Purification methods of mammalian catechol-O-methyltransferases

doi:10.1016/0378-4347(96)00117-X

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 684, Issues 1–2, 20 September 1996, Pages 147-161

https://doi.org/10.1016/0378-4347(96)00117-X Get rights and content

Abstract

The protein purification strategies used for obtaining homogeneous rat and human soluble catechol-O-methyltransferase (S-COTM) polypeptides are reviewed. Expression and purification of recombinant rat and human S-COMT in Escherichia coli and for human S-COMT in baculovirus-infected insect cells made it possible to elucidate the S-COMT polypeptides in more detail. The application of these purification methods has allowed the crystallization of the rat S-COMT protein and the analysis of the kinetic properties of the enzyme in great detail. The availability of the pure S-COMT protein together with the structural data has also greatly enhanced the development of more potent COMT inhibitors.

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Unveiling the biopathway for the design of novel COMT inhibitors
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Citation Excerpt :
During the previous century, pioneering studies used COMT extracted from mammalian tissues, mainly from the brain and rat liver.18 This procedure involved several preliminary steps to recover a highly pure protein fraction,75 namely consecutive centrifugations, acid and salt precipitation steps, and hydroxyapatite adsorption combined with chromatographic techniques (Table 1).75 However, the enzyme yields and bioactivity levels obtained were not suitable for bio-interaction and structural analysis studies.75–77
Catechol-O-methyltransferase (COMT) is an enzyme responsible for the O-methylation of biologically active catechol-based molecules. It has been associated with several neurological disorders, especially Parkinson’s disease (PD), because of its involvement in catecholamine metabolism, and has been considered an important therapeutic target for central nervous system disorders. In this review, we summarize the biophysical, structural, and therapeutical relevance of COMT; the medicinal chemistry behind the development of COMT inhibitors and the application of computer-aided design to support the design of novel molecules; current methodologies for the biosynthesis, isolation, and purification of COMT; and revise existing bioanalytical approaches for the assessment of enzymatic activity in several biological matrices.
Applications of gellan natural polymer microspheres in recombinant catechol-O-methyltransferase direct capture from a Komagataella pastoris lysate
2021, International Journal of Biological Macromolecules
The present work shows the application of nickel- and magnesium-crosslinked gellan microspheres in ionic and affinity capture strategies to directly extract hSCOMT from the complex Komagataella pastoris lysate through a simple batch method. Both formulations present similar morphology, but nickel–crosslinked microspheres present higher crosslinker content and smaller diameters. Four different capture strategies were established, by manipulating the ionic strength, pH, temperature and competing agents' presence. The most promising results for hSCOMT capture and clarification were obtained employing an ionic strategy with nickel-crosslinked microspheres and an affinity strategy with magnesium-crosslinked microspheres at 4 °C. The bioactivity results (200%) and purification degree (70%) of hSCOMT captured by the ionic strategy were more satisfactory probably due to the soft ionic conditions used (100 mM NaCl). For the first time, the gellan polysaccharide versatility was demonstrated in the microsphere application for the direct capture of hSCOMT from a complex lysate, simplifying isolation biotechnological procedures.
Bioavailability of the flavonol quercetin in cows after intraruminal application of quercetin aglycone and rutin
2012, Journal of Dairy Science
Citation Excerpt :
Ader et al. (2000) assumed that postabsorptive methylation of quercetin at position C-3′ (isorhamnetin) was more likely than methylation at C-4′ (tamarixetin). Tilgmann and Ulmanen (1996) reported similar findings for the methylation of catechols. Whereas methylation of quercetin absorbed might occur in the liver (Ader et al., 2000; Reinboth et al., 2010), the origin of kaempferol after i.v. application of quercetin is difficult to explain.
The bioavailability of quercetin has been intensively investigated in monogastric species, but knowledge about its bioavailability in ruminants does not exist. Thus, the aim of the present study was to determine the bioavailability of quercetin in nonlactating cows equipped with indwelling catheters placed in one jugular vein after intraruminal and additionally after i.v. application, respectively. Quercetin was administered intraruminally in equimolar amounts, either in the aglycone form or as its glucorhamnoside rutin, each at 2 dosages [10 and 50 mg of quercetin/kg of body weight (BW)]. In a second trial, 0.8 mg of quercetin aglycone/kg of BW was applied i.v. Blood samples were drawn 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, and 24 h after intraruminal application and every 5 min (first hour), every 10 min(second hour), and at 3 and 6 h after i.v. bolus application, respectively. Quercetin and quercetin metabolites with an intact flavonol structure (isorhamnetin, tamarixetin, and kaempferol) in plasma samples were analyzed by HPLC with fluorescence detection. After intraruminal application of quercetin and rutin, respectively, quercetin and its methylated (isorhamnetin, tamarixetin) and dehydroxylated (kaempferol) derivatives were present in plasma mainly as conjugated forms, whereas free quercetin and its derivatives were scarcely detected. For rutin, the relative bioavailability of total flavonols (sum of conjugated and nonconjugated quercetin and its conjugated and nonconjugated derivatives after intake of 50 mg/kg of BW) was 767.3% compared with quercetin aglycone (100%). Absolute bioavailability of total flavonols was only 0.1 and 0.5% after quercetin aglycone and rutin applications, respectively. Our data demonstrate that bioavailability of quercetin from rutin is substantially higher compared with that from quercetin aglycone in cows after intraruminal (or oral) application, unlike in monogastric species.
Crystal structures of the Apo and Holo form of rat catechol-O-methyltransferase
2009, Journal of Structural Biology
Catechol-O-methyltransferase (COMT, EC 2.1.1.6) is a monomeric enzyme that catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (AdoMet) to the phenolic oxygen of substituted catechols. Although the inhibitor recognition pattern and AdoMet site have already been studied crystallographically, structural information on the catalytic cycle of COMT has not yet been obtained. In this study, comparison of the co-factor and inhibitor-bound structures revealed that the Apo form of COMT shows a conformational change and there was no cleft corresponding to the AdoMet-binding site; the overall structure was partially open form and the substrate recognition site was not clearly defined. The Holo form of COMT was similar to the quaternary structure except for the β6–β7 and α2–α3 ligand recognition loops. These conformational changes provide a deeper insight into the structural events occurring in reactions catalyzed by AdoMet.
Crystal structures of rat catechol-O-methyltransferase complexed with coumarine-based inhibitor
2009, Biochemical and Biophysical Research Communications
Citation Excerpt :
Protein expression and purification. Protein expression and purification methods were referenced by the previously published method [11,12]. A truncate form of rat S-COMT was designed as amino acids 1–221 having an N-terminal GST-tag fusion protein.
In human, catechol-O-methyltransferase (COMT: E.C. 2.1.1.6) is responsible for metabolism of catechol neurotransmitter and xenobiotics. The main clinical interest in COMT results from the possibility of using COMT inhibitors as adjuncts in the therapy of Parkinson’s disease (PD) with l-DOPA. COMT is therefore a target for inhibitor development aiming at PD treatment and has been submitted to extensive structure-based drug design. Recently reported inhibitors have nitrocatechol structure that may inhibit oxidative phosphorylation and uncouple mitochondrial energy production. This work reports the first crystallographic study of Rat COMT complexed with non-nitrocatechol inhibitor. Analysis of the structural differences among the previously reported inhibitor complexes, coumarine-based inhibitor (4-phenyl-7, 8-dihydroxycoumarine: 4PCM) bound structure provides the explanation for inhibitor binding and can be used for future inhibitor design.
A new approach on the purification of recombinant human soluble catechol-O-methyltransferase from an Escherichia coli extract using hydrophobic interaction chromatography
2008, Journal of Chromatography A
Catechol-O-methyltransferase (COMT) is a significant target in protein engineering due to its role not only in normal brain function but also to its possible involvement in some human disorders. In this work, a new approach was employed for the purification of recombinant human soluble COMT (hSCOMT) using hydrophobic interaction chromatography, as the main isolation method, from an Escherichia coli culture broth. A simplified overall process flow is proposed. Indeed, with an optimized heterologous expression system for recombinant hSCOMT production, such as E. coli, it was possible to produce and recover the active monomeric enzyme directly from the cell crude culture broth either by a freeze/thaw or ultrasonication lysis step. The recombinant enzyme present in the bacterial soluble fraction, exhibited similar affinity for epinephrine (K_m 276 [215; 337] μM) and the methyl donor (S-adenosyl-l-methionine, SAMe) (K_m 36 [30; 41] μM) as human SCOMT. After the precipitation step by 55% of ammonium sulphate, a HIC step on the butyl-sepharose resin was found to be highly effective in selectively eluting a range of contaminating key proteins present in the concentrate soluble extract. Consequently, the partially purified eluate from HIC could then be loaded and polished by gel filtration in order to increase the process efficiency. The final product appeared as a single band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The procedure resulted in a global 10.9-fold purification with a specific activity of 5500 nmol/h/mg of protein. The widespread applicability of the process, here described, to different COMT sources could make this protocol highly useful for all studies requiring purified and active COMT proteins.

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ReviewPurification methods of mammalian catechol-O-methyltransferases

Abstract

Biochim. Biophys. Acta

J. Biol. Chem.

Trends. Pharmacol. Sci.

Gene

Genomics

Rev. Physiol. Biochem. Pharmacol.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

J. Biol. Chem.

Clin. Chim. Acta

Life Sci.

Biochem. Pharmacol.

Clin. Chim. Acta

Anal. Biochem.

J. Chromatogr.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

Life Sci.

Life Sci.

Clin. Immunol. Immunopathol.

Am. J. Obstet. Gynecol.

Brain Res.

Life Sci.

Biochim. Biophys. Acta

Biochem. Biophys. Res. Commun.

Life Sci.

Biochem. Pharmacol.

FEBS Lett.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Science

Naunyn-Schmiedeberg's Arch. Pharmacol.

Acta. Obstet. Gynec. Scand.

J. Clin. Endocrinol. Met.

Pharmacol. Rev.

Ann. Rev. Pharmacol. Toxicol.

Neurology

DNA and Cell Biol.

Review
Purification methods of mammalian catechol-O-methyltransferases