A fluorometric assay for cholesterol reductase activity.
A fluorometric method for the assay of cholesterol reductase activity from pea leaves (Pisum sativum) is presented. This method is based on the decrease in relative fluorescence occurring as a result of the oxidation of NADH when cholesterol is reduced catalytically to coprostanol by cholesterol reductase. The reaction mixture consisted of micellar cholesterol, NADH, and cytosol of pea leaves in a phosphate buffer. After incubation for 1 h, the reaction mixture were diluted with 2-(N-cyclohexylamino)ethanesulfonic acid buffer (50 mM, pH 10.0) to an appropriate concentration for NADH quantification. The relative fluorescence was measured at an excitation wavelength of 360 nm and at an emission wavelength of 460 nm. This fluorometric method is relatively rapid, simple, and inexpensive. The results obtained show close correlation (R = 0.997) with those obtained by the more time-consuming and expensive radiometric method for assay of cholesterol reductase activity. Results suggest that the fluorometric method is useful for the accurate determination of cholesterol reductase activity in biological specimens.
Yang
J B
JB
Department of Animal Science, Iowa State University, Ames 50011.
Beitz
D C
DC
D002784:Cholesterol / Q000378:metabolism; D007887:Fabaceae / Q000201:enzymology*; D007202:Indicators and Reagents; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D010088:Oxidoreductases / Q000032:analysis* / Q000378:metabolism; D010946:Plants, Medicinal; D011865:Radioisotope Dilution Technique; D012588:Scintillation Counting / Q000379:methods; D013050:Spectrometry, Fluorescence / Q000379:methods
D007202:Indicators and Reagents; D009243:NAD; D002784:Cholesterol; D010088:Oxidoreductases; C078268:cholesterol reductase