Micro-radiometricCholesterolEsterHydrolaseAssay in Rat LutealTissue

A micro-radiometric assay for measurement of rat luteal cholesterol ester hydrolase has been developed. The assay measures conversion of (4-'4C1 -cholesteryl oleate into (4-'4C1 -cholesterol within the 149,000 X g supernatant. Specificity studies indicated no significant conversion of (4-'4C) -cholesterol product into either (4-'4C1 -cholesterol esters, or (4-'4C1 -progesterone. Assay precision, as measured by the coefficient of variability, was 10.75%. Sensitivity of the assay was 12.9 @tg of 149,000 X g supernatant protein. Utilization of this assay to characterize luteal choles terol ester hydrolase in adult, pseudopregnant rats indicated that cholesterol ester hydrolase had an apparent Km of 18.3 @M, and a maximal rate (Vmax) of 754 dpm/min. Time period of initial velocity was 5 mm, and rate of product formation was linear between 65 and 260 j@gof protein. Development of this assay allows measurement of hormonal effects on cholesterol ester hydrolase in the small quantities of luteal tissue present in mature, pseudopregnant or pregnant rats.

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