Fatty Acid Ethyl and Methyl Ester Synthases, and Fatty Acid Anilide Synthase in HepG2 and AR42J Cells: Interrelationships and Inhibition by Tri-o-tolyl Phosphate

Kaphalia, Bhupendra S.; Green, Steven M.; Ansari, Ghulam

doi:10.1006/taap.1999.8724

Cited by 22 publications

(19 citation statements)

References 43 publications

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“…Therefore, esterase activity can be used to monitor the purification of esterases in general, including ChE. Among ethanol, methanol, and aniline, methanol was found to be a better substrate as previously reported by us [6,12]. Structural properties of the rat pancreatic FAEE synthase, purified in this study, suggest that the purified protein is identical to pancreatic ChE.…”

Section: Discussionmentioning

confidence: 49%

“…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. Therefore, in this study, we purified and characterized rat pancreatic microsomal FAEE synthase, and determined its relationship with pancreatic ChE.…”

Section: Introductionmentioning

confidence: 88%

“…Purification of rat pancreatic FAEE synthase was done according to the method of Jacobson et al [23], a scheme used for the purification of ChE with modifications. FAEE synthase activity was monitored using ethanol, methanol, or aniline in the pooled fractions at each step of purification, while esterase activity was measured using PNPA as substrate to identify the active fractions during various column and gel filtration chromatography as our previous studies indicate that hepatic and pancreatic FAEE synthase also express PNPA-hydrolyzing activity [5][6][7].…”

Section: Purification Of Pancreatic Faee Synthasementioning

confidence: 99%

“…Esterase activity was determined according to Erlanson [24], using PNPA as substrate. FAEE synthase activity was measured using [1-14 C]oleic acid and ethanol, methanol, or aniline as substrate as described earlier by us [5,6,16].…”

Section: Ammonium Sulfate Precipitationmentioning

confidence: 99%

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

Kaphalia

Ansari

2003

J Biochem & Molecular Tox

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Formation of fatty acid ethyl esters (FAEEs, catalyzed by FAEE synthase) has been implicated in the pathogenesis of chronic pancreatitis. In previous studies, we demonstrated that FAEE synthase, purified from rat liver microsomes, is identical to rat liver carboxylesterase (pI 6.1), and structurally and functionally different than that from pancreas. In this study, we purified and characterized rat pancreatic microsomal FAEE synthase, and determined its relationship with rat pancreatic cholesterol esterase (ChE). Since most of the serine esterases express p-nitrophenyl acetate (PNPA)-hydrolyzing activity as well as synthetic activity to form fatty acid esters or amides with a wide spectrum of alcohols and amines, respectively, we used PNPA-hydrolyzing activity to monitor the purification of FAEE synthase during various chromatographic purification steps. Synthesizing activity towards FAEEs, fatty acid methyl esters, and fatty acid anilides was measured only in the pooled fractions. At each step of purification (ammonium sulfate saturation, Q Sepharose XL, and heparin-agarose column chromatographies, and high performance liquid chromatography (anion exchange and gel filtration)) synthetic as well as hydrolytic activities copurified. Using ethanol, methanol, or aniline as substrates, the ester or anilide synthesizing activity of the purified protein was found to be 8709, 13000, and 2201 nmol/h/mg protein, respectively. The purified protein displayed a single band with an estimated molecular mass of approximately 68 kD upon SDS-PAGE under reduced denaturing conditions, cross-reacted with antisera against rat pancreatic ChE and showed 100% N-terminal sequence homology of the first 15 amino acids to that of rat pancreatic ChE. These results suggest that the purified protein has broad substrate specificity towards the conjugation of endogenous long chain fatty acids with substrates having hydroxyl and amino groups and is identical to ChE.

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Section: Discussionmentioning

confidence: 49%

Section: Introductionmentioning

confidence: 88%

Section: Purification Of Pancreatic Faee Synthasementioning

confidence: 99%

Section: Ammonium Sulfate Precipitationmentioning

confidence: 99%

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

Kaphalia

Ansari

2003

J Biochem & Molecular Tox

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“…The nonoxidative metabolism of ethanol results from esterification of fatty acid and ethanol, with the production of fatty acid ethyl esters (FAEE) (Dan et al 1998;Kaphalia et al 1999;Laposata 1999). This reaction is catalyzed by the enzyme FAEE synthase, which is present in organs commonly damaged by ethanol abuse, including liver and pancreas (Doyle et al 1994;Gorski et al 1996;Wilson and Apte 2003).…”

mentioning

confidence: 97%

Role of undecan-2-one on ethanol-induced apoptosis in HepG2 cells

et al. 2007

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Based on the reduced expression of ethanol-oxidizing enzymes in human hepatocellular carcinoma (HepG2) cells, we analyzed the role of nonoxidative metabolites in ethanol-induced apoptosis in HepG2 cells. For this purpose, an analysis of volatile metabolites of ethanol using ion-mobility spectrometry and gas chromatography-mass spectrometry was performed. HepG2 cells exposed to 1 mmol/L ethanol exhibited significant synthesis of undecan-2-one compared to untreated cells. Undecan-2-one is a fatty acid ethyl ester metabolite synthesized through a nonoxidative pathway. Undecan-2-one had a dose-dependent cytotoxic effect on HepG2 cells as shown by release of lactate dehydrogenase (LDH). The most notable finding of this study was the potentiation of ethanol-induced apoptosis demonstrated by an increased apoptotic rate induced by undecan-2-one in ethanol-treated HepG2 cells. The data presented in this study contribute to the better understanding of the molecular mechanisms of ethanol exposure at low concentration in HepG2 cells, a human hepatocellular carcinoma-derived cell line.

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

Kaphalia

Ansari

2001

J Biochem & Molecular Tox

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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.

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Fatty Acid Ethyl and Methyl Ester Synthases, and Fatty Acid Anilide Synthase in HepG2 and AR42J Cells: Interrelationships and Inhibition by Tri-o-tolyl Phosphate

Cited by 22 publications

References 43 publications

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

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

Role of undecan-2-one on ethanol-induced apoptosis in HepG2 cells

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

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