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Alpha-class Glutathione Transferases from Pig: a Comparative Study
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Glutathione transferases (GSTs, EC 2.5.1.18) possess multiple functions and have potential applications in biotechnology. This thesis contributes to knowledge about glutathione transferases from Sus scrofa (pig). The study is needed for better understanding of biochemical processes in this species and is desirable for drug development, for food industry research and in medicine.

A primary role of GSTs is detoxication of electrophilic compounds. Our study presents porcine GST A1-1 as a detoxication enzyme expressed in many tissues, in particular adipose tissue, liver and pituitary gland. Based on comparison of activity and expression profiles, this enzyme can be expected to function in vivo similarly to human GST A2-2 (Paper II).

In addition to its protective function, human GST A3-3 is an efficient steroid isomerase and contributes to the biosynthesis of steroid hormones in vivo. We characterized a porcine enzyme, pGST A2-2, displaying high steroid-isomerase activity and resembling hGST A3-3 in other properties as well. High levels of pGST A2-2 expression were found in ovary, testis and liver. The properties of porcine enzyme strengthen the notion that particular GSTs play an important role in steroidogenesis (Paper I).

Combination of time-dependent and enzyme concentration-dependent losses of activity as well as the choice of the organic solvent for substrates were found to cause irreproducibility of activity measurements of GSTs. Enzyme adsorption to surfaces was found to be the main explanation of high variability of activity values of porcine GST A2-2 and human Alpha-class GSTs reported in the literature. Several approaches to improved functional comparison of highly active GSTs were proposed (Paper III).

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2011. , p. 43
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 803
Keyword [en]
glutathione transferase, substrate selectivity, steroidogenesis, Sus scrofa, functional comparison, irreproducibility, reproducible assays
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-144119ISBN: 978-91-554-7994-7 (print)OAI: oai:DiVA.org:uu-144119DiVA, id: diva2:392594
Public defence
2011-03-11, B7:113a, Biomedical Center, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Funder
Swedish Research Council
Note
Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 733Available from: 2011-02-18 Created: 2011-01-27 Last updated: 2011-03-21Bibliographically approved
List of papers
1. Porcine glutathione transferase Alpha 2-2 is a human GST A3-3 analogue that catalyses steroid double-bond isomerization
Open this publication in new window or tab >>Porcine glutathione transferase Alpha 2-2 is a human GST A3-3 analogue that catalyses steroid double-bond isomerization
2010 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 431, no 1, p. 159-167Article in journal (Refereed) Published
Abstract [en]

A primary role of GSTs (glutathione transferases) is detoxication of electrophilic compounds. In addition to this protective function, hGST (human GST) A3-3, a member of the Alpha class of soluble GSTs, has prominent steroid double-bond isomerase activity. The isomerase reaction is an obligatory step in the biosynthesis of steroid hormones, indicating a special role of hGST A3-3 in steroidogenic tissues. An analogous GST with high steroid isomerase activity has so far not been found in any other biological species. In the present study, we characterized a Sus scrofa (pig) enzyme, pGST A2-2, displaying high steroid isomerase activity. High levels of pGST A2-2 expression were found in ovary, testis and liver. In its functional properties, other than steroid isomerization, pGST A2-2 was most similar to hGST A3-3. The properties of the novel porcine enzyme lend support to the notion that particular GSTs play an important role in steroidogenesis.

Keyword
Delta(5)-androstene-3, 17-dione, androstenedione, double-bond isomerization, glutathione transferase Alpha 2-2 (GST A2-2), steroidogenesis, tributyltin
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-134177 (URN)10.1042/BJ20100839 (DOI)000282852800017 ()20673231 (PubMedID)
Available from: 2010-11-22 Created: 2010-11-22 Last updated: 2017-12-12Bibliographically approved
2. Characterization of porcine Alpha-class glutathione transferase A1-1
Open this publication in new window or tab >>Characterization of porcine Alpha-class glutathione transferase A1-1
2011 (English)In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 507, no 2, p. 205-211Article in journal (Refereed) Published
Abstract [en]

An Alpha-class glutathione transferase (GST) has been cloned from pig gonads. In addition to two conservative point mutations our nucleotide sequence presents a frame shift resulting from a missing A as compared to a previously published porcine GST A1-1 sequence. The deduced C-terminal amino-acid segment of the protein differs between the two variants. Repeated sequencing of cDNA isolated from different tissuesand animals ruled out the possibility of a cloning artifact, and the deduced amino acid sequence ofour clone showed higher similarity to related mammalian GST sequences. Hereafter, we refer to ourcloned enzyme as GST A1-1 and to the previously published enzyme as GST A1-1*. The study of the tissue distribution of the GSTA1 mRNA revealed high expression levels in many organs, in particular adipose tissue, liver, and pituitary gland. Porcine GST A1-1 was expressed in Escherichia coli and its kinetic properties were determined using alternative substrates. The catalytic activity in steroid isomerization reactionswas at least 10-fold lower than the corresponding values for porcine GST A2-2, whereas the activity with 1-chloro-2,4-dinitrobenzene was approximately 8-fold higher. Differences in the H-site residues of mammalian Alpha-class GSTs may explain the catalytic divergence.

Keyword
Sus scrofa, glutathione transferase A1-1, alternative sequence, substrate selectivity
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-140698 (URN)10.1016/j.abb.2010.12.015 (DOI)000288058000001 ()21172301 (PubMedID)
Available from: 2011-01-07 Created: 2011-01-07 Last updated: 2017-12-11Bibliographically approved
3. Experimental Conditions Affecting Functional Comparison of Highly Active Glutathione Transferases
Open this publication in new window or tab >>Experimental Conditions Affecting Functional Comparison of Highly Active Glutathione Transferases
2011 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 413, no 1, p. 16-23Article in journal (Refereed) Published
Abstract [en]

Glutathione transferases (GSTs, EC 2.5.1.18) possess multiple functions and have potential applications in biotechnology. Direct evidence of underestimation of activity of human GST A3-3 and porcine GST A2-2 measured at submicromolar enzyme concentrations is reported here for the first time. Combination of time-dependent and enzyme concentration-dependent loss of activity as well as the choice of the organic solvent for substrates were found to cause irreproducibility of activity measurementsof GSTs. These effects contribute to high variability of activity values of porcine GST A2-2 and human Alpha-class GSTs reported in the literature. Adsorption of GSTs to surfaces was found to be the main explanation of the observed phenomena. Several approaches to improved functional comparison of highly active GSTs are proposed.

Keyword
concentration-dependent instability, functional comparison, glutathione transferase, reproducibility, time-dependent instability
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-144107 (URN)10.1016/j.ab.2011.01.041 (DOI)000289607900003 ()21295006 (PubMedID)
Funder
Swedish Research Council
Available from: 2011-01-27 Created: 2011-01-27 Last updated: 2017-12-11Bibliographically approved

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