Unusual properties of retinyl palmitate hydrolase activity in rat liver.

These studies report the hydrolysis of retinyl palmitate with liver homogenates and homogenate fractions from retinol-depleted rats. The studies utilized an effective in vitro assay for retinyl palmitate hydrolase (RPH) activity, in which microgram amounts of retinyl palmitate were employed as substrate, followed by the chromatographic separation and fluorescence assay of free and esterified retinol. RPH activity was maximal near pH 8 in Tris-maleate buffers, and required a bile salt for stimulation. Both cholate and taurocholate stimulated the reaction, whereas a number of other detergents tested were ineffective. The enzymatic activity showed an unusual subcellular distribution, with about 40% of total RPH activity recovered in the washed "nuclear" fraction (1,500 g pellet) and about 30--35% in the 105,000 g supernatant. This unusual distribution was not observed for marker constituents for plasma membranes, nuclei, mitochondria, lysosomes, Golgi apparatus, or endoplasmic reticulum. Despite its enrichment in the "nuclear" fraction, RPH activity was not enriched in purified preparations of nuclei or plasma membranes. Thus, RPH activity was not localized in any single, characterized subcellular structure. Another striking feature of the hepatic RPH activity was its extreme variability from rat to rat as assayed in vitro. Both the unusual subcellular distribution and the marked variability in activity were not observed for a variety of other hepatic ester hydrolase activities examined. Of ten lipid and nonlipid esters tested as substrates, only the hydrolytic activities against cholesteryl oleate and phytyl oleate correlated with, and partly resembled, RPH activity in these respects. The results suggest that the observed RPH activity is relatively specific for the hydrolysis of retinyl palmitate, and may therefore be significantly involved in hepatic retinyl ester metabolism.