A continuous fluorescence assay for lecithin cholesterol acyltransferase

Bonelli, Frank S.; Kézdy, Katherine E.; Jonas, Ana

doi:10.1016/0003-2697(87)90564-1

Cited by 7 publications

(8 citation statements)

References 14 publications

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“…68,69 The reaction mixture contained 50 μL crude enzyme extract, 10 μL PTAE (final concentrations: 0, 0.01, 0.02, 0.76, 1.14 and 1.52 mg mL −1 ), 100 μL reaction liquid, 100 μL PNPB and 790 μL buffer. Esterase activity was determined by the hydrolysis of a watersoluble substrate, ρ-nitrophenyl butyrate (PNPB), as previously described.…”

Section: Lcat Activitymentioning

confidence: 99%

Effects of Pu-erh tea aqueous extract (PTAE) on blood lipid metabolism enzymes

et al. 2015

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Disorders of blood lipid metabolism are the primary risk factors for many diseases. Recently, the effect of Pu-erh tea on blood lipid metabolism has received increasing attention. However, the mechanism underlying its ability to regulate blood lipid metabolism is unclear. We set out to study this through assessing the effects of Pu-erh tea aqueous extract (PTAE) on the central enzymes of blood lipid metabolism, including lipoprotein-associated phospholipase A2 (Lp-PLA2), lecithin: cholesterol acyltransferase (LCAT), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and pancreatic lipase (PL). We find that the Lp-PLA2, HMRG and PL activities are inhibited by PTAE in a dose-dependent manner and that the LCAT activity tends to increase with increasing PTAE concentrations. Lineweaver-Burk plot analyses reveal that PTAE acts as a competitive inhibitor for HMGR and PL and as a noncompetitive inhibitor for Lp-PLA2. Moreover, we determine that its active ingredients include catechins, gallic acid, caffeine, free amino acids, and soluble sugar. However, the effect of each ingredient and whether any of them have synergistic effects are still unknown. The results suggest that Pu-erh tea has a potent ability to regulate blood lipid metabolism and knowledge of the mechanisms provides insights into its potential therapeutic application as an alternative hypolipidemic drug.

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Section: Lcat Activitymentioning

confidence: 99%

Effects of Pu-erh tea aqueous extract (PTAE) on blood lipid metabolism enzymes

et al. 2015

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“…The amount of cholesteryl esters generated during the reaction was quantified by HPLC (Vercaemst et al, 1989;Vanloo et al, 1992). The phospholipase activity on a monomeric substrate was assayed using 1 ,2-bis-(l-pyrene-butanoyl)-sn-glycero-3-phosphocholine (Bonelli &Jonas, 1992). LCAT mass was assayed by solid-phase enzyme immunoassay using chicken antibodies specific to LCAT, and purified human LCAT as a standard, as follows: 100 pL culture medium was coated onto titer plates for 3 h at 37 "C and overnight at 4°C.…”

Section: Lcat Activity and Mass Measurementsmentioning

confidence: 99%

A proposed architecture for lecithin cholesterol acyl transferase (LCAT): Identification of the catalytic triad and molecular modeling

et al. 1998

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The enzyme cholesterol lecithin acyl transferase (LCAT) shares the Ser/Asp‐Glu/His triad with lipases, esterases and proteases, but the low level of sequence homology between LCAT and these enzymes did not allow for the LCAT fold to be identified yet. We, therefore, relied upon structural homology calculations using threading methods based on alignment of the sequence against a library of solved three‐dimensional protein structures, for prediction of the LCAT fold. We propose that LCAT, like lipases, belongs to the α/β hydrolase fold family, and that the central domain of LCAT consists of seven conserved parallel beta‐strands connected by four α‐helices and separated by loops. We used the conserved features of this protein fold for the prediction of functional domains in LCAT, and carried out site‐directed mutagenesis for the localization of the active site residues. The wild‐type enzyme and mutants were expressed in Cos‐1 cells. LCAT mass was measured by ELISA, and enzymatic activity was measured on recombinant HDL, on LDL and on a monomelic substrate. We identified D345 and H377 as the catalytic residues of LCAT, together with F103 and L182 as the oxyanion hole residues. In analogy with lipases, we further propose that a potential “lid” domain at residues 50‐74 of LCAT might be involved in the enzyme‐substrate interaction. Molecular modeling of human LCAT was carried out using human pancreatic and Candida antarctica lipases as templates. The three‐dimensional model proposed here is compatible with the position of natural mutants for either LCAT deficiency or Fish‐eye disease. It enables moreover prediction of the LCAT domains involved in the interaction with the phospholipid and cholesterol substrates.

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“…One type employs a water soluble, fluorescently-labeled phosphatidylcholine derivative which serves to detect the phospholipase activity of LCAT [20]. The assay is uncomplicated and the fluorescence-based analysis provides much greater sensitivity than the colorimetric assays.…”

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confidence: 99%

A fluorescence method to detect and quantitate sterol esterification by lecithin:cholesterol acyltransferase

Homan

Esmaeil

Mendelsohn

et al. 2013

Analytical Biochemistry

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We describe a simple but sensitive fluorescence method to accurately detect the esterification activity of lecithin:cholesterol acyltransferase (LCAT). The new assay protocol employs a convenient mix, incubate and measure scheme. This is possible by using the fluorescent sterol, dehydroergosterol (DHE) in place of cholesterol as the LCAT substrate. The assay method is further enhanced by incorporation of an amphiphilic peptide in place of apolipoprotein A-I as the lipid emulsifier and LCAT activator. Specific fluorescence detection of DHE ester synthesis is achieved by employing cholesterol oxidase to selectively render unesterified DHE non-fluorescent. The assay accurately detects LCAT activity in buffer and in plasma that is depleted of apolipoprotein B lipoproteins by selective precipitation. Analysis of LCAT activity in plasmas from control subjects and sickle cell disease (SCD) patients confirms previous reports of reduced LCAT activity in SCD and demonstrates a strong correlation between plasma LCAT activity and LCAT content. The fluorescent assay combines the sensitivity of radiochemical assays with the simplicity of non-radiochemical assays to obtain accurate and robust measurement of LCAT esterification activity.

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A continuous fluorescence assay for lecithin cholesterol acyltransferase

Cited by 7 publications

References 14 publications

Effects of Pu-erh tea aqueous extract (PTAE) on blood lipid metabolism enzymes

Effects of Pu-erh tea aqueous extract (PTAE) on blood lipid metabolism enzymes

A proposed architecture for lecithin cholesterol acyl transferase (LCAT): Identification of the catalytic triad and molecular modeling

A fluorescence method to detect and quantitate sterol esterification by lecithin:cholesterol acyltransferase

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