Abstract
Kinetic analyses of HIF prolyl 4-hydroxylases (HIF-P4Hs) allow determination of substrate, cosubstrate and cofactor requirements, analysis of the reaction rate, and inhibitory properties of the isoenzymes in vitro. Here we describe an assay measuring the substrate hydroxylation-coupled decarboxylation of radioactive 2-oxoglutarate to radioactive carbon dioxide as a fast, efficient, and diverse method to analyze the enzyme kinetics of HIF-P4Hs.
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Acknowledgments
This work was supported by Academy of Finland through Grants 120156, 140765 and 218129 (P.K.), 200471, 202469, and 296498 and Center of Excellence 2012–2017 Grant 251314 (J.M.) and by the S. Jusélius Foundation (P.K., J.M.), the Emil Aaltonen Foundation (P.K.), the Jane and Aatos Erkko Foundation (P.K., J.M.), and FibroGen, Inc. (J.M.).
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Koivunen, P., Myllyharju, J. (2018). Kinetic Analysis of HIF Prolyl Hydroxylases. In: Huang, L. (eds) Hypoxia. Methods in Molecular Biology, vol 1742. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7665-2_2
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DOI: https://doi.org/10.1007/978-1-4939-7665-2_2
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