Purification and characterization of hypoxia-inducible factor 1 - PubMed (original) (raw)
. 1995 Jan 20;270(3):1230-7.
doi: 10.1074/jbc.270.3.1230.
Affiliations
- PMID: 7836384
- DOI: 10.1074/jbc.270.3.1230
Free article
Purification and characterization of hypoxia-inducible factor 1
G L Wang et al. J Biol Chem. 1995.
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Abstract
Hypoxia-inducible factor 1 (HIF-1) is a DNA-binding protein that activates erythropoietin (Epo) gene transcription in Hep3B cells subjected to hypoxia or cobalt chloride treatment. HIF-1 DNA binding activity is also induced by hypoxia or cobalt in non-Epo-producing cells, suggesting a general role for HIF-1 in hypoxia signal transduction and transcriptional regulation. Here we report the biochemical purification of HIF-1 from Epo-producing Hep3B cells and non-Epo-producing HeLa S3 cells. HIF-1 protein was purified 11,250-fold by DEAE ion-exchange and DNA affinity chromatography. Analysis of HIF-1 isolated from a preparative gel shift assay revealed four polypeptides. Peptide mapping of these HIF-1 components demonstrated that 91-, 93-, and 94-kDa polypeptides had similar tryptic maps, whereas the 120-kDa polypeptide had a distinct profile. Glycerol gradient sedimentation analysis suggested that HIF-1 exists predominantly in a heterodimeric form and to a lesser extent as a heterotetramer. Partially purified HIF-1 bound specifically to the wild-type HIF-1 binding site from the EPO enhancer but not to a mutant sequence that lacks hypoxia-inducible enhancer activity. UV cross-linking analysis with purified HIF-1 indicated that both subunits of HIF-1 contact DNA directly. We conclude that in both cobalt chloride-treated HeLa cells and hypoxic Hep3B cells HIF-1 is composed of two different subunits: 120-kDa HIF-1 alpha and 91-94-kDa HIF-1 beta.
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