Identification of rat liver carboxylesterase isozymes (EC 3.1.1.1) using polyacrylamide gel electrophoresis and isoelectric focusing

Simon, Babette; Looze, Stephen de; Ronai, Adam; Deimling, Otto von

doi:10.1002/elps.1150061202

Cited by 18 publications

(8 citation statements)

References 27 publications

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“…This low abundance makes their purification challenging. Moreover, the identification of rat liver carboxylesterases based on pI values is difficult because of the complexity of their banding patterns and the variation in the reported values from one laboratory to another [30]. These carboxylesterase isoenzymes have overlapping substrate specificities so it is equally difficult to differentiate them by substrate specificity [28,16].…”

Section: Discussionmentioning

confidence: 99%

Identification of microsomal rat liver carboxylesterases and their activity with retinyl palmitate

Sanghani

Davis

Dumaual

et al. 2002

European Journal of Biochemistry

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Retinyl esters are a major endogenous storage source of vitamin A in vertebrates and their hydrolysis to retinol is a key step in the regulation of the supply of retinoids to all tissues. Some members of nonspecific carboxylesterase family (EC 3.1.1.1) have been shown to hydrolyze retinyl esters. However, the number of different isoenzymes that are expressed in the liver and their retinyl palmitate hydrolase activity is not known. Six different carboxylesterases were identified and purified from rat liver microsomal extracts. Each isoenzyme was identified by mass spectrometry of its tryptic peptides. In addition to previously characterized rat liver carboxylesterases ES10, ES4, ES3, the protein products for two cloned genes, AB010635 and D50580 (GenBank accession numbers), were also identified. The sixth isoenzyme was a novel carboxylesterase and its complete cDNA was cloned and sequenced (AY034877). Three isoenzymes, ES10, ES4 and ES3, account for more than 95% of rat liver microsomal carboxylesterase activity. They obey MichaelisMenten kinetics for hydrolysis of retinyl palmitate with K m values of about 1 lM and specific activities between 3 and 8 nmolAEmin )1 AEmg )1 protein. D50580 and AY034877 also hydrolyzed retinyl palmitate. Gene-specific oligonucleotide probing of multiple-tissue Northern blot indicates differential expression in various tissues. Multiple genes are highly expressed in liver and small intestine, important tissues for retinoid metabolism. The level of expression of any one of the six different carboxylesterase isoenzymes will regulate the metabolism of retinyl palmitate in specific rat cells and tissues.Keywords: retinyl palmitate hydrolase, carboxylesterase, mass spectrometry, rat, retinol, vitamin A.Vitamin A metabolism [1,2] is a significant area of research because of its diverse role in the regulation of gene expression through retinoic acid receptors. Dietary intake of vitamin A from animal food products is mainly in the form of retinyl esters and retinol, and from plant food products such as provitamin A or b-carotenes. Retinyl esters are converted to retinol in the intestine. After dietary uptake, retinol is converted to retinyl esters in intestinal mucosa and packaged into chylomicrons. These are partially processed during circulation to chylomicron remnants, which contain retinyl esters. Chylomicron remnants are rapidly cleared from circulation by liver hepatocytes where the retinyl esters are hydrolyzed by retinyl ester hydrolases to retinol. The retinol product can either undergo oxidation to retinoic acid for signaling or be secreted into circulation as a complex with retinol binding protein. After meeting the tissue needs, excess retinol is stored in hepatic stellate cells by conversion to retinyl esters (mostly as retinyl palmitate). The stored retinyl esters are the primary vitamin A reservoir in the body and can be mobilized by hydrolysis to retinol by retinyl ester hydrolases. Hence, retinyl ester hydrolases play very important roles in a variety of cells and tissues to...

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Section: Discussionmentioning

confidence: 99%

Identification of microsomal rat liver carboxylesterases and their activity with retinyl palmitate

Sanghani

Davis

Dumaual

et al. 2002

European Journal of Biochemistry

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“…To overcome these and other difficulties with the previous classification, a new but conventional nomenclature of Culex esterases was introduced by . This, in keeping with the rat and mouse esterase nomenclature (Zutphen, 1983(Zutphen, , 1987Bender et al,1984;Simon et al, 1985), refers to enzymes specific for αand β-naphthyl acetate as Estα and Estβ, respectively, with the genes italicised. Numbers for electrophoretically distinct enzymes are allocated historically, Culex tarsalis (Coquillett) CtaEstα1 Current A 3 (Prabhaker et al, 1987) Estβ Series Culex quinquefasciatus (Say) TEMR Estβ1 1 B 1 (Pasteur et al, 1981b;Raymond et al, 1987) MRES Estβ1 2 B 1 & B 8 (Bisset et al, 1995;Guillemaud et al, 1996) .…”

Section: Introductionmentioning

confidence: 97%

Mosquito carboxylesterases: a review of the molecular biology and biochemistry of a major insecticide resistance mechanism

Hemingway

Karunaratne

1998

Medical Vet Entomology

278

176

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The major mechanism of organophosphorus insecticide resistance in Culex mosquitoes involves the elevation of one or more esterases. The general mechanism underlying this resistance is the amplification of the structural genes. This review covers the classification of the mosquito esterases in the context of classical esterase nomenclature. The function of the amplified esterases and the structure of the amplified DNA on which they occur are also described. Implications of information on the esterase amplicons are discussed in relation to the evolution and migration of insecticide resistance in Culex.

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“…They are thus good model proteins to investigate the mechanism of protein retention in the ER lumen. Their primary structure is encoded in four different genes (Simon et al, 1985). They are glycosylated, some show structural microheterogeneity (Mentlein et al, 1980;Robbi & Beaufay, 1983;Simon et al, 1985), and all migrate in SDS/polyacrylamide gels as -60 kDa polypeptides.…”

Section: Introductionmentioning

confidence: 99%

Immunochemical characterization and biosynthesis of pI-6.4 esterase, a carboxylesterase of rat liver microsomal extracts

Robbi

Beaufay

1988

Biochemical Journal

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Rat liver pI-6.4 esterase was purified from microsomes (microsomal extracts) and used to generate antibodies in the rabbit. Two active enzyme forms, similarly sensitive to endo-H (endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96), but differing slightly in polypeptide chain length, were present in the preparation. In microsomes, immunoblots revealed a single form, with Mr congruent to 62,000, identical with the large component of the purified enzyme, indicating that the second component is an artefact. Rabbit reticulocyte lysates and wheat germ extracts programmed with RNA extracted from total or bound polysomes synthesized a single immunoreactive 61 kDa polypeptide, which was not formed with RNA extracted from free polysomes. The immunoreactive product synthesized in the presence of dog pancreas microsomes was slightly larger (62 kDa); like the authentic enzyme, it bound to concanavalin A and was decreased in molecular size to 60 kDa by the action of endo-H. Thus the enzyme is synthesized with a short cleavable sequence and bears at least one high-mannose oligosaccharide chain. Metabolic labelling in hepatocytes cultured with [35S]methionine also generated a single immunoreactive polypeptide of 62 kDa, which was decreased to 60 kDa in size by treatment with endo-H or addition of tunicamycin to the culture medium. This confirms the molecular homogeneity and the glycosylation of the enzyme in the intact cell. Culture media contained no pI-6.4-esterase-related protein, whether tunicamycin was present or not. The processing steps in the synthesis of pI-6.4 esterase are thus, as for other esterases of the endoplasmic reticulum [Robbi & Beaufay (1986) Eur. J. Biochem. 158, 187-194; (1987) Biochem. J. 248, 545-550] indistinguishable from those occurring early in the synthesis of secretory proteins. Glycosylation is apparently not the sorting signal responsible for their retention in the endoplasmic reticulum.

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Identification of rat liver carboxylesterase isozymes (EC 3.1.1.1) using polyacrylamide gel electrophoresis and isoelectric focusing

Cited by 18 publications

References 27 publications

Identification of microsomal rat liver carboxylesterases and their activity with retinyl palmitate

Identification of microsomal rat liver carboxylesterases and their activity with retinyl palmitate

Mosquito carboxylesterases: a review of the molecular biology and biochemistry of a major insecticide resistance mechanism

Immunochemical characterization and biosynthesis of pI-6.4 esterase, a carboxylesterase of rat liver microsomal extracts

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