Abstract
This chapter discusses 25-hydroxylation of vitamin D 3 in liver microsomes, and their smooth and rough subfractions. In 1973, Bhattacharyya and DeLuca described an assay for calciferol 25-hydroxylase activity in liver homogenates. The activity of this enzyme in D-deficient chicken and rat liver homogenates, and in rat liver microsomes has been studied. This chapter describes a modification of the above procedure to study the properties of the enzyme system in rat liver microsomes and in smooth- and rough-surfaced subfractions. The principle of this method, utilizing the conversion of a radioactive substrate to an end product, is applicable to the study of any enzymic step in the metabolism of D. Although activity can be demonstrated only in preparations obtained from rats maintained on D-deficient diet and allowed to become rachitic, the method provides a consistently reproducible and sensitive means for determining microsomal 25-hydroxylase activity, as well as that of the smooth and rough microsomal subfractions. The identity of the product of the enzymic reaction [ 3 H]25-OH-D 3 is established by additional chromatography on silicic acid, celite liquid–liquid partition, and high-pressure liquid chromatography. Confirmation of this identity is further established by comparative binding of the isolated metabolite and pure standard [ 3 H]25-OH-D 3 to its serum transport protein.
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