Method for Detecting NADPH-Cytochrome P450 Reductase in Liver Microsomal Fractions by Using Native Polyacrylamide Gel Electrophoresis and NADPH-Diaphorase Staining
- 1 Department of Internal Medicine, School of Medicine, Keio University, Shinanomachi Shinjukuku, Tokyo, Japan
- 2 Department of Internal Medicine, School of Medicine, Keio University, Shinanomachi Shinjukuku, Tokyo, Japan
- 3 Department of Internal Medicine, School of Medicine, Keio University, Shinanomachi Shinjukuku, Tokyo, Japan
Abstract
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 mo bility 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 kD a , 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 in creased 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 na ive PAGE/NADPH-diaphorase staining method, NADPH-cytochrome P450 reductase is detected in rat liver micro somes. This method is beneficial because compared with the conventional SDS-PAGE/Western blot analysis, the quan tification of NADPH-cytochrome P450 reductase in tissue samples is allowed to be more easily done.
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