Purification and characterization of rat hepatic microsomal low molecular weight fatty acid ethyl ester synthase and its relationship to carboxylesterases

Abstract

We reported purification of a high molecular weight (HMW) (ca. 180 kD) and a low molecular weight (LMW) (ca. 60 kD) protein fractions from digitonized rat liver microsomes using ammonium sulfate precipitation followed by ion exchange and gel filtration column chromatography. Both fractions expressed fatty acid ethyl ester (FAEE) synthase as well as p-nitrophenyl acetate (PNPA)-hydrolyzing (esterase) activities. The HMW fraction was found to be a trimer with subunit molecular weight ca. 60 kD and structurally and functionally similar to rat hepatic microsomal carboxylesterase (CE, pI 6.1) and adipose tissue FAEE synthase. In this article, we report further purification and characterization of the LMW (minor) fraction expressing FAEE synthase activity and its structural and functional relationship to hepatic microsomal CEs. Using isoelectric focusing (IEF) followed by gel filtration-high-performance liquid chromatography (GF-HPLC), five proteins were purified, which expressed FAEE synthase as well as PNPA-hydrolyzing activity. The isoelectric point values of 6.5, 5.8, 5.6, 5.3, and 5.0 were found for the purified LMW proteins by IEF and each showed a peak corresponding to ca. 60 kD molecular weight by GF-HPLC, which expressed FAEE synthase as well as PNPA-hydrolyzing activity. Sodium dodecyl sulfate-polyacrylamide gel elecrophoresis (SDS-PAGE) analysis of the GF-HPLC purified LMW proteins revealed that these proteins are monomers (ca. 60 kD). All the purified LMW proteins cross-reacted with antibodies to rat adipose tissue FAEE synthase. Coelution of PNPA-hydrolyzing and FAEE synthase activity at each step of purification and cross-reactivity with rat adipose tissue FAEE synthase antibodies suggest that the purified proteins are related to various hepatic microsomal CEs. This conclusion is further supported by the homology of N-terminal amino acid sequence of the purified LMW proteins to various hepatic microsomal CEs and protease precursors. Therefore, LMW FAEE synthase activity most probably is expressed by various isozymes of hepatic microsomal CEs, which are also involved in the biotransformation of xenobiotic alcohols and amines.

Purification and characterization of rat pancreatic fatty acid ethyl ester synthase and its structural and functional relationship to pancreatic cholesterol esterase

“…Similarly, other rat liver microsomal low molecular weight FAEE synthase were also found to have close structural and functional relationships to rat hepatic microsomal CEs [7]. Our recent studies on differential inhibition of FAEE synthase activity using trio-tolyl phosphate (␤-esterase inhibitor) in HepG2 and AR42J cells [6] and in vivo in rats [12] indicate that hepatic and pancreatic FAEE synthase are different.…”

“…In the past 20 years, FAEE synthase has been purified and characterized from human and animal tissues, and it has been suggested that FAEE synthase is structurally and functionally different in the liver and pancreas [1,5,12]. It has also been shown that FAEE synthase is involved in the synthesis of fatty acid esters with other alcohols and amines [1,[4][5][6][7]16]. Surprisingly, pancreatic FAEE synthase activity is up-regulated following chronic ethanol exposure [17].…”

“…Such activity is found in almost all the mammalian organs and tissues with the highest being in the pancreas [1][2][3][4][5][6][7]9,12].…”

Altered hepatic lipid metabolism in C57BL/6 mice fed alcohol: a targeted lipidomic and gene expression study

“…FAEEs have also been associated with disease development in the alcoholic liver, heart, and pancreas ( 36,38,39 ). The molecular identities of enzymes that synthesize and degrade FAEEs are currently not established ( 40,41 ). It is thought that multiple hepatic microsomal carboxyesterases (CES) are able to catalyze the synthesis of FAEE; but because of this uncertainty, we did not exhaustively study expression of CES that might be involved in FAEE synthesis ( 41 ).…”

“…The molecular identities of enzymes that synthesize and degrade FAEEs are currently not established ( 40,41 ). It is thought that multiple hepatic microsomal carboxyesterases (CES) are able to catalyze the synthesis of FAEE; but because of this uncertainty, we did not exhaustively study expression of CES that might be involved in FAEE synthesis ( 41 ). Our results, however, identify ethyl oleate (C18:1) as the most abundant FAEE in the plasma and liver of alcohol-fed mice.…”