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Title: ISOLATION AND CHARACTERIZATION OF AN ACID, BILE SALT-INDEPENDENT, RETINYL ESTER HYDROLASE FROM RAT LIVER MICROSOMES

Author
item Linke, Thomas
item Harrison, Earl

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/21/2004
Publication Date: 6/17/2005
Citation: Linke, T.N., Harrison, E.H. 2005. Isolation and characterization of an acid, bile salt-independent, retinyl ester hydrolase from rat liver microsomes. Journal of Biological Chemistry. 280(24):23287-23294.

Interpretive Summary: We are studying the liver enzymes that hydrolyze retinyl esters, the form in which newly absorbed dietary vitamin A is delivered to the liver. These enzymes (retinyl ester hydrolases or REHs) are important in both the uptake of dietary vitamin A by the liver and in the mobilization of stored vitamin A from the liver when dietary input is inadequate to meet the body's need for this essential nutrient. Although a number of liver enzymes can catalyze the hydrolysis of vitamin A esters the identity and physiological role of these enzymes is unclear. Our research attempts to fill these gaps in knowledge by isolating these enzymes in pure form to understand more about them. In the study reported here we have isolated and characterized an enzyme that is likely involved in the initial uptake and storage of dietary vitamin A in liver. This work will enhance our understanding of vitamin A metabolism in humans and will benefit other research scientists and human nutritionists.

Technical Abstract: Previous work in this laboratory has revealed the presence of both acidic and neutral bile-salt independent retinyl ester hydrolase activities in rat liver homogenates. Here we present the purification, identification and characterization of an acid retinyl ester hydrolase activity from solubilized rat liver microsomes. Purification to apparent homogeneity was achieved by sequential chromatography using SP-Sepharose cation exchange, Phenyl-Sepharose hydrophobic interaction, Concanavalin A-Sepharose affinity and Superose 12 gel filtration chromatography. The isolated protein had a monomer molecular weight of 62 kDa, as measured by mass spectrometry. Analytical gel filtration chromatography of the purified protein revealed a native molecular weight of approximately 172 kDa, indicating that the protein exists as a homotrimeric complex in solution. The purified protein was identified as carboxylesterase ES-10 (EC 3.1.1.1) by N-terminal Edman sequencing and extensive LC-MS/MS sequence analysis and cross-reacted with anti-ES-10 antibodies. Glycosylation analysis revealed that only one of two potential N-linked glycosylation sites is occupied by a high mannose-type carbohydrate structure. Using retinyl palmitate in a micellar assay system the enzyme was active over a broad pH range and displayed Michaelis-Menten kinetics with a Km of 86 mM. Thus, carboxyl esterase ES-10 may catalyze the hydrolysis of newly-endocytosed, chylomicron retinyl esters in both neutral and acidic membrane compartments of liver cells.