Purification, Characterization of NADPH-Cytochrome P450 Reductase from Turkey Liver and Effects of Some Metal Ions on Enzyme Activity

Year 2018, , 183 - 189, 30.09.2018

Ebru Akkemik Mehmet Çiftçi

https://doi.org/10.21597/jist.458623

Abstract

NADPH-Cytochrome P450 reductase (CPR) is a key enzyme catalyzing electron transfer in

detoxification metabolism. In this study, two methods were employed to purify CPR enzyme from turkey liver

microsomes. In the first method, NADPH-Cytochrome P450 reductase was purified using 2ʹ, 5ʹ-ADP Sepharose 4B

affinity column with ~114 purification fold and ~23% yield. In the second method, CPR was purified using DE-52

Cellulose anion exchange column and 2ʹ, 5ʹ-ADP Sepharose 4B affinity column with 124 purification fold and 8%

yield. Enzyme purity was checked in both methods with SDS-PAGE. Characteristics kinetic features of the purified

enzyme were identified. The effects of some metal ions on purified PCR enzyme activity have been investigated

in vitro conditions. It has been found that Ag+, Hg2+ and Cu2+ metal ions have an inhibitory effect on CPR enzyme

activity.

Keywords

Characterization, cytochrome P450 reductase, inhibition, metal ions, turkey liver

Hindi Karaciğerinden NADPH-Sitokrom P450 Redüktaz’ın Saflaştırılması, Karakterizasyonu ve Bazı Metal İyonlarının Enzim Aktivitesi Üzerindeki Etkileri

Abstract

NADPH-Sitokrom P450 redüktaz (CPR) enzimi, detoksifikasyon metabolizmasında elektron transferini

katalizleyen anahtar bir enzimdir. Bu çalışmada, hindi karaciğer mikrozomlarından CPR enziminin saflaştırılması

için iki yöntem kullanılmıştır. Birinci yöntemde, CPR enzimi ~114 saflaştırma katsayısıyla ve ~%23 verimle

2’, 5’-ADP Sefaroz 4B afinite kolonu kullanılarak saflaştırılmıştır. İkinci yöntemde, CPR, DE-52 selüloz anyon

değişim kolonu ve 2’, 5’-ADP Sefaroz 4B afinite kolonu kullanılarak, ~124’lük saflaştırma katsayısı ve %8

verimle saflaştırılmıştır. Enzim saflığı her iki yöntemde de SDS-PAGE ile kontrol edilmiştir. Saflaştırılmış enzimin

karakteristik kinetik özellikleri belirlenmiştir. Bazı metal iyonlarının saflaştırılmış CPR enzim aktivitesi üzerindeki

etkileri in vitro şartlarda araştırılmıştır. Ag+, Hg2+ ve Cu2+ metal iyonlarının CPR enzim aktivitesi üzerinde inhibisyon

etkisi gösterdiği tespit edilmiştir.

Keywords

Hindi karaciğeri, inhibisyon, karakterizasyon, metal iyonları, sitokrom P450 redüktaz

References

• Aoi K, Fujii-Kuryama Y, Tashiro Y, 1981. Intracellular Distribution of NADPH- Cytocrome C Reductase İn Rat Hepatocytes Studied By Direct Ferritin-Immunoelectro Mıcroscopy. Journal of Cell Science, 50: 181-198.
• Bozcaarmutlu A, Arinc E, 2007. Effect of mercury, cadmium, nickel, chromium and zinc on kinetic properties of NADPH-cytochrome P450 reductase purifed from leaping mullet (Liza saliens). Toxicology in Vitro, 21: 408–416.
• Bozcaarmutlu A, Celik H, Arinc E, 2011. Inhibition mechanisms of lead, cadmium and nickel for the purified bovine liver NADPH-cytochrome P450 reductase. Toxicology Letters, 205: 192-192.
• Crankshaw DL, Hetnarski K, Wilkinson CF, 1979. Purification and Characterization of NADPH-Cytochrome c Reductase from the Midgut of the Southern Armyworm (Spodoptera eridania). Biochem Journal, 181: 593-605.
• Çelik H, 2002. Biochemical and Immunological Charachterization of Beef Liver NADPH-cytochrome P450 Reductase, The Graduate School of Natural and Applied Sciences of Middle East Technical University, Degree of Master. 136s.
• Dalloul RA, Long JA., Zimin VA, Aslam L, Bea K, et al, 2010. Multi-Platform Next-Generation Sequencing of the Domestic Turkey (Meleagris gallopavo): Genome Assembly and Analysis. PLOS Biology, 9: e1000475.
• Danişan A, Ceyhan D, Öğüs¸ IH. Özer N, 2004. Purification and Characterization of Glucose-6-Phosphate Dehydrogenase from Rat Small Intestine. Protein Journal, 23: 317-324.
• Dignam JD, Strobel HW, 1977. NADPH-Cytochrome P-450 reductase from rat liver: Purification by affinity chromatography and characterization. Biochemistry, 16: 1116-1123.
• Halliwell B, Auroma OI, 1991. DNA damage by oxygen-derived species. Its mechanism and measurement in mammalian systems. FEBS Letters, 281: 9–19.
• Iscan MY, Arinc E, 1986. Kinetic and structural properties of biocatalytically active sheep lung microsomal NADPH-cytocrome c reductase. International Journal of Biochemistry, 18: 731-741.
• Iscan MY, Arinc E, 1988. Comparison of highly purified sheep liver and lung NADPH-cytochrome P450 reductases by the analysis of kinetic and catalytic properties. International Journal of Biochemistry, 20: 1189–1196.
• Kappus H, 1986. Overview of enzyme systems involvedin bioreduction of drugs and in redox cycling. Biochemical Pharmacology, 35: 1-6
• Kayaalp O, 2002. İlaçların biyotransformasyonu; Tıbbi Farmakoloji, Feryal Matbaacılık San. ve Tic. Ltd. Şti, Ankara, Yedinci baskı, İstanbul, Türkiye, 1: 92-129.
• Keha E, Küfrevioğlu Öİ, 2009. Biyokimya. Aktif yayınevi, Dokuzuncu baskı, Erzurum, Türkiye, 653s
• Kim JS, Ahn T, Yim SK, Yun CH, 2002. Differential Effect of Copper (II) on the Cytochrome P450 Enzymes and NADPH-Cytochrome P450 Reductase: Inhibition of Cytochrome P450-Catalyzed Reactions by Copper (II) Ion. Biochemistry, 41: 9438-9447.
• Klotz AV, Stegeman JJ, Walsh C, 1983. An aryl hydrocarbon hydroxylating hepatic cytochrome P450 from the marine fish Stenotomus chrysops. Archives of Biochemistry and Biophysics, 226: 578–592.
• Kong S, Ngo SNT, McKinnon RA, Stupans I, 2009. Cloning and expression of koala (Phascolarctos cinereus) liver cytochrome P450 reductase. Comparative Biochemistry and Physiology, Part C 150: 1–9.
• Laemmli DK, 1970. Cleavage of structural proteins during in assembly of the heat of bacteriophage T4. Nature, 227: 680-685.
• Lineweaver H, Burk D, 1934. The determination of enzyme dissocation constants. Journal of the American Chemical Society, 57: 658-666.
• Lu AYH, 1991. NADPH-Dependent Cytochrome P450 Reductase. In: Arinç E., Schenkman J.B., Hodgson E. (eds) Molecular Aspects of Monooxygenases and Bioactivation of Toxic Compounds. NATO ASI Series Advanced Science Institutes Series (Series A: Life Sciences), Springer, Boston, MA, 202: 135-147p.
• Masters BSS, 1980. In Enzymatic Basis of Detoxication (Jakoby, W. B., Ed.), Academic Press, New York. 1: 183-200p
• Masters BSS, Williams CH, Kamin H, 1967. The preparation and properties of microsomal TPNH-cytochrome c reductase from pig liver. In Methods in Enzymology (Edited by Colowick S. P. And Kaplan N. O.), Academic Press, New York. 10: 565-573p.
• Menting JGT, Cornish E, Scopes RK, 1994. Purification and partial characterization of NADPH-cytochrome c reductase from Petunia hybrida flowers. Plant Physiology, 106: 643–650.
• Murray RK, Granner DK, Mayes PA, Rodwell VW, 2004. Harper’ın Biyokimyası, Nobel Tıp Kitapevleri, Yirmibeşinci baskı. İstanbul, Türkiye, 816s
• Nelson DL, Cox MM, 2005. Lehninger Biyokimyanın İlkeleri. (Çeviren: N. KILIÇ), Palme Yayıncılık, Ankara, Türkiye, 243-293s.
• Özbolat G, Tuli A, 2016. Ağır Metal Toksisitesinin İnsan Sağlığına Etkileri, Arşiv Kaynak Tarama Dergisi, 25: 502-521
• Özerol E, 1996. Cytochrom P 450 containing monooksigenase enzym systems, Journal of Tugut Özal Medical Center, 33: 257-275.
• Porter TD, Kasper CB, 1986. NADPHcytochrome P-450 oxidoreductase: Flavin mononucleotide and flavin adenine dinucleotide domains evolved from different flavoproteins. Biochemistry, 25: 1682-1687.
• Segel IH, 1968. Biochemical Caculations, Inc,New York, 403p.
• Sen A, Arinc E, 1998. Purification Characterization of Cytochrome P450 Reductase from Liver Microsomes of Feral Leaping Mullet (Liza saliens), J. Biochem Molecular Toxicology, 12: 103-113.
• Shet, MS, Sathasiva K, Arlotto MA, Mehdy MC, Estabrook RW, 1993. Purification, characterization and cDNA cloning of an NADPH-cytochrome P450 reductase from mung bean, Proceedings of the National Academy of Sciences, 90: 2890–2894.
• Sinha BK, 1989. Free radicals in anticancer drug pharmacology. Chemico-Biological Interactions, 69: 293-317.
• Soyut H, 2006. Purification And Characterization of Carbonic Anhydrase from Rainbow Trout (Oncorhynchus Mykiss) Tıssues And Inhibition Effects of Some Antibiotics on Enzyme Activity. Ph. D. Thesis, Graduate School of Science and Arts Faculty, Department of Chemistry, Atatürk University, Erzurum, 174p.
• Wang M, Roberts DL, Paschke R, Shea, TM, Masters BSS, Kım JJP, 1997. Three-Dimensional Structure of NADPH–Cytochrome P450 Reductase: Prototype For FMN-And FAD-Containing Enzymes. Biochemistry, 94: 8411–8416.
• Wilson TG, Hodgson E, 1971. Microsomal NADPH-cytochrome c reductase from the house fly, Musca domestica: properties of the purified enzyme, Insect Biochemistry. 1: 171-180.