Abstract
Previous work demonstrated both acid 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 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 mass of approximately 62 kDa, as measured by mass spectrometry. Gel filtration chromatography of the purified protein revealed a native molecular mass of approximately 176 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-reaction with an anti-ES-10 antibody. 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 K(m) of 86 microm. Substrate specificity studies showed that ES-10 is also able to catalyze hydrolysis of triolein. Cholesteryl oleate was not a substrate for ES-10 under these assay conditions. Real time reverse transcriptase-PCR and Western blot analysis revealed that ES-10 is highly expressed in liver and lung. Lower levels of ES-10 mRNA were also found in kidney, testis, and heart. A comparison of mRNA expression levels in liver demonstrated that ES-10, ES-4, and ES-3 were expressed at significantly higher levels than ES-2, an enzyme previously thought to play a major role in retinyl ester metabolism in liver. Taken together these data indicate that carboxylesterase ES-10 plays a major role in the hydrolysis of newly-endocytosed, chylomicron retinyl esters in both neutral and acidic membrane compartments of liver cells.
Highlights
Higher eukaryotic organisms require dietary vitamin A, which gives rise to a variety of active metabolites
Trial experiments showed that binding of AREH activity to Concanavalin A Sepharose was inhibited by the presence of Triton X-100 in the binding buffer; increased affinity of AREH activity to Con A-Sepharose was observed when Triton X-100 was replaced by OG in the binding buffer
4 preparations of the enzyme have been carried out according to the described protocol with similar results in specific activtiy, yield and enrichment to those shown in Identification of AREH as carboxylesterase ES-10 — In order to identify the AREH activity, the purified protein was subjected to in-gel tryptic digestion, followed by MALDI-TOF analysis of the resulting peptides
Summary
Higher eukaryotic organisms require dietary vitamin A (retinol), which gives rise to a variety of active metabolites. Preformed vitamin A is found in the diet mainly in the form of long-chain fatty acid esters (retinyl esters), with minor amounts being found in the form of retinol and retinoic acid [1]. Dietary retinyl esters are hydrolyzed to retinol in the lumen of the small intestine. This activity was previously thought to be catalyzed by pancreatic carboxyl ester lipase [2]; experiments with a carboxyl ester lipase knock-out mouse model indicated that the luminal hydrolysis of retinyl esters is catalyzed by pancreatic triglyceride lipase [3]. Rigtrup et al identified an enzyme with alkaline retinyl ester hydrolase activity in intestinal brush border membranes that is thought to be phospholipase B [4, 5]. Liberated retinol is subsequently absorbed by enterocytes, reesterified with long-chain fatty acids primarily by lecithin:retinol acyltransferase, incorporated into chylomicrons and secreted into the lymphatic system
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