An improved assay for cholesterol 7 alpha-hydroxylase activity using phospholipid liposome solubilized substrate.
A persistent problem in measurement of cholesterol 7 alpha-hydroxylase (7 alpha-OHase) activity by isotope incorporation has been solubilization of cholesterol substrate. Solubilization with Tween 20, for example, resulted in a 75% reduction in 7 alpha-OHase activity after a 60 min incubation of substrate with microsomes. Incorporation of cholesterol substrate into small, unilamellar phospholipid vesicles (liposomes) prevented this effect, resulting in a 50% increase in activity over the same 60 min incubation at optimal concentrations. Using cholesterol in liposomes as substrate, standard assay conditions were determined to be: preparation of liposomes with 180 microM cholesterol substrate and 0.5 mg phospholipid/assay; incubation of these liposomes with 0.5 mg microsomal protein at 37 C for 60 min; addition of a NADPH generating system to start the reaction, and incubation at 37 C for 30 min before stopping the reaction and determining the amount of 7 alpha-hydroxycholesterol formed. In addition to preventing the detergent-related inhibition of the enzyme, liposome-solubilized substrate also reduced the variation among replicates from a coefficient of 45% with Tween 20 to 4.2% with phospholipid. This method provides a sensitive and reliable alternative to methods which require more sophisticated equipment and allows total control of substrate concentration in a form readily accessible to the enzyme.
D000818:Animals; D002250:Carbon Radioisotopes; D002790:Cholesterol 7-alpha-Hydroxylase / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D010713:Phosphatidylcholines; D010743:Phospholipids; D011865:Radioisotope Dilution Technique; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D013250:Steroid Hydroxylases / Q000378:metabolism*
D002250:Carbon Radioisotopes; D008081:Liposomes; D010713:Phosphatidylcholines; D010743:Phospholipids; C024990:asolectin; D013250:Steroid Hydroxylases; D002790:Cholesterol 7-alpha-Hydroxylase