Determination and comparison of specific activity of the HIF-prolyl hydroxylases.

Journal article

Hypoxia-inducible factor (HIF) is a transcriptional complex that is regulated by oxygen sensitive hydroxylation of its alpha subunits by the prolyl hydroxylases PHD1, 2 and 3. To better understand the role of these enzymes in directing cellular responses to hypoxia, we derived an assay to determine their specific activity in both native cell extracts and recombinant sources of enzyme. We show that all three are capable of high rates of catalysis, in the order PHD2=PHD3>PHD1, using substrate peptides derived from the C-terminal degradation domain of HIF-alpha subunits, and that each demonstrates similar and remarkable sensitivity to oxygen, commensurate with a common role in signaling hypoxia.

Authors: Tuckerman, JR; Zhao, Y; Hewitson, K; Tian, Y; Pugh, C

• Publication status: Published
• Journal: FEBS letters
• Volume: 576
• Issue: 1-2
• Pages: 145-150
• Publication date: 2004-10-01
• DOI: 10.1016/j.febslet.2004.09.005
• EISSN: 1873-3468
• ISSN: 0014-5793
• Language: English
• Keywords: Cell Line; Kinetics; Recombinant Proteins; Procollagen-Proline Dioxygenase; Sensitivity and Specificity; Substrate Specificity; Humans; Protein Structure, Tertiary; Isoenzymes; Amino Acid Sequence; Protein Subunits; Hydroxyproline; Transcription Factors; Baculoviridae; Catalysis; Hydroxylation; RNA Interference; Oxygen; Animals; Spodoptera; Peptides; Hypoxia-Inducible Factor 1, alpha Subunit; Cell Extracts; Cell Hypoxia