Enzymatic esterification of vitamin A in the pigment epithelium of bovine retina.

The kinetic properties and subcellular distribution of an esterifying enzyme in the pigment epithelium of bovine retina have been studied using both [1-3H]retinol and [3H]retinol bound to cellular retinol-binding protein as substrates. The most active esterifying fraction in pigment epithelial cell preparations was the microsomes, but the lysosome plus mitochondria fraction also showed some activity, probably due to endoplasmic reticulum present as an impurity. The microsomal enzyme showed optimum activity at pH 7.5, and the reaction was linear up to 30 microgram protein and for the first 10-15 min. The apparent Km values were 16.6 . 10(-6) and 5.5 . 10(-6) M for [3H]retinol and bound [3H]retinol, respectively. This is the first time that retinol bound to cellular retinol-binding protein has been shown to undergo metabolic transformation. The microsomal esterifying activity was destroyed by boiling for 1 min, or after freezing for 2 months. No clear requirement for ATP, CoA or fatty acid could be demonstrated. Of all the other tissues examined under the same experimental conditions as those used for the pigment epithelium, only intestine showed measurable activity. With larger amounts of tissue protein and longer incubation periods, activity was also detectable in microsomes of liver, testis and retina.