NADPH-Sitokrom P450 redüktaz enziminin sığır karaciğer mikrozomlarından preparatif ölçekte saflaştırılması, antikor elde edilmesi ve enzimin immünolojik, kinetik, biyokatalitik ve diğer fonksiyonel özelliklerinin karakterizasyonu

Arınç, Emel
Çelik, Haydar
NADPH-cytochrome P450 reductase is an obligatory component of the microsomal cytochrome P450 dependent monoxygenase system where it catalyzes the transfer of reducing equivalents from NADPH to cytochrome P450. The cytochrome P450 reductase, by itself, also plays an important role in the reductive metabolism of some anticancer drugs and antibiotics as well as in lipid peroxidation and production of reactive species that can result in genotoxicity and cytotoxicity. The cytochrome P450 reductase has been purified from small laboratory animals such as the rat, rabbit, guinea-pig and mouse in a biocatalytically active form and characterized. On the other hand, studies related with the characterization of cytochrome P450 reductase and its role in the metabolism of drugs and xenobiotics in veterinary animals like beef have remained limited. In our laboratory, cytochrome P450 reductase was purifed from beef liver tissue in a biocatallytically active amphipathic form for the first time and some biocatalytic and spectral properties were characterized (Arınç and Çelik, 2002; J. Biochem. Mol. Toxicol., 16, 286- 297). However, these studies can be considered as preliminary studies. In this study, immunologic, biocatalytic, kinetic and other funcitonal properties of beef liver reductase were further characterized in detail. The enzyme was purified from beef liver tissue on a preparative-scale and with a higher specific activity by optimizing the previously described method. The specific activity was found as twice of the previous preparation. The molecular weight of the purified enzyme was found to be between 76.000-78.000 dalton as the P450 reductase obtained previously and its absorption spectrum showed the characteristics of flavoproteins. The in vitro effects of detergents Emulgen 913, cholate and Triton X-100 on the activity of beef liver P450 reductase were evaluated using both purified enzyme and microsomes. The results obtained demonstrated that, at various concentrations and mixtures tested, these detergents increased the activity of beef liver P450 reductase between 17% and 40%. These results demonstrated the appropriate concentrations of Emulgen 913 and cholate required for the solubilization of P450 reductase from beef liver microsomes during the purification proceess. The purified enzyme was found to be in biocatalytically active form as shown by its ability to catalyze the benzphetamine Ndemethylation reaction in reconstituted systems containing purified phenobarbital-treated rabbit liver cytochrome P4502B4 and phosphatidylcholine dilauroyl as a synthetic lipid. Since the aminoacid sequence of rabbit liver P4502B is known and it is classified as P4502B4 (Arınc, 1993), this P450 was used in reconstitution studies of benzphetamine Ndemethylation enzyme activity. Stability of cytochrome c reductase activity of the purified 16 enzyme was determined at 37°C in the presence and absence of 20% glycerol and the results obtained were compared with sheep lung P450 reductase. It was shown that glycerol enhanced the stability of both enzyme and beef liver P450 reductase was found to be much more stable compared to sheep lung P450 reductase. The in vitro effects of the metal ions Ni+2, Cd+2 and Pb+2 on the activity of the purified beef liver P450 reductase were elucidated. All the metals ions tested inhibited the activity of the purified enzyme in a noncompetitive manner. Of the metal ions tested, Pb+2 (Ki value: 0.03 mM) was the most potent inhibitor of the purified enzyme, which was followed by cadmium (Ki value: 0.04 mM). Thus, it was demonstrated that lead and cadmium have more serious toxic effects on the purified cytochrome P450 reductase with respect to nickel metal. In this project, policlonal antibodies were produced against beef liver cytochrome P450 reductase for the first time. Thus, an industrial product with many uses in biochemistry, pharmacology, toxicology, pharmaceutical industry and in some biotechnological applications was obtained. The Western-blot experiments using the polyclonal antibodies raised against both purified beef liver P450 reductase and purified sheep lung P450 reductase showed that beef liver and sheep lung P450 reductases are immunologically similar and share common epitopes or antigenic determinants. In this study, it was tried to clarify the action mechanism of anticancer drug idarubicin at the molecular level by using purified beef liver P450 reductase and other purified P450 reductases (sheep lung P450 reductase and phenobarbital-treated rabbit liver P450 reductase). The experiments conducted for this purpose were repeated with mitomycin C, used as a model compound, and the results obtained were compared. It was demonstrated, for the first time, that cytochrome P450 reductase can catalyze the reductive bioactivation of idarubicin with resulting formation of DNA single strand breaks. The mechanism of DNA damage involved the redox cycling of idarubicin with cytochrome P450 reductase under aerobic conditions to generate “reactive oxygen species” (ROS). Thus, the action mechanism of idarubicin at the molecular level was demonstrated for the first time within this study. This pathway with cytochrome P450 reductase was proposed to potentially contribute to the antitumor effect of this anticancer agent. (Çelik and Arınç 2008; J. Pharm. Pharmaceut. Sci., 11(4), 68-82).


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Citation Formats
E. Arınç and H. Çelik, “NADPH-Sitokrom P450 redüktaz enziminin sığır karaciğer mikrozomlarından preparatif ölçekte saflaştırılması, antikor elde edilmesi ve enzimin immünolojik, kinetik, biyokatalitik ve diğer fonksiyonel özelliklerinin karakterizasyonu,” 2009. Accessed: 00, 2020. [Online]. Available: