Determination and comparison of specific activity of the HIF-prolyl hydroxylases.
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.
Henry Wellcome Building for Genomic Medicine, Roosevelt Drive, Oxford OX3 7BN, UK.
Jason R
JR
Tuckerman
Yuguang
Y
Zhao
Kirsty S
KS
Hewitson
Ya-Min
YM
Tian
Christopher W
CW
Pugh
Peter J
PJ
Ratcliffe
David R
DR
Mole
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002384:Catalysis; D002457:Cell Extracts; D015687:Cell Hypoxia; D002460:Cell Line; D006801:Humans; D006900:Hydroxylation; D006909:Hydroxyproline / Q000032:analysis; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D010100:Oxygen / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011393:Procollagen-Proline Dioxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry; D034622:RNA Interference; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity; D018411:Spodoptera / Q000166:cytology; D013379:Substrate Specificity; D014157:Transcription Factors
D002457:Cell Extracts; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007527:Isoenzymes; D010455:Peptides; D021122:Protein Subunits; D011994:Recombinant Proteins; D014157:Transcription Factors; D011393:Procollagen-Proline Dioxygenase; D006909:Hydroxyproline; D010100:Oxygen