New strategies for the isolation and activity determination of naturally occurring type-4 glutathione peroxidase.

Protein Expression and Purification
Robert M Kernstock, Albert W Girotti

Abstract

Type 4 glutathione peroxidase (GPx4) is a widely expressed mammalian selenoenzyme known to play a vital role in cytoprotection against lipid hydroperoxide (LOOH)-mediated oxidative stress and regulation of oxidative signaling cascades. Since prokaryotes are not equipped to express mammalian selenoproteins, preparation of recombinant GPx4 via commonly used bacterial transformation is not feasible. A published procedure for isolating the enzyme from rat testis employs affinity chromatography on bromosulfophthalein-glutathione-linked agarose as the penultimate step in purification. Since this resin is no longer commercially available and preparing it in satisfactory operational form is tedious, we have developed an alternative purification approach based on sequential anion exchange, size exclusion, and cation exchange chromatography. Final preparations were found to be essentially homogeneous in GPx4 (M(r) approximately 20 kDa), as demonstrated by SDS-PAGE with protein staining and immunoblotting. Specific enzymatic activity was determined using a novel thin-layer chromatographic approach in which the kinetics of phosphatidylcholine hydroperoxide loss or cholesterol-7alpha-hydroperoxide loss was monitored. A >400-fold purificatio...Continue Reading

References

Jul 1, 1987·Chemistry and Physics of Lipids·F Ursini, A Bindoli
Nov 1, 1969·Proceedings of the Society for Experimental Biology and Medicine·G WhelanB Combes
Nov 24, 1999·Free Radical Biology & Medicine·R Brigelius-Flohé
Jul 25, 2000·Methods in Enzymology·A W Girotti, W Korytowski
Jul 11, 2002·Free Radical Biology & Medicine·Hartmut Kühn, Astrid Borchert
Oct 7, 1964·Annals of the New York Academy of Sciences·H KEBERLE
Feb 6, 2004·BioFactors·Leopold FlohéBirgit Hofmann
Oct 22, 2004·The Journal of Biological Chemistry·Qitao RanHolly Van Remmen
May 3, 2005·Nucleic Acids Research·Dan SuVadim N Gladyshev
May 19, 2005·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Tamas Kriska, Albert W Girotti
May 5, 2006·Journal of Neuroscience Research·Qitao RanArlan Richardson
May 19, 2007·Antioxidants & Redox Signaling·Laura Vanda PappKum Kum Khanna

❮ Previous
Next ❯

Citations

Oct 28, 2011·The Journal of Experimental Biology·Jeffrey M GrimElizabeth L Crockett
Jun 9, 2009·Free Radical Biology & Medicine·Etsuo Niki
Jan 1, 2013·Journal of Nutritional Science·S A MattmillerL M Sordillo
Feb 10, 2021·Acta Crystallographica. Section D, Structural Biology·Dieter MoosmayerRoman C Hillig
Nov 17, 2021·Molecular and Cellular Biochemistry·Mo LiYu Bai

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.