP-Sepharose 4B afinite kromatografisi ile 252 kat saflık, 27.04 EU/mg.protein spesifik aktivite ve % 27.42 verim
ile saflaştırılmıştır. Elde edilen enzim preparatı için optimum sıcaklık, pH ve iyonik güç sırasıyla 40 ºC, pH 8, ve 0.1 M olarak
bulunmuştur. Enzimin kinetik davranışı gerek glukoz-6-fosfat (G6P) gerekse NADP+ için değerlendirilmiştir. Elde edilen verilere
göre, Km ve Vmax değerleri NADP+ için sırasıyla 0.14 mM ve 3.40 EU/mL olarak belirlenirken, G6P için sırasıyla 0.23 mM ve 2.62
EU/mL olduğu bulunmuştur. Bu veriler, substrat olarak G6P ile karşılaştırıldığında, enzimin NADP+ için daha yüksek bir afinite
sergilediğini önermektedir. Saflaştırılan enzim preparatında SDS-PAGE analizi sonucunda, molekül kütlelerinin yaklaşık olarak 108
kDa ve 60 kDa olan iki protein bandının olduğu görülmüştür. Elde edilen sonuçlar, kefal türlerinden glukoz-6-fosfat dehidrojenazın
saflaştırılmasında ek kromatografik adımların gerekli olduğunu önermektedir.

Keywords

Chelon auratus (Risso, 1810) glukoz-6-fosfat dehidrojenaz saflaştırma kinetik karakterizasyon

Supporting Institution

Sivas Cumhuriyet Üniversitesi

Project Number

F-625

Thanks

Bu çalışma Sivas Cumhuriyet Üniversitesi Bilimsel Araştırma Projeleri (CÜBAP) Birimi tarafından F-625 No’lu proje olarak desteklenmiştir. Bu nedenle, teşekkürlerimizi sunuyoruz.

References

  • Adem, Ş., Çiftçi, M. 2012. Purification of rat kidney glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase enzymes using 2′5′-ADP sepharose4B affinity in a single chromatography step. Protein Expr. Purif. 81(1): 1-4. Doi: 10.1016/j.pep.2011.08.031
  • Adem, Ş., Çiftçi, M. 2016 a. Purification and characterization of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase from rat heart and inhibition effects of furosemide, digoxin, and dopamine on the enzymes activities. J. Biochem. Mol. Toxicol. 30(6): 295-301. Doi: 10.1002/jbt.21793
  • Adem, Ş., Çiftçi, M. 2016 b. Purification and biochemical characterization of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione reductase from rat lung and inhibition effects of some antibiotics. J. Enzyme Inhib. Med. Chem. 31(6): 1342-1348. Doi: 10.3109/14756366.2015.1132711
  • Beutler, E. 1984. Red cell metabolism: a manual of biochemical methods. Academic Press, London, 1971, pp 68-71
  • Bollag, DM., Rozycki, MD., Edelstein, SJ. 1996. Protein methods, A John Wıley and Sons, Inc., Publication, 57-82.
  • Ceyhan, D., Danişan, A., Öğüş, H., Özer, N. 2005. Purification and kinetic properties of 6-Phosphogluconate dehydrogenase from rat small intestine. The Protein Journal. 24(5): 293-301. Doi: 10.1007/s10930-005-6750-z
  • Çiftçi, M., Beydemir, Ş., Yılmaz, H., Altıkat, S. 2003. Purification of glucose 6-phosphate dehydrogenase from buffalo (Bubalus bubalis) erythrocytes and investigation of some kinetic properties. Protein Expr. Purif. 29(2): 304-310. Doi: 10.1016/s1046-5928(03)00073-1
  • Çiftçi, M., Çiltaş, A., Erdoğan, O. 2004. Purification and characterization of glucose 6-phosphate dehydrogenasefrom rainbow trout (Oncorhynchus mykiss) erythrocytes. Vet. Med.-Czech. 49(9): 327-333. Doi: 10.17221/5712-VETMED.
  • Çomaklı, V., Akkemik, E., Çiftçi, M., Küfrevioğlu, Öİ. 2015. Purification and characterization of glucose 6-phosphate dehydrogenase enzyme from rainbow trout (Oncorhynchus mykiss) liver and investigation of the effects of some metal ions on enzyme activity. Toxicol. Ind. Health. 31(5): 403-411. Doi: 10.1177/0748233713475514
  • Frederiks, WM., Vizan, P., Bosch, KS., Vereeling-Sindelarova, H., Boren, J., Cascante, M. 2008. Elevated activity of the oxidative and non-oxidative pentose phosphate pathway in (pre)neoplastic lesions in rat liver. Int. J. Exp. Pathol. 89(4): 232-240. Doi: 10.1111/j.1365-2613.2008.00582.x
  • Hasmann, FA., Gurpilhares, DB., Roberto, IC., Coverti, A., Pessoa Jr., A. 2007. New combined kinetic and thermodynamic approach to model glucose 6-phosphate dehydrogenase activity and stability. Ezyme Microb. Tech. 40(4): 849-858. Doi: 10.1016/j.enzmictec.2006.06.017
  • Hochachka, PW., Clayton-Hochachka, B. 1973. Glucose-6-phosphate dehydrogenase and thermal acclimation in the mullet fish. Mar. Biol. 18: 251–259. Doi: 10.1007/BF00347787
  • Hu, W., Zhi, L., Zhuo, M-Q., Zhu, Q-L., Zheng, J-L., Chen, Q-L., Gong, Y., Liu, C-X. 2013. Purification and characterization of glucose 6-phosphate dehydrogenase (G6PD) from grass carp (Ctenopharyngodon idella) and inhibition effetcs of several metal ions on G6PD activity in vitro. Fish Physiol. Biochem. 39: 637-647. Doi: 10.1007/s10695-012-9726-x.
  • İbrahim, MA., Ghazy, A-HM., Salem, AMH, Ghazy, MA., Abdel-Monsef, MM. 2014. Purification and characterization of glucose 6-phosphate dehydrogenase from camel liver. Enzyme Res. 2014: 1-10. Doi: 10.1155/2014/714054
  • Kuzu, M., Aslan, A., Ahmed, I., Çomaklı, V., Demirdağ, R., Uzun, N. 2016. Purification of glucose-6-phosphate dehydrogenase and glutathione reductase enzymes from the gill tissue of Lake Van fish and analyzing the effects of some chalcone derivatives on enzyme activities. Fish Physiol. Biochem. 42: 483-491. Doi: 10.1007/s10695-015-0153-7
  • Laemmli, DK. 1970. Cleavage of structural proteins during in assembly of the heat of bacteriophage T4. Nature 227: 680–685. Doi: 10.1038/227680a0
  • Meloni, L., Manca, MR., Loddo, I., Cioglia, G., Cocco, P., Schwartz, A., Muntoni, S., Muntoni, Sa. 2008. Glucose 6-phosphate dehydrogenase deficiency protects against coronary heart disease. J. Inherit. Metab. Dis. 31: 412-417. Doi: 10.1007/s10545-008-0704-5
  • Ninfali, P., Orsenigo, I., Baronciani, L., Rapa, S. 1990. Rapid purification of glucose-6-phosphate dehydrogenase from mammal’s erytrocytes. Prep. Biochem. 20(3-4): 297-309. Doi: 10.1080/00327489008050202
  • Ribeiro, MZ., Silva, DP., Vitolo, M., Roberto, IC., Pessoa-Jr., A. 2007. Partial purification of glucose 6-phosphate dehydrogenase by aqueous two-phase poly(ethyleneglycol)/phosphate systems. Braz. J. Microbiol. 38(1): 78-83. Doi: 10.1590/S1517-83822007000100016
  • Ulusu, NN., Kus, MS., Acan, NL., Tezcan, EF. 1999. A rapid method for the purification of glucose 6-phosphate dehydrogenase from bovine lens. Int. J. Biochem. Cell Biol. 31(7): 787-796. Doi: 10.1016/s1357-2725(99)00019-9
  • Yoshida, A. 1967. Glucose 6-phosphate dehydrogenase of human erytrocytes: I. Purification and characterization of normal (B+) enzyme. J. Biol. Chem. 241(21): 4966-4976. Doi: 10.1016/S0021-9258(18)99658-4

Purification and Characterization of Glucose-6-Phosphate Dehydrogenase from the Liver of Chelon Auratus (Risso, 1810)

Year 2021, Volume: 11 Issue: 2, 138 - 144, 15.12.2021

Salih Görgün Emre Koç

Abstract

In this study, glucose-6-phospahte dehydrogenase (EC. 1.1.1.49) from the liver of Chelon auratus (Risso, 1810), an economically
important mullet species, were purified with purification parameters of 252-fold purity, a specific activity of 27.04 EU/mg.protein
and a yield of 27.42%. The optimum temperature, pH and ionic strength were found to be 40 ºC, pH 8 and 0.1 M, respectively, for
the enzyme preparation obtained. The kinetic behavior of the enzyme was evaluated for both G6P and NADP+. According to the
data obtained, Km and Vmax values were determined as 0.14 mM and 3.40 EU/mL for NADP+, respectively, while it was found to be
0.23 mM and 2.62 EU/mL for G6P, respectively. These data have been suggested that the enzyme has higher affinity for the NADP+,
when compared with G6P as a substrate. According to the SDS-PAGE analyses of the purified enzyme, it was observed that there
were two protein bands having molecular weight of 108 kDa and 60 kDa. The results obtained have been suggested that additional
chromatographic steps might be required for the purification of glucose-6-phospahte dehydrogenase from the mullet species.

Keywords

Chelon auratus Glucose-6-phosphate dehydrogenase Kinetic characterization Purification

Project Number

F-625

References

  • Adem, Ş., Çiftçi, M. 2012. Purification of rat kidney glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase enzymes using 2′5′-ADP sepharose4B affinity in a single chromatography step. Protein Expr. Purif. 81(1): 1-4. Doi: 10.1016/j.pep.2011.08.031
  • Adem, Ş., Çiftçi, M. 2016 a. Purification and characterization of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase from rat heart and inhibition effects of furosemide, digoxin, and dopamine on the enzymes activities. J. Biochem. Mol. Toxicol. 30(6): 295-301. Doi: 10.1002/jbt.21793
  • Adem, Ş., Çiftçi, M. 2016 b. Purification and biochemical characterization of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione reductase from rat lung and inhibition effects of some antibiotics. J. Enzyme Inhib. Med. Chem. 31(6): 1342-1348. Doi: 10.3109/14756366.2015.1132711
  • Beutler, E. 1984. Red cell metabolism: a manual of biochemical methods. Academic Press, London, 1971, pp 68-71
  • Bollag, DM., Rozycki, MD., Edelstein, SJ. 1996. Protein methods, A John Wıley and Sons, Inc., Publication, 57-82.
  • Ceyhan, D., Danişan, A., Öğüş, H., Özer, N. 2005. Purification and kinetic properties of 6-Phosphogluconate dehydrogenase from rat small intestine. The Protein Journal. 24(5): 293-301. Doi: 10.1007/s10930-005-6750-z
  • Çiftçi, M., Beydemir, Ş., Yılmaz, H., Altıkat, S. 2003. Purification of glucose 6-phosphate dehydrogenase from buffalo (Bubalus bubalis) erythrocytes and investigation of some kinetic properties. Protein Expr. Purif. 29(2): 304-310. Doi: 10.1016/s1046-5928(03)00073-1
  • Çiftçi, M., Çiltaş, A., Erdoğan, O. 2004. Purification and characterization of glucose 6-phosphate dehydrogenasefrom rainbow trout (Oncorhynchus mykiss) erythrocytes. Vet. Med.-Czech. 49(9): 327-333. Doi: 10.17221/5712-VETMED.
  • Çomaklı, V., Akkemik, E., Çiftçi, M., Küfrevioğlu, Öİ. 2015. Purification and characterization of glucose 6-phosphate dehydrogenase enzyme from rainbow trout (Oncorhynchus mykiss) liver and investigation of the effects of some metal ions on enzyme activity. Toxicol. Ind. Health. 31(5): 403-411. Doi: 10.1177/0748233713475514
  • Frederiks, WM., Vizan, P., Bosch, KS., Vereeling-Sindelarova, H., Boren, J., Cascante, M. 2008. Elevated activity of the oxidative and non-oxidative pentose phosphate pathway in (pre)neoplastic lesions in rat liver. Int. J. Exp. Pathol. 89(4): 232-240. Doi: 10.1111/j.1365-2613.2008.00582.x
  • Hasmann, FA., Gurpilhares, DB., Roberto, IC., Coverti, A., Pessoa Jr., A. 2007. New combined kinetic and thermodynamic approach to model glucose 6-phosphate dehydrogenase activity and stability. Ezyme Microb. Tech. 40(4): 849-858. Doi: 10.1016/j.enzmictec.2006.06.017
  • Hochachka, PW., Clayton-Hochachka, B. 1973. Glucose-6-phosphate dehydrogenase and thermal acclimation in the mullet fish. Mar. Biol. 18: 251–259. Doi: 10.1007/BF00347787
  • Hu, W., Zhi, L., Zhuo, M-Q., Zhu, Q-L., Zheng, J-L., Chen, Q-L., Gong, Y., Liu, C-X. 2013. Purification and characterization of glucose 6-phosphate dehydrogenase (G6PD) from grass carp (Ctenopharyngodon idella) and inhibition effetcs of several metal ions on G6PD activity in vitro. Fish Physiol. Biochem. 39: 637-647. Doi: 10.1007/s10695-012-9726-x.
  • İbrahim, MA., Ghazy, A-HM., Salem, AMH, Ghazy, MA., Abdel-Monsef, MM. 2014. Purification and characterization of glucose 6-phosphate dehydrogenase from camel liver. Enzyme Res. 2014: 1-10. Doi: 10.1155/2014/714054
  • Kuzu, M., Aslan, A., Ahmed, I., Çomaklı, V., Demirdağ, R., Uzun, N. 2016. Purification of glucose-6-phosphate dehydrogenase and glutathione reductase enzymes from the gill tissue of Lake Van fish and analyzing the effects of some chalcone derivatives on enzyme activities. Fish Physiol. Biochem. 42: 483-491. Doi: 10.1007/s10695-015-0153-7
  • Laemmli, DK. 1970. Cleavage of structural proteins during in assembly of the heat of bacteriophage T4. Nature 227: 680–685. Doi: 10.1038/227680a0
  • Meloni, L., Manca, MR., Loddo, I., Cioglia, G., Cocco, P., Schwartz, A., Muntoni, S., Muntoni, Sa. 2008. Glucose 6-phosphate dehydrogenase deficiency protects against coronary heart disease. J. Inherit. Metab. Dis. 31: 412-417. Doi: 10.1007/s10545-008-0704-5
  • Ninfali, P., Orsenigo, I., Baronciani, L., Rapa, S. 1990. Rapid purification of glucose-6-phosphate dehydrogenase from mammal’s erytrocytes. Prep. Biochem. 20(3-4): 297-309. Doi: 10.1080/00327489008050202
  • Ribeiro, MZ., Silva, DP., Vitolo, M., Roberto, IC., Pessoa-Jr., A. 2007. Partial purification of glucose 6-phosphate dehydrogenase by aqueous two-phase poly(ethyleneglycol)/phosphate systems. Braz. J. Microbiol. 38(1): 78-83. Doi: 10.1590/S1517-83822007000100016
  • Ulusu, NN., Kus, MS., Acan, NL., Tezcan, EF. 1999. A rapid method for the purification of glucose 6-phosphate dehydrogenase from bovine lens. Int. J. Biochem. Cell Biol. 31(7): 787-796. Doi: 10.1016/s1357-2725(99)00019-9
  • Yoshida, A. 1967. Glucose 6-phosphate dehydrogenase of human erytrocytes: I. Purification and characterization of normal (B+) enzyme. J. Biol. Chem. 241(21): 4966-4976. Doi: 10.1016/S0021-9258(18)99658-4
There are 21 citations in total.

Details

Primary Language Turkish
Subjects Health Care Administration
Journal Section Research Article
Authors

Salih Görgün 0000-0002-2121-2577

Emre Koç 0000-0002-5942-5287

Project Number F-625
Publication Date December 15, 2021
Published in Issue Year 2021 Volume: 11 Issue: 2

Cite

APA Görgün, S., & Koç, E. (2021). Chelon auratus (Risso, 1810)’un Karaciğerinden Glukoz-6-Fosfat Dehidrojenaz’ın Saflaştırılması ve Kinetik Karakterizasyonu. Karaelmas Fen Ve Mühendislik Dergisi, 11(2), 138-144. https://doi.org/10.7212/karaelmasfen.858431
AMA Görgün S, Koç E. Chelon auratus (Risso, 1810)’un Karaciğerinden Glukoz-6-Fosfat Dehidrojenaz’ın Saflaştırılması ve Kinetik Karakterizasyonu. Karaelmas Fen ve Mühendislik Dergisi. December 2021;11(2):138-144. doi:10.7212/karaelmasfen.858431
Chicago Görgün, Salih, and Emre Koç. “Chelon Auratus (Risso, 1810)’un Karaciğerinden Glukoz-6-Fosfat Dehidrojenaz’ın Saflaştırılması Ve Kinetik Karakterizasyonu”. Karaelmas Fen Ve Mühendislik Dergisi 11, no. 2 (December 2021): 138-44. https://doi.org/10.7212/karaelmasfen.858431.
EndNote Görgün S, Koç E (December 1, 2021) Chelon auratus (Risso, 1810)’un Karaciğerinden Glukoz-6-Fosfat Dehidrojenaz’ın Saflaştırılması ve Kinetik Karakterizasyonu. Karaelmas Fen ve Mühendislik Dergisi 11 2 138–144.
IEEE S. Görgün and E. Koç, “Chelon auratus (Risso, 1810)’un Karaciğerinden Glukoz-6-Fosfat Dehidrojenaz’ın Saflaştırılması ve Kinetik Karakterizasyonu”, Karaelmas Fen ve Mühendislik Dergisi, vol. 11, no. 2, pp. 138–144, 2021, doi: 10.7212/karaelmasfen.858431.
ISNAD Görgün, Salih - Koç, Emre. “Chelon Auratus (Risso, 1810)’un Karaciğerinden Glukoz-6-Fosfat Dehidrojenaz’ın Saflaştırılması Ve Kinetik Karakterizasyonu”. Karaelmas Fen ve Mühendislik Dergisi 11/2 (December 2021), 138-144. https://doi.org/10.7212/karaelmasfen.858431.
JAMA Görgün S, Koç E. Chelon auratus (Risso, 1810)’un Karaciğerinden Glukoz-6-Fosfat Dehidrojenaz’ın Saflaştırılması ve Kinetik Karakterizasyonu. Karaelmas Fen ve Mühendislik Dergisi. 2021;11:138–144.
MLA Görgün, Salih and Emre Koç. “Chelon Auratus (Risso, 1810)’un Karaciğerinden Glukoz-6-Fosfat Dehidrojenaz’ın Saflaştırılması Ve Kinetik Karakterizasyonu”. Karaelmas Fen Ve Mühendislik Dergisi, vol. 11, no. 2, 2021, pp. 138-44, doi:10.7212/karaelmasfen.858431.
Vancouver Görgün S, Koç E. Chelon auratus (Risso, 1810)’un Karaciğerinden Glukoz-6-Fosfat Dehidrojenaz’ın Saflaştırılması ve Kinetik Karakterizasyonu. Karaelmas Fen ve Mühendislik Dergisi. 2021;11(2):138-44.
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