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Glutathione transferases immobilized on nanoporous alumina: Flow system kinetics, screening, and stability
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-6258-1443
Stockholm University, Faculty of Science, Department of Neurochemistry. Uppsala University, Sweden.ORCID iD: 0000-0002-6416-064X
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2014 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 446, 59-63 p.Article in journal (Refereed) Published
Abstract [en]

The previously uncharacterized Drosophila melanogaster Epsilon-class glutathione transferases E6 and E7 were immobilized on nanoporous alumina. The nanoporous anodized alumina membranes were derivatized with 3-aminopropyl-triethoxysilane, and the amino groups were activated with carbonyldiimidazole to allow coupling of the enzymes via c-amino groups. Kinetic analyses of the immobilized enzymes were carried out in a circulating flow system using CDNB (1-chloro-2,4-dinitrobenzene) as substrate, followed by specificity screening with alternative substrates. A good correlation was observed between the substrate screening data for immobilized enzyme and corresponding data for the enzyme in solution. A limited kinetic study was also carried out on immobilized human GST S1-1 (also known as hematopoietic prostaglandin D synthase). The stability of the immobilized enzymes was virtually identical to that of enzymes in solution, and no leakage of enzyme from the matrix could be observed.

Place, publisher, year, edition, pages
2014. Vol. 446, 59-63 p.
Keyword [en]
Immobilization, Glutathione transferase, Kinetics, Screening, Enzyme reactor, Drosophila
National Category
Biochemistry and Molecular Biology Analytical Chemistry
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-100853DOI: 10.1016/j.ab.2013.10.004ISI: 000329949500010OAI: oai:DiVA.org:su-100853DiVA: diva2:698344
Note

AuthorCount:5;

Available from: 2014-02-21 Created: 2014-02-17 Last updated: 2017-12-05Bibliographically approved
In thesis
1. The fruit fly Drosophila melanogaster GSTE6 and E7; characterization, immobilization and transgenic overexpression
Open this publication in new window or tab >>The fruit fly Drosophila melanogaster GSTE6 and E7; characterization, immobilization and transgenic overexpression
2014 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Glutathione transferases (GSTs) are multifunctional enzymes that are universally distributed in most eukaryotes and prokaryotes. They play a pivotal role in the metabolism and detoxication of numerous endogenous and exogenous electrophiles by conjugating them with ubiquitous tripeptide glutathione. In this study we have immobilized two heterologously expressed and purified Epsilon-class enzymes, GSTE6 and GSTE7, from of Drosophila melanogaster on nanoporous alumina membranes. The membranes were derivatized with 3-aminopropyl-triethoxysilane and the amino groups were activated with carbonyldiimidazole to allow coupling of the enzymes. Kinetic analyses of the immobilized enzymes were carried out in a circulating flow system using CDNB (1-chloro-2,4-dinitrobenzene) as substrate, followed by specificity screening with alternative substrates. A good correlation was observed between the substrate screening data for immobilized enzyme and corresponding data for the enzymes in solution. The stability of the immobilized enzymes was virtually identical to that for the enzymes in solution and no leakage of enzyme from the matrix could be observed.

Additionally, we have investigated the catalytic activities of GSTE7 with organic isothiocyanates (ITCs). These reactive compounds are strong electrophilic molecules produced in plants by the hydrolysis of glucosinolates and exert toxicity in biological tissues.  Our in vitro studies, showed high catalytic activity of GSTE7 towards ITCs. We have then explored the in vivo effect of phenethyl isothiocyanate (PEITC) and allyl isothiocyanate (AITC) in transgenic fruit flies overexpressing GSTE7. A concentration of 0.25 mM PEITC in standard fly food was shown to be toxic to flies and significantly shortened the lifespan. We noticed that overexpression of GSTE7 could protect females from the initial acute toxic effects, but had no positive effect on long term exposure. The effect on males seems to be the opposite to that of females, where a higher mortality was seen in fly males overexpressing GST E7 after one week of exposure.  On the other hand 1mM concentration of AITC showed no toxic effects, but dramatically reduced the oviposition activity of wild-type flies in comparison to the transgenic flies.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University, 2014
Keyword
Glutathione transferases, drosophila melanogaster, isothiocyanates, toxicity, immobilization, kinetics
National Category
Neurosciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-102003 (URN)978-91-7447-888-4 (ISBN)
Presentation
2014-04-08, Heilbronnsalen, C 458, Svante Arrhenius väg 16 B, Stockholm, 12:15 (English)
Opponent
Supervisors
Note

At the time of the seminar the following paper was unpublished and had a status as follows: Paper 2: Submitted manuscript.

Available from: 2014-04-01 Created: 2014-03-20 Last updated: 2015-03-09Bibliographically approved
2. Studies on Human and Drosophila melanogaster Glutathione Transferases of Biomedical and Biotechnological Interest
Open this publication in new window or tab >>Studies on Human and Drosophila melanogaster Glutathione Transferases of Biomedical and Biotechnological Interest
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Glutathione transferases (GSTs, EC.2.5.1.18) are multifunctional enzymes that are universally distributed in all cellular life forms. They play important roles in metabolism and detoxication of endogenously produced toxic compounds and xenobiotics. GSTs have gained considerable interest over the years for biomedical and biotechnological applications due to their involvement in the conjugation of glutathione (GSH) to a vast array of chemical species. Additionally, the emergence of non-detoxifying functions of GSTs has further increased their biological significance. The present work encompasses four scientific studies aimed at investigating human as well as fruit fly Drosophila melanogaster GSTs.

Paper I presents the immobilization of GSTs on nanoporous alumina membranes. Kinetic analyses with 1-chloro-2,4-dinitrobenzene followed by specificity screening with alternative substrates showed a good correlation between the data obtained from immobilized enzymes and the enzymes in solution. Furthermore, immobilization showed no adverse effects on the stability of the enzymes. Paper II presents inhibition studies of human hematopoietic prostaglandin D2 synthase (HPGDS), a promising therapeutic target for anti-allergic and anti-inflammatory drugs. Our screening results with an FDA-approved drug library revealed a number of effective inhibitors of HPGDS with IC50 values in the low micromolar range. Paper III concerns the toxicity of organic isothiocyanates (ITCs) that showed high catalytic activities with GSTE7 in vitro. The in vivo results showed that phenethyl isothiocyanate (PEITC) and allyl isothiocyanate in millimolar dietary concentrations conferred toxicity to the adult fruit flies leading to death or shortened life-span. The transgenic female flies overexpressing GSTE7 showed increased tolerance against PEITC toxicity compared to the wild-type. However, the effect was opposite in male flies overexpressing GSTE7 after one week exposure. Notably, the transgene enhanced the oviposition activity of flies with and without ITCs exposure. Paper IV highlights Drosophila GSTs as efficient catalysts of the environmental pollutant and explosive 2,4,6-trinitrotoluene and the related 2,4-dinitrotoluene degradation. This result suggests the potential of GST transgenes in plants for biotransformation and phytoremediation of these persistent environmental pollutants. 

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University, 2016. 43 p.
Keyword
glutathione transferases, detoxication, hpgds inhibition, immobilization, isothiocyanates, drosophila GSTs, environmental pollutants
National Category
Chemical Sciences Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-126723 (URN)978-91-7649-349-6 (ISBN)
Public defence
2016-03-18, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2016-02-24 Created: 2016-02-12 Last updated: 2017-02-23Bibliographically approved

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