Kinetic Analysis of HIF Prolyl Hydroxylases

Peppi Koivunen; Johanna Myllyharju

doi:10.1007/978-1-4939-7665-2_2

Kinetic Analysis of HIF Prolyl Hydroxylases

Methods Mol Biol. 2018:1742:15-25. doi: 10.1007/978-1-4939-7665-2_2.

Authors

Peppi Koivunen¹, Johanna Myllyharju²

Affiliations

¹ Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine, Oulu Center for Cell-Matrix Research, Biocenter Oulu, University of Oulu, Oulu, Finland. peppi.karppinen@oulu.fi.
² Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine, Oulu Center for Cell-Matrix Research, Biocenter Oulu, University of Oulu, Oulu, Finland.

PMID: 29330786
DOI: 10.1007/978-1-4939-7665-2_2

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.

Keywords: 2-oxoglutarate; HIF-P4H; Inhibitor; Kinetics; Substrate.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Biochemistry / methods*
Carbon Radioisotopes / metabolism
Decarboxylation
Humans
Hydroxylation
Hypoxia-Inducible Factor 1, alpha Subunit / metabolism
Hypoxia-Inducible Factor-Proline Dioxygenases / chemistry*
Isoenzymes / chemistry
Ketoglutaric Acids / chemistry
Ketoglutaric Acids / metabolism
Kinetics

Substances

Carbon Radioisotopes
HIF1A protein, human
Hypoxia-Inducible Factor 1, alpha Subunit
Isoenzymes
Ketoglutaric Acids
Hypoxia-Inducible Factor-Proline Dioxygenases