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 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.
Highlights
Nicotinamide adenine dinucleotide phosphate (NADPH)cytochrome P450 reductase (EC 1.6.2.4) is a microsomal flavoprotein expressed in various organs including liver [1]
When microsomal fractions prepared from rat livers were subjected to native polyacrylamide gel electrophoresis (PAGE) followed by nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining, several bands with different mobility including a prominent one appeared in the most anodal position were visualized (Figure 1(a))
By using the native PAGE/NADPH-diaphorase staining method, we examined levels of NADPH-diaphorase staining assigned to NADPH-cytochrome P450 reductase in liver microsomal fractions prepared from rats fed ethanol chronically and controls (Figure 2(a))
Summary
Nicotinamide adenine dinucleotide phosphate (NADPH)cytochrome P450 reductase (EC 1.6.2.4) is a microsomal flavoprotein expressed in various organs including liver [1]. It extracts electrons from NADPH and transfers them to various other microsomal cytochrome P450 enzymes which contribute to the metabolisms of steroids, heme groups, and xenobiotics in electron dependent manners [2,3]. NADPH-cytochrome P450 reductase is not an exclusive NADPH dependent electron donor In some tissues, this enzyme and the other ones acting as NADPH dependent electron donors are co-expressed. This enzyme and the other ones acting as NADPH dependent electron donors are co-expressed For detecting the former by NADPH-diaphorase staining in such tissues, it must be distinguished from the others
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