Isolation of two forms of carboxylester lipase (cholesterol esterase) from porcine pancreas

Rudd, Edwin A.; Mizuno, Nancy K.; Brockman, Howard L.

doi:10.1016/0005-2760(87)90184-6

Cited by 60 publications

(37 citation statements)

References 15 publications

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“…The purified protein is different from hepatic CE as pancreatic ChE does not cross-react with antibodies to rat hepatic or adipose tissue FAEE synthase [5]. The calculated molecular weight of the purified protein by gel-filtration HPLC showed that the protein could be a dimer as reported by Rudd et al [36].…”

Section: Discussionmentioning

confidence: 68%

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: 68%

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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“…One cholesteryl ester hydrolytic enzyme synthesized by human macrophages is identical to the bile salt-stimulated cholesterol esterase, previously thought to be synthesized only in pancreas and in lactating mammary glands (35). This conclusion was based on results of several studies.…”

Section: Expression Of Cholesteryl Ester Hydrolytic Enzymes In Mousementioning

confidence: 89%

Modified Low Density Lipoprotein Enhances the Secretion of Bile Salt-stimulated Cholesterol Esterase by Human Monocyte-Macrophages

Hui

1997

Journal of Biological Chemistry

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Reverse transcriptase-polymerase chain reaction was used to study the biosynthesis of two different cholesteryl ester hydrolases by human and mouse macrophages. Oligonucleotide primers for bile salt-stimulated cholesterol esterase yielded positive reactions with RNA isolated from human peripheral blood monocytes, monocyte-derived macrophages, the human monocytic THP-1 cells, and phorbol ester-induced THP-1 macrophages. In contrast, oligonucleotide primers for hormone-sensitive lipase yielded positive reactions only with RNA isolated from non-differentiated human THP-1 monocytic cells and peripheral blood monocytes, but not those obtained from differentiated THP-1 macrophages or monocyte-derived macrophages. Thus, while human monocytes were capable of synthesizing both enzymes, human macrophages synthesized only bile salt-stimulated cholesterol esterase and not the hormone-sensitive lipase. The synthesis of bile salt-stimulated cholesterol esterase by human macrophages was confirmed by detection of bile salt-stimulated cholesteryl ester hydrolytic activity in conditioned media of differentiated THP-1 cells and human peripheral blood monocyte-derived macrophages. Moreover, incubating human macrophages with oxidized low density lipoprotein (LDL) or acetylated LDL increased bile salt-stimulated cholesterol esterase activity in the conditioned media of these cells. These results with human macrophages were contrasted with results of studies with mouse macrophages, which showed the presence of hormone-sensitive lipase mRNA but not the bile salt-stimulated cholesterol esterase mRNA. Taken together, these results demonstrated species-specific differences in expression of cholesteryl ester hydrolytic enzymes in macrophages. The expression of bile salt-stimulated cholesterol esterase by human macrophages, in a process inducible by modified LDL, suggests a role of this protein in atherogenesis.A major characteristic of atherosclerosis is the accumulation of lipid-laden foam cells in the arterial wall. The foam cells are derived mainly from macrophages that have endocytosed modified lipoproteins through the scavenger receptor-mediated pathways (1). The cytoplasmic lipid droplets are mostly cholesteryl esters, which exist in a dynamic equilibrium with unesterified cholesterol and undergo continues hydrolysis and re-esterification (1). In the presence of exogenous cholesterol acceptors such as high density lipoproteins, the equilibrium favors cholesteryl ester hydrolysis with a net efflux of cholesterol from cells. In such circumstances, very little amount of cholesterol is stored in the macrophages. However, in the absence of extracellular cholesterol acceptors, or when acceptor concentration is low, equilibrium of the cholesteryl ester cycle favors esterification and cholesteryl esters accumulate in the cytosol.Cholesteryl ester can also accumulate in macrophages due to different levels of the esterification and de-esterification enzymes. Macrophages with high neutral cholesteryl ester hydrolytic activity were shown to accu...

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“…[1 ␣ ,2 ␣ ( n )-3 H]cholesteryl oleate (specific activity of 49.6 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). Porcine pancreatic CEL was purified as reported previously (27). Reagents for lipoprotein electrophoresis were obtained from Beckman (Fullerton, CA).…”

Section: Introductionmentioning

confidence: 99%

Pancreatic carboxyl ester lipase: a circulating enzyme that modifies normal and oxidized lipoproteins in vitro.

Shamir¹,

Johnson²,

Morlock-Fitzpatrick³

et al. 1996

J. Clin. Invest.

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Pancreatic carboxyl ester lipase (CEL) hydrolyzes cholesteryl esters (CE), triglycerides (TG), and lysophospholipids, with CE and TG hydrolysis stimulated by cholate. Originally thought to be confined to the gastrointestinal system, CEL has been reported in the plasma of humans and other mammals, implying its potential in vivo to modify lipids associated with LDL, HDL (CE, TG), and oxidized LDL (lysophosphatidylcholine, lysoPC). We measured the concentration of CEL in human plasma as 1.2 Ϯ 0.5 ng/ml (in the range reported for lipoprotein lipase). Human LDL and HDL 3 reconstituted with radiolabeled lipids were incubated with purified porcine CEL without or with cholate (10 or 100 M, concentrations achievable in systemic or portal plasma, respectively). Using a saturating concentration of lipoprotein-associated CE (4 M), with increasing cholate concentration there was an increase in the hydrolysis of LDL-and HDL 3 -CE; at 100 M cholate, the percent hydrolysis per hour was 32 Ϯ 2 and 1.6 Ϯ 0.1, respectively, indicating that CEL interaction varied with lipoprotein class. HDL 3 -TG hydrolysis was also observed, but was only ‫ف‬ 5-10% of that for HDL 3 -CE at either 10 or 100 M cholate. Oxidized LDL (OxLDL) is enriched with lysoPC, a proatherogenic compound. After a 4-h incubation with CEL, the lysoPC content of OxLDL was depleted 57%. Colocalization of CEL in the vicinity of OxLDL formation was supported by demonstrating in human aortic homogenate a cholate-stimulated cholesteryl ester hydrolytic activity inhibited by anti-human CEL IgG. We conclude that CEL has the capability to modify normal human LDL and HDL composition and structure and to reduce the atherogenicity of OxLDL by decreasing its lysoPC content.

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Isolation of two forms of carboxylester lipase (cholesterol esterase) from porcine pancreas

Cited by 60 publications

References 15 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

Modified Low Density Lipoprotein Enhances the Secretion of Bile Salt-stimulated Cholesterol Esterase by Human Monocyte-Macrophages

Pancreatic carboxyl ester lipase: a circulating enzyme that modifies normal and oxidized lipoproteins in vitro.

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