Purification and molecular cloning of porcine intestinal glycerol‐ester hydrolase

David, Laetitia; Guo, Xin; Villard, Claude; Moulin, A.; Puigserver, Antoine

doi:10.1046/j.1432-1327.1998.2570142.x

Cited by 24 publications

(32 citation statements)

References 25 publications

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“…[1] So far, PLE can only be obtained by the extraction of pig liver with organic solvents and, as a consequence, an illdefined preparation is provided. [6] This hydrolase was found to share as much as 97 % amino acid sequence identity with that reported by Matsushima et al [4] We now describe for the first time the cloning and functional expression of a PLE subunit ± -which represents the major esterase in commercial PLE preparationsÐin the methylotrophic yeast Pichia pastoris. [2] Some fractions separated by isoelectric focusing preferred methyl butyrate or butanilicain (N-butylglycyl-2-chloro-6-methylanilide hydrochloride), whereas others hydrolyzed butyrylcholine and proline-bnaphthylamide with high activity.…”

Section: Introductionsupporting

confidence: 58%

“…As already outlined in the introduction, two groups reported the cloning of putative pig liver [4] or intestinal [6,12] esterase genes, but they did not report the functional expression of active enzymes. Initially, we also failed in functional expression of PLE in either E. coli or P. pastoris, despite the use of various plasmid constructs and replacement of the 18 amino acid N-terminal leader sequence of PLE with the ompA signal sequence or the afactor leader sequence, respectively.…”

Section: Discussionmentioning

confidence: 99%

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Cloning, Functional Expression, and Characterization of Recombinant Pig Liver Esterase

et al. 2001

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The N-terminal amino acid sequence of pig liver esterase (PLE) from a commercial sample was determined and shown to match closely to a published sequence encoding a proline-beta-naphthylamidase from pig liver. Next, mRNA isolated from pig liver was transcribed into cDNA and primers deduced from the N-terminal sequence were used to clone the 1698 base pairs of PLE cDNA. Initial attempts to express the cDNA in Escherichia coli and Pichia pastoris with different expression vectors and secretion signal sequences failed. Only after deletion of the putative C-terminal sequence His-Ala-Glu-Leu, usually considered as an endoplasmic reticulum retention signal, could heterologous expression of PLE be readily achieved in the methylotrophic yeast P. pastoris. Recombinant PLE (rPLE) was secreted into the medium and exhibited a specific activity of approximately 600 Umg(-1) and a Vmax/Km value of 139 micromolmin(-1)mM(-1) with p-nitrophenyl acetate as a substrate. Activity staining of renatured sodium dodecylsulfate-polyacrylamide gels gave a single band with esterolytic activity for rPLE, whereas several bands are visible in crude commercial PLE preparations. This was confirmed by native gels, which also show that rPLE is active as a trimer. Biochemical characterization of the recombinant enzyme and comparison with properties of commercial PLE preparations as well as with published data confirmed that we expressed a single PLE isoenzyme which showed a high preference for proline-beta-naphthylamide. This is a substrate specificity for the so-called gamma subunit of PLE. The optimum pH value and temperature for the recombinant PLE were 8.0 and 60 degrees C, respectively. The determined molecular weight of the secreted enzyme was approximately 61-62 kDa, which closely matches the calculated value of 62.419 kDa. The active site residues are located at Ser203, His448, and Asp97, and the typical consensus sequence motif for hydrolases was found around the active site serine (Gly-Glu-Ser-Ala-Gly).

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Section: Introductionsupporting

confidence: 58%

Section: Discussionmentioning

confidence: 99%

Cloning, Functional Expression, and Characterization of Recombinant Pig Liver Esterase

et al. 2001

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“…Although these enzymes are yet to be characterized, various esterases with sequence identities to PMPMEase in excess of 70% have been reported from the intestines [29], human brain [30], human alveolar macrophages [31], human monocytes and macrophages [31, 32]. …”

Section: Discussionmentioning

confidence: 99%

Porcine Liver Carboxylesterase Requires Polyisoprenylation for High Affinity Binding to Cysteinyl Substrates

Lamango

Duverna²,

Zhang³

et al. 2009

TOEIJ

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Abstract:The polyisoprenylation pathway enzymes have been the focus of numerous studies to better understand the roles of polyisoprenylated proteins in eukaryotic cells and to identify novel targets against diseases such as cancer. The final step of the pathway is a reversible reaction catalyzed by isoprenyl carboxylmethyl transferase (icmt) whose products are then hydrolyzed by polyisoprenylated methylated protein methyl esterase (PMPMEase). Unlike the other pathway enzymes, the esterase has received little attention. We recently purified PMPMEase from porcine liver using an S-polyisoprenylated cysteine methyl ester substrate-dependent screening assay. However, no data is available showing its relative interaction with structurally diverse substrates. As such, its role as the putative endogenous PMPMEase has not been demonstrated. A series of substrates with S-alkyl substituents ranging from 2 to 20 carbons, including the two moieties found in polyisoprenylated proteins, were synthesized. Enzyme kinetics analysis revealed a 33-fold increase in affinity (K M values) from ethyl-(C-2, 505±63 μM), prenyl-(C-5, 294±25 μM), trans-geranyl-(C-10, 87±12 μM), trans, trans-farnesyl-(C-15, 29±2.2 μM) to all trans-geranylgeranyl-(C-20-, 15±2.7 μM) based analogs. Comparative molecular field analysis of the data yielded a cross-validated q 2 of 0.863±0.365 and a final R 2 of 0.995. Since the substrates with the S-trans, trans-farnesyl and S-all trans-geranylgeranyl moieties that occur in proteins show the highest affinity towards PMPMEase and are not hydrolyzed by the cholinesterases, the results suggest that polyisoprenylated proteins are the endogenous substrates of this esterase. The results suggest design strategies for high affinity and selective inhibitors of PMPMEase.

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“…Two carboxylesterases have been identified in the pig, and they have been classified as CES1 and CES (Satoh and Hosokawa, 2006). David et al (1998) reported a pig intestinal carboxylesterase that showed high similarity with that of rat and human intestinal carboxylesterases. Clark et al (1993) and Hewitt et al (2000) showed that esters applied to the skin surface were hydrolyzed during dermal absorption using skin in vitro in a diffusion cell.…”

mentioning

confidence: 99%

Specificity of Procaine and Ester Hydrolysis by Human, Minipig, and Rat Skin and Liver

Jewell

Ackermann²,

Payne³

et al. 2007

Drug Metab Dispos

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ABSTRACT:The capacity of human, minipig, and rat skin and liver subcellular fractions to hydrolyze the anesthetic ester procaine was compared with carboxylesterase substrates 4-methylumbelliferyl-acetate, phenylvalerate, and para-nitrophenylacetate and the arylesterase substrate phenylacetate. Rates of procaine hydrolysis by minipig and human skin microsomal and cytosolic fractions were similar, with rat displaying higher activity. Loperamide inhibited procaine hydrolysis by human skin, suggesting involvement of human carboxylesterase hCE2. The esterase activity and inhibition profiles in the skin were similar for minipig and human, whereas rat had a higher capacity to metabolize esters and a different inhibition profile. Minipig and human liver and skin esterase activity was inhibited principally by paraoxon and bis-nitrophenyl phosphate, classical carboxylesterase inhibitors. Rat skin and liver esterase activity was inhibited additionally by phenylmethylsulfonyl fluoride and the arylesterase inhibitor mercuric chloride, indicating a different esterase profile. These results have highlighted the potential of skin to hydrolyze procaine following topical application, which possibly limits its pharmacological effect. Skin from minipig used as an animal model for assessing transdermal drug preparations had similar capacity to hydrolyze esters to human skin.

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Purification and molecular cloning of porcine intestinal glycerol‐ester hydrolase

Cited by 24 publications

References 25 publications

Cloning, Functional Expression, and Characterization of Recombinant Pig Liver Esterase

Cloning, Functional Expression, and Characterization of Recombinant Pig Liver Esterase

Porcine Liver Carboxylesterase Requires Polyisoprenylation for High Affinity Binding to Cysteinyl Substrates

Specificity of Procaine and Ester Hydrolysis by Human, Minipig, and Rat Skin and Liver

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