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 sou rce 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 puri fied to apparent homogeneity by a three-column procedure to obtain a p rotein of apparent molecular weight of 130 000. The purified protein r etained the pattern of bile salt stimulation, specificity for the acyl moiety of the retinyl ester, and the K-m values previously observed f or the activity present in the isolated brush border membrane. This pr otein also had a potent phospholipase activity, while having little me asurable ability to hydrolyze triacylglyceride and cholesteryl ester s ubstrates. The retinyl ester hydrolase enzyme was localized to the dis tal two-thirds of the small intestine. A polyclonal antiserum against rat brush border phospholipase B reacted with the purified retinyl est er hydrolase, strongly suggesting that this enzyme was the same as tha t previously purified and characterized as a calcium-independent brush border phospholipase B [Pind, S., & Kuksis, A. (1991) Biochem. Cell B iol. 69, 346-357]. Detailed kinetic studies revealed lower K-m values for retinyl palmitate substrate compared to phosphatidylcholine substr ate, with all tested bile salts. The K-m values for each substrate wer e bile salt dependent and differently altered when bile salts were cha nged. V-max values were also bile salt dependent. Retinyl palmitate wa s hydrolyzed most rapidly in the presence of deoxycholate and least ra pidly in taurocholate. Interestingly, the opposite was true for phosph olipid substrate, with taurocholate resulting in the highest V-max and deoxycholate the lowest. These studies indicated a specific bile salt -enzyme interaction that affected the catalytic activity of the enzyme and altered the kinetic parameters differently for the two preferred substrates retinyl ester and phosphatidylcholine.