Improved assay for cholesterol 7 alpha-hydroxylase activity using phospholipid liposome solubilized substrate

Journal Article · Tue Oct 01 00:00:00 EDT 1985 · Lipids; (United States)

OSTI ID:6045534

Junker, L H; Story, J A

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. 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.

• Research Organization: Purdue Univ., W. Lafayette, IN
• OSTI ID: 6045534
• Journal Information: Lipids; (United States), Vol. 10
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
CARBON 14 COMPOUNDS
ISOTOPE DILUTION
HYDROXYLASES
BIOCHEMISTRY
KINETICS
LIPOSOMES
PHOSPHOLIPIDS
RATS
SOLUBILITY
ANIMALS
CELL CONSTITUENTS
CHEMISTRY
ENZYMES
ESTERS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LIPIDS
MAMMALS
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
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OXIDOREDUCTASES
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TRACER TECHNIQUES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques