TITLE:
Method for Detecting NADPH-Cytochrome P450 Reductase in Liver Microsomal Fractions by Using Native Polyacrylamide Gel Electrophoresis and NADPH-Diaphorase Staining
AUTHORS:
Hirokazu Yokoyama, Yukishige Okamura, Toshifumi Hibi
KEYWORDS:
NADPH-Cytochrome p450 Reductase; Native PAGE; NADPH-Diaphorase Staining; Chronic Ethanol Consumption
JOURNAL NAME:
American Journal of Analytical Chemistry,
Vol.4 No.6,
June
20,
2013
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.