Purification and Characterization of 6-Phosphogluconate Dehydrogenase from Japanese Quail (Coturnix, coturnix japonica) Erythrocytes

Year 2020, Volume: 10 Issue: 2, 1180 - 1190, 01.06.2020

Hardi Rafat Baqı Yusuf Temel Mehmet Çiftci

https://doi.org/10.21597/jist.625729

Abstract

In this study, the 6PGD enzyme from Japanese quail erythrocytes was purified with specific activity of 52.84 EU/mg and 69% yield of purification by 2ˈ, 5ˈ- ADP Sepharose 4B affinity gel in a single chromatographic method. The purification folds of the enzyme were 4984 folds. The purified enzyme was checked using SDS polyacrylamide gel electrophoresis (SDS-PAGE) method; the result of gel showed a single band. The subunit molecular weight of the enzyme was calculated as 81 kDa by the SDS-PAGE method. The characterization studies of the 6PGD enzyme from erythrocytes of Japanese quail showed: the optimum ionic strength to be at 0.5 M Tris-HCl, optimum and stable pH values to be at 0.5 M Tris-HCl buffers pH 8.0. The optimal temperature for the enzyme activity was found at 60 ˚C. Finally, the KM and Vmax values for the 6PGD enzyme from Japanese quail’s erythrocytes were calculated respectively for the 6PGA the KM value found as 0.120 mM, Vmax value as 0.191 EU/mL and for NADP+ the KM value as 0.017 mM and Vmax value as 0.228 EU/mL.

Keywords

Quail, 6PGD, erythrocytes, purification, characterization

Supporting Institution

Bingol University Research Project Department

Project Number

BÜBAP, Project number FEF.3.16.003.

Thanks

Authors are indebted to Bingol University Research Project Department (BÜBAP, Project number FEF.3.16.003.) for their finical support .

6-Fosfoglukonat Dehidrogenaz Enziminin Japon Bıldırcın (Coturnix Coturnix Japonica) Eritrositlerinden Saflaştırılması ve Karakterizasyonu

Abstract

Bu çalışmada Japon bıldırcın eritrosit dokularından 6-fosfoglukonat dehidrogenaz enzimi (6PGD) 52,84 EU.mg-1 spesifik aktiviteyle ve % 69 verimle 2ˈ, 5ˈ-ADP Sepharose 4B afinite kromatografisi kullanılarak saflaştırıldı. Japon bıldırcın eritrositlerinden saflaştırılan 6PGD enzimi için saflaştırma katsayısı 4984 olarak bulundu. Enzim saflığının kontrolü için SDS-poliakrilamid jel elektroforezi (SDS-PAGE) yapıldı ve tek bant gözlendi. SDS-PAGE yöntemi kullanılarak enzimin alt birimlerinin molekül kutlesi 81 kDa olarak hesaplandı. Optimum iyonik şiddet 0,5 M Tris-HCl, optimum pH ve stabil pH 0,5 M Tris-HCl tamponu pH 8,0 olarak bulundu. Optimum sıcaklık 60 ºC olarak bulundu. Ayrica Japon bıldırcın 6PGD enziminin KM ve Vmax değerleri Lineweaver-Burk grafiklerinden sırasıyla 6PGA substratı için KM değeri 0,120 mM, Vmax değeri 0,191 EU/mL, NADP+ substratı için KM değeri 0,017 mM, Vmax değerise 0,228 EU/mL olarak hesaplandı.

Keywords

Bıldırcın, 6PGD, eritrosit, saflaştırma, karakterizasyon

Project Number

BÜBAP, Project number FEF.3.16.003.

References

• Adams MJ, Archibald IG, Bugg CE, Carne A, Gover S, Helliwell JR, Pickersgill RW, White SW, 1983. The three dimensional structure of sheep liver 6-phosphogluconate dehydrogenase at 2.6 A resolution. The EMBO Journal 2(6): 1009–1014.
• Adem S, 2010. Siçan kalp ve akciger dokularindan glukoz–6 fosfat dehidrogenaz, 6 fosfoglukanat dehidrogenaz, glutatyon redüktaz enzimlerinin saflastirilmasi, karakterizasyonu, kotinin ve bazi ilaçlarin enzimlerin aktiviteleri üzerine etkilerinin incelenmesi. PhD thesis, Atatürk University, Institute of science.
• Akkoyun MB, Bengü AŞ, Temel Y, Akkoyun HT, Ekin S, Ciftci M, 2018. The effect of astaxanthin and cadmium on rat erythrocyte G6PD, 6PGD, GR, and TrxR enzymes activities in vivo and on rat erythrocyte 6PGD enzyme activity in vitro. Journal of biochemical and molecular toxicology, 32(8), e22170.
• Akyüz M, Erat M, Çiftçi M, Gümüştekin K, Bakan N, 2004. Effects of some antibiotics on human erythrocyte 6-phosphogluconate dehydrogenase: an in vitro and in vivo study. Journal of enzyme inhibition and medicinal chemistry, 19(4): 361-365.
• Altikat S, Çiftci M, Buyukokuroglu ME, 2002. "In vitro effects of some anesthetic drugs on enzymatic activity of human red blood cell glucose 6-phosphate dehydrogenase." Polish journal of pharmacology 54(1): 67-72.
• Bayindir S, Ayna A, Temel Y, Ciftci M, 2018. The synthesis of new oxindoles as analogs of natural product 3,3-bis(indolyl)oxindole and in vitro evaluation of the enzyme activity of G6PD and 6PGD. Turkish Journal of Chemistry. 42(2): 332-345.
• Betts SA, Mayer RJ, 1975. Purification and properties of 6-phosphogluconate dehydrogenase from rabbit mammary gland. The Biochemical Journal. 151(2): 263–270. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1172355&tool=pmcentrez&rendertype=abstract.
• Beutler E, 1971. Red cell metabolism manual of biochemical methods. London Academic Press: 19-68.
• Beydemir S, Ciftci M, Yilmaz H, Kufrevioglu OI, 2004. 6-phosphogluconate dehydrogenase: purification, characterization and kinetic properties from rat erythrocytes. Turkish Journal of Veterinary and Animal Science 28: 707–714. http://mistug.tubitak.gov.tr/bdyim/abs.php?dergi=vet&rak=0212-4.
• Bianchi D, Bertrand O, Haupt K, Coello N, 2001. Effect of gluconic acid as a secondary carbon source on non-growing l-lysine producers cells of corynebacterium glutamicum. purification and properties of 6-phosphogluconate dehydrogenase. Enzyme and Microbial Technology 28(9–10): 754–759. doi:10.1016/S0141-0229(01)00310-6.
• Bradford MM, 1976. A Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72(1–2): 248–254. doi:10.1016/0003-2697(76)90527-3.
• Carnes A, Walker JE, 1983. Amino acid sequence of ovine 6-phosphogluconate dehydrogenase. Journal of Biological Chemistry 258(21): 12895–12906.
• Ceyhan D, Danişan A, Oğüş IH, Ozer 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.
• Demir H, Çiftçi M, Küfrevioğlu Öİ, 2003. Purification of 6-phosphogluconate dehydrogenase from parsley (petroselinum hortense) leaves and investigation of some kinetic properties. Preparative Biochemistry and Biotechnology 33(1): 39–52. doi:10.1081/PB-120018368.
• Erat M, 2005. Purification of 6‐phosphogluconate dehydrogenase from chicken liver and investigation of some kinetic properties. Preparative Biochemistry and Biotechnology 35(1): 53–69. doi:10.1081/PB-200041446.
• Fujita Y, Fujita T, 1987. The gluconate operon gnt of bacillus subtilis encodes its own transcriptional negative regulator. Proceedings of the National Academy of Sciences of the United States of America 84(July): 4524–4528. doi:10.1073/pnas.84.13.4524.
• Goulielmos GN, Eliopoulos E, Loukas M, Tsakas S, 2004. Functional constraints of 6-phosphogluconate dehydrogenase (6-pgd) based on sequence and structural information. Journal of Molecular Evolution 59(3): 358–71. doi:10.1007/s00239-004-2630-y.
• Harbitz I, Chowdhary B, Chowdhary R, Kran S, Frengen E, Gustavsson I, Davies W, 1990. Isolation, characterization and chromosomal assignment of a partial cdna for porcine 6-phosphogluconate dehydrogenase. Hereditas 112(1): 83–88. https://www.scopus.com/inward/record.uri?eid=2-s2.0-0025295377&partnerID=40&md5=2544cf498948e719ae6880b6c889628a.
• Human M, Kahlers SG, Kirkman HN, 1983. Intracellular glucose-6-phosphate dehydrogenase does not monomerize in human erythrocytes. Journal of Biological Chemistry 11:717–18.
• Kiliç S, 2007. Van kedisi’nden 6 fosfoglukanat dehidrogenaz enziminin saflastirilmasi karekterizasyonu , kinetigi,inhibisyonu ve hplc ile tayin yöntenteminin gelistirilmesi. PhD thesis,Yüzüncü Yil University, chemistry department.
• Kwok S, Tsui W, Yuet J, Chan W, Miu M, Waye Y, Fung KP, Lee CY, 1996. Identification of a cDNA encoding 6-phosphogluconate dehydrogenase from a human heart cdna library 1. Biochemical Genetics 34: 365–73.
• Laemmli UK, 1970. Cleavage of structural proteins during assembly of head of bacteriophage‐T4. Nature 227: 680–85,. doi:10.1038/227680a0.
• Morelli A, Bennati U, Gaetani GF, De Flora A, 1979. Biochemical mechanisms of glucose-6-phosphate dehydrogenase deficiency. Proceedings of the National Academy of Sciences 75(4): 1979–1983, 1978. doi:10.1073/pnas.75.4.
• Murray RK, Granner DK, Mayes PA, Rodwel. VW, 2003. Harper’s Illustrated Biochemistry. McGraw-Hill Companies, New York, Twenty-sixth Edition. Vol. 16: 693. doi:10.1016/0307-4412(88)90029-5.
• Nasoff MS, Baker HV, Wolf RE, 1984. DNA sequence of the Eschericia coli gene, Gnd, for 6- phosphoglueonate dehydrogenase. Gene 21(27): 253–264.
• Nelson D, Cox M, 2005. Lehninger principles of biochemistry. WH. Freeman, New York, Volume 33, Fourth edition: 190-225. doi:10.1002/bmb.2005.494033010419.
• Ninfali P, Orsenigo I, Baronciani L, Rapa S, 1990. Rapid purification of glucose-6-phosphate dehydrogenase from mammal’s erythrocytes. Preparative Biochemistry 20(3–4): 297–309,. doi:10.1080/00327489008050202.
• Özabacigil F, 2005. Insan eritrosit 6-fosfoglukanat dehidrogenaz enziminin saflastirilmasi, bazi ilaçlarin enzim aktivitesi üzerine in vitro ve tavsanlarda in vivo etkisinin incelenmesi. PhD thesis, Atatürk University, Health institute.
• Reizer A, Deutscher J, Saier MH, Reizer J, 1991. Analysis of the gluconate (gnt) operon of Bacillus subtilis. Molecular Microbiology 5(5): 1081–89.
• Segel IH, 1968. Biochemical calculations. New York: 212-337.
• Silverberg M, Dalziel K, 1973. Crystalline 6-phosphogluconate dehydrogenase from sheep liver. European Journal of Biochemistry 38(2): 229–38,. doi:10.1111/j.1432-1033.1973.tb03054.x.
• Somers DON, Hajdu J, Adams MJ, 1991. A two-step purification procedure for sheep liver 6-phosphogluconate dehydrogenase. Protein Expression and Purification 389(2): 385–389.
• Srivastava SK, Beutler E, 1970. Glutathione metabolism of the erythrocyte. the enzymic cleavage of glutathione-haemoglobin preparations by glutathione reductase. Biochemical Journal 119(3): 353–357,. http://www.biomednet.com/db/medline/71112233.
• Taranci A, 2011. Purification and characterization of 6-phosphogluconate dehydrogenase (e.c. 1.1.1.44) enzyme from rainbow trout (Oncorhynchus mykiss) erythrocytes and investigation of the effects of some metals on enzyme activity. Master thesis, Ataturk University, Institute of science: 93.
• Temel Y, Bayındır S, 2019. The Synthesis of Thiosemicarbazone-Based Aza-Ylides as Inhibitors of Rat Erythrocyte Glucose 6-Phosphate Dehydrogenase Enzyme. Iğdır Üniversitesi Fen Bilimleri Enstitüsü Dergisi, 9(3): 1503-1512.
• Temel Y, Bozkuş T, Karagözoğlu Y, Çiftçi M, 2017b. Glutatyon Redüktaz (GR) Enziminin Japon Bıldırcın (Coturnix coturnix japanica) Eritrositlerinden Saflaştırılması ve Karakterizasyonu. Iğdır Üniversitesi Fen Bilimleri Enstitüsü Dergisi, 7(3): 143-150.
• Temel Y, Küfrevioğlu Öİ, Ciftci M, 2017a. Investigation of the effects of purification and characterization of turkey (Meleagris gallopavo) liver mitochondrial thioredoxin reductase enzyme and some metal ions on enzyme activity. Turkish Journal of Chemistry, 41(1): 48-60. Temel Y, Kocyigit UM, 2017. Purification of glucose‐6‐phosphate dehydrogenase from rat (Rattus norvegicus) erythrocytes and inhibition effects of some metal ions on enzyme activity. Journal of biochemical and molecular toxicology, 31(9): e21927.
• Temel Y, Koçyigit UM, Taysı MŞ, Gökalp F, Gürdere MB, Budak Y, Ceylan M, Gülçin İ, Çiftci M, 2018. Purification of glutathione S‐transferase enzyme from quail liver tissue and inhibition effects of (3aR, 4S, 7R, 7aS)‐2‐(4‐((E)‐3‐(aryl) acryloyl) phenyl)‐3a, 4, 7, 7a‐tetrahydro‐1H‐4, 7‐methanoisoindole‐1, 3 (2H)‐dione derivatives on the enzyme activity. Journal of biochemical and molecular toxicology, 32(3): e22034.
• Toews ML, Kanji MI, Carpe, WR, 1976. 6-Phosphogluconate dehydrogenase. The Journal of Biochemical Chemistry 251(22): 7121–31.
• Villet RH, Dalziel K, 1969. Purification and properties of sheep liver phosphofructokinase. The Biochemical Journal 113(2): 235–242,.
• http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1184628&tool=pmcentrez&rendertype=abstract.
• Villet RH, Dalziel K, 1972. Studies of 6-phosphogluconate dehydrogenase from sheep liver: 1. kinetics of the reductive carboxylation reaction. European Journal of Biochemistry 27(2): 244–250,. doi:10.1111/j.1432-1033.1972.tb01833.x.
• Wang Y, Zhang YHP, 2009. Overexpression and simple purification of the Thermotoga maritima 6-phosphogluconate dehydrogenase in Escherichia coli and its application for NADPH regeneration. Microbial Cell Factories 8: 30,. doi:10.1186/1475-2859-8-30.
• Weisz KS, Schofield PJ, Edwards MR, 1985. Human brain 6-phosphogluconate dehydrogenase: purification and kinetic properties. Journal of Neurochemistry 44 (2): 510–517.
• Zera AJ, Wehrkamp C, Schilder R, Black C, Gribben P, 2014. Purification and characterization of 6-phosphogluconate dehydrogenase from the wing-polymorphic cricket, gryllus firmus, and assessment of causes of morph-differences in enzyme activity. Comparative Biochemistry and Physiology Part - B: Biochemistry and Molecular Biology 172–173(1): Elsevier Inc. 29–38,. doi:10.1016/j.cbpb.2014.04.001.