Pseudo-esterase activity of human albumin - Slow turnover on tyrosine 411 and stable acetylation of 82 residues including 59 lysines
Eppley Institute, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA. Journal of Biological Chemistry
(Impact Factor: 4.57).
07/2008; 283(33):22582-90. DOI: 10.1074/jbc.M802555200
Human albumin is thought to hydrolyze esters because multiple equivalents of product are formed for each equivalent of albumin. Esterase activity with p-nitrophenyl acetate has been attributed to turnover at tyrosine 411. However, p-nitrophenyl acetate creates multiple, stable, acetylated adducts, a property contrary to turnover. Our goal was to identify residues that become acetylated by p-nitrophenyl acetate and determine the relationship between stable adduct formation and turnover. Fatty acid-free human albumin was treated with 0.5 mm p-nitrophenyl acetate for 5 min to 2 weeks, or with 10 mm p-nitrophenyl acetate for 48 h to 2 weeks. Aliquots were digested with pepsin, trypsin, or GluC and analyzed by mass spectrometry to identify labeled residues. Only Tyr-411 was acetylated within the first 5 min of reaction with 0.5 mm p-nitrophenyl acetate. After 0.5-6 h there was partial acetylation of 16-17 residues including Asp-1, Lys-4, Lys-12, Tyr-411, Lys-413, and Lys-414. Treatment with 10 mm p-nitrophenyl acetate resulted in acetylation of 59 lysines, 10 serines, 8 threonines, 4 tyrosines, and Asp-1. When Tyr-411 was blocked with diisopropylfluorophosphate or chlorpyrifos oxon, albumin had normal esterase activity with beta-naphthyl acetate as visualized on a nondenaturing gel. However, after 82 residues had been acetylated, esterase activity was almost completely inhibited. The half-life for deacetylation of Tyr-411 at pH 8.0, 22 degrees C was 61 +/- 4 h. Acetylated lysines formed adducts that were even more stable. In conclusion, the pseudo-esterase activity of albumin is the result of irreversible acetylation of 82 residues and is not the result of turnover.
Available from: Jean-François Cavalier
- "As a typical example of this, p-nitrophenyl acyl esters (pNP esters) are widely used as " lipase " substrates because of their high detection sensitivity [20e22], but they are not hydrolysed by many true lipases. Moreover, pseudoenzyme activity has been recorded with these substrates and non-enzymatic proteins like serum albumin  . "
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ABSTRACT: Lipases play various roles in fat digestion, lipoprotein metabolism, and in the mobilization of fat stored in lipid bodies in animals, plants and microorganisms. In association with these physiological functions, there is an important field of research for discovering lipase inhibitors and developing new treatments of diseases such as obesity, atherosclerosis, diabetes and tuberculosis. In this context, the development of convenient, specific and sensitive analytical methods for the detection and assay of lipases and/or lipase inhibitors is of major importance. It is shown here that purified triacylglycerols (TAGs) from Punica granatum (Pomegranate) seed oil coated on microtiter plates can be used for the continuous assay of lipase activity by recording the variations with time of the UV absorption spectra at 275 nm. UV absorption is due the release of punicic acid (9Z,11E,13Z-octadeca-9,11,13-trienoic acid), a conjugated triene contained in Pomegranate oil. This new microtiter plate assay allows to accurately measure the activity of a wider range of lipases compared to the similar assay previously developed with Tung oil containing α-eleostearic acid (9Z,11E,13E-octadeca-9,11,13-trienoic acid), including the LipY lipase from Mycobacterium tuberculosis. Although punicic acid is a diastereoisomer of α-eleostearic acid, the Δ(13)cis double bound found in punicic acid gives a different structure to the acyl chain that probably favours the interaction of Pomegranate TAGs with the lipase active site. The microplate lipase assay using Pomegranate TAGs shows high sensitivity, reproducibility and remarkable relevance for the high-speed screening of lipases and/or lipase inhibitors directly from raw culture media without any purification step.
Biochimie 09/2015; DOI:10.1016/j.biochi.2015.09.004 · 2.96 Impact Factor
Available from: sciencedirect.com
- "This esterase-like activity of sera albumin has been endorsed to the presence of arginine and tyrosine residues at 410 and 411 positions (Watanabe et al., 2000). This has been confirmed that esteraselike activity of sera albumin is inhibited by various drugs and actually the acetylation of lysine residues results in the inhibition of esterase-like activity of sera albumin (Lockridge et al., 2008). "
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ABSTRACT: Albumin is a globular and un-glycosylated multifunctional plasma protein and thus correlated with several human diseases. Owing to esterase contamination, albumin levels are usually misleading. In this study, we propose methodical accuracy for albumin estimation taking healthy and fibrotic rats. Liver fibrosis in rats was generated by N′-Nitrosodimethylamine (NDMA) (10 mg/kg body weight) within three weeks followed by its confirmation through H&E and immunohistochemical staining for α-SMA expression. Animal sera were screened by native polyacrylamide gel electrophoresis (native-PAGE) (7.5%). In-gel esterase-like albumin activity was detected using α- and β-naphthyl acetate (5.58 × 10−3 mM; pH 7.5) as substrate. Sera albumin was purified from unstained PA gel-slices through electroelution. Subsequent to conformation of albumin purity by its molecular weight determination using SDS–PAGE (10%) and peptide mass fingerprinting by MALDI-TOF-MS, samples were treated with different concentrations of urea. Urea-treated albumins were screened for esterase activity, conformational change and, albumin levels by immunoblotting. Our results demonstrate that esterase-like albumin activity in rat sera albumin is located in domain-III. The esterase-like activity remains detectable up to 4 M urea, which diminishes with increasing urea concentrations. Further, immunoblotting of urea-treated albumin samples displays a significant decline in purified protein bands, indicating hypoalbuminemia during hepatic fibrosis in rats. In conclusion, the present approach of albumin separation and estimation is of potential interest and may be recommended for diagnostic purposes.
Arabian Journal of Chemistry 10/2014; 1. DOI:10.1016/j.arabjc.2014.10.016 · 3.73 Impact Factor
Available from: Yasaman Delaviz
- "The substrate combines rapidly and reversibly to Tyr-411 and Arg-410, and the hydroxyl group of Ser-489 and the aromatic residue of Phe-488 help to stabilize the albumin-substrate intermediate. Modification of those sites or changes within the microenvironments of those sites will change the esterase activity of albumin    . In addition , seven fatty acid binding sites are distributed asymmetrically across all of the above three domains of human serum albumin, and three of them overlap with the two primary drug-binding sites, IIA and IIIA. "
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The ester linkages contained within dental resin monomers (such as Bisphenol A-glycidylmethacrylate (BisGMA) and triethylene glycol dimethacrylate (TEGDMA)) are susceptible to hydrolytic degradation by salivary esterases, however very little is known about the specific esterase activities implicated in this process. The objective of this work was to isolate and identify the dominant proteins from saliva that are associated with the esterase activities shown to be involved in the degradation of BisGMA.
Human whole saliva was collected and processed prior to separation in a HiPrep 16/60 Sephacryl S-200 HR column. The fraction with the highest esterase activity was further separated by an anion exchange column (Mono-Q (10/100G)). Isolated fractions were then separated by gel electrophoresis, and compared to a common bench marker esterase, cholesterol esterase (CE), and commercial albumin which has been reported to express esterase activity. Proteins suspected of containing esterase activity were analyzed by Mass Spectroscopy (MS). Commercially available proteins, similar to the salivary esterase proteins identified by MS, were used to replicate the enzymatic complexes and confirm their degradation activity with respect to BisGMA.
MS data suggested that the enzyme fraction with the highest esterase activity was contained among a group of proteins consisting of albumin, Zn-α2-glycoprotein, α-amylase, TALDO1 protein, transferrin, lipocalin2, and prolactin-induced protein. Studies concluded that the main esterase bands on the gels in each fraction did not overlap with CE activity, and that albumin activity emerged as a lead candidate with significant esterase activity relative to BisGMA degradation, particularly when it formed a complex with Zn-α2-glycoprotein, under slightly basic conditions.
These enzyme complexes can be used as a physiologically relevant formulation to test the biostability of composite resins.
Dental materials: official publication of the Academy of Dental Materials 06/2014; 30(8). DOI:10.1016/j.dental.2014.05.031 · 3.77 Impact Factor
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