AJAC  Vol.4 No.6 , June 2013
Method for Detecting NADPH-Cytochrome P450 Reductase in Liver Microsomal Fractions by Using Native Polyacrylamide Gel Electrophoresis and NADPH-Diaphorase Staining

By combining native polyacrylamide gel electrophoresis (PAGE) and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining, a simple method for detecting NADPH-cytochrome P450 reductase in tissue samples was established. When rat liver microsomal fractions were examined by this method, several bands with different mobility were visualized. Western blot analysis indicated that the band which appeared in the most anodal position among them represented NADPH-cytochrome P450 reductase. SDS-PAGE/Western blot analysis revealed that the molecular weight of the protein forming the band was around 80 kDa, which was identical to that of rat NADPH-cytochrome P450 reductase. The intensity level of NADPH-diaphorase staining assigned to this enzyme estimated by this method increased four times in microsomal fractions prepared from rat fed ethanol chronically compared to that from controls. When a dilution series of a rat liver microsomal fraction was examined by this method and SDS-PAGE/Western blot analysis, their staining intensities representing this enzyme were significantly correlated with each other. Using the naive PAGE/NADPH-diaphorase staining method, NADPH-cytochrome P450 reductase is detected in rat liver microsomes. This method is beneficial because compared with the conventional SDS-PAGE/Western blot analysis, the quantification of NADPH-cytochrome P450 reductase in tissue samples is allowed to be more easily done.

Cite this paper: H. Yokoyama, Y. Okamura and T. Hibi, "Method for Detecting NADPH-Cytochrome P450 Reductase in Liver Microsomal Fractions by Using Native Polyacrylamide Gel Electrophoresis and NADPH-Diaphorase Staining," American Journal of Analytical Chemistry, Vol. 4 No. 6, 2013, pp. 301-305. doi: 10.4236/ajac.2013.46038.

[1]   A. Shen and C. B. Kasper, “Mechanistic Studies on the Reductive Half-reaction of NADPH-Cytochrome P450 Oxidoreductase,” In: J. B. Schenknan and H. Greim, Eds., Handbook of Experimental Pharmacology, Springer-Verlag Inc., New York, 1933, pp. 35-59.

[2]   B. S. Masters and C. C Marohnic, “Cytochromes P450— A Family of Proteins and Scientists-Understanding Their Relationships,” Drug Metabolism Reviews, Vol. 38, No. 1-2, 2006, pp. 209-225. doi:10.1080/03602530600570065

[3]   D. L. Roerig, L. Mascaro and S. D. Aust Jr., “Microsomal Electron Transport: Tetrazolium Reduction by Rat Liver Microsomal NADPH-Cytochrome c Reductase,” Archives of Biochemistry and Biophysics, Vol. 153, No. 2, 1972, pp. 475-479. doi:10.1016/0003-9861(72)90365-7

[4]   P. J. Norris, J. P. Hardwick and P. C. Emson, “Localization of NADPH Cytochrome P450 Oxidoreductase in Rat Brain by Immunohistochemistry and in Situ Hybridization and a Comparison with the Distribution of Neuronal NADPH-Diaphorase Staining,” Neuroscience, Vol. 61, No. 2, 1994, pp. 331-350. doi:10.1016/0306-4522(94)90235-6

[5]   B. T. Hope, G. J. Michael, K. M. Knigge and S. R. Vincent, “Neuronal NADPH Diaphorase Is a Nitric Oxide Synthase,” Proceedings of National Academy Science of the USA, Vol. 88, No. 7, 1991, pp. 2811-2814. doi:10.1073/pnas.88.7.2811

[6]   M. Belinsky and A. K. Jaiswal, “NAD(P)H:quinone Oxidoreductase1 (DT-Diaphorase) Expression in Normal and Tumor Tissues,” Cancer and Metastasis Reviews, Vol. 12, No. 2, 1993, pp. 103-117. doi:10.1007/BF00689804

[7]   C. S. Lieber, D. P. Jones and L. M. Decarli, “Effects of Prolonged Ethanol Intake; Production of Fatty Liver Despite Ad-Equate Diets,” The Journal of Clinical Investigation, Vol. 44, No. 6, 1965, pp. 1009-1021. doi:10.1172/JCI105200

[8]   J. D. Dignam and H. W. Strobel, “NADPH-Cytochrome p-450 Reductase from Rat Liver: Purification by Affinity Chromatography and Characterization,” Biochemistry, Vol. 16, No. 6, 1997, pp. 1116-1123.

[9]   A. A. Nanji, S. S. Greenberg, S. R. Tahan, F. Fogt, J. Loscalzo, S. M. Sadrzadeh, J. Xie and J. S. Stamler, “Nitric Oxide Production in Experimental Alcoholic Liver Disease in the Rat: Role in Protection from Injury,” Gastroenterology, Vol. 109, No. 3, 1995, pp. 899-907. doi:10.1016/0016-5085(95)90400-X

[10]   J. G. Joly, H. Ishii, H. R. Teschke, Y, Hasumura and C. S. Lieber, “Effect of Chronic Feeding on the Activities and Submicrosomal Distribution of Reduced Nicotinamide Adenine Dinucleotide Phosphate-Cytochrome P-450 Reductase and the Demethylases for Aminopyrine and Ethylmorphine,” Biochemical Pharmacology, Vol. 22, No. 12, 1973, pp. 1532-1535. doi:10.1016/0006-2952(73)90334-1