Purification and partial characterization of a retinyl ester hydrolase from the brush border of rat small intestine mucosa: probable identity with brush border phospholipase B.

Retinol esterified with long-chain fatty acids is a common dietary source of vitamin A, that is hydrolyzed prior to absorption. An intrinsic brush border membrane retinyl ester hydrolase activity had previously been demonstrated for rat small intestine [Rigtrup, K. M., & Ong, D. E. (1992) Biochemistry 31, 2920-2926]. This activity has now been purified to apparent homogeneity by a three-column procedure to obtain a protein of apparent molecular weight of 130,000. The purified protein retained the pattern of bile salt stimulation, specificity for the acyl moiety of the retinyl ester, and the Km values previously observed for the activity present in the isolated brush border membrane. This protein also had a potent phospholipase activity, while having little measurable ability to hydrolyze triacylglyceride and cholesteryl ester substrates. The retinyl ester hydrolase enzyme was localized to the distal two-thirds of the small intestine. A polyclonal antiserum against rat brush border phospholipase B reacted with the purified retinyl ester hydrolase, strongly suggesting that this enzyme was the same as that previously purified and characterized as a calcium-independent brush border phospholipase B [Pind, S., & Kuksis, A. (1991) Biochem. Cell Biol. 69, 346-357]. Detailed kinetic studies revealed lower Km values for retinyl palmitate substrate compared to phosphatidylcholine substrate, with all tested bile salts. The Km values for each substrate were bile salt dependent and differently altered when bile salts were changed. Vmax values were also bile salt dependent. Retinyl palmitate was hydrolyzed most rapidly in the presence of deoxycholate and least rapidly in taurocholate.(ABSTRACT TRUNCATED AT 250 WORDS)