©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Characterization of Hypoxia-inducible Factor 1 (*)

(Received for publication, August 18, 1994; and in revised form, November 10, 1994)

Guang L. Wang Gregg L. Semenza (§)

From the Center for Medical Genetics, Departments of Pediatrics and Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-3914

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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-1alpha and 91-94-kDa HIF-1beta.


INTRODUCTION

Gene transcription is regulated by transcription factors that can recognize specific DNA sequences and modulate the assembly of an active transcription initiation complex in response to various extracellular and intracellular signals(1) . Oxygen (O(2)) is the final electron acceptor in respiratory redox reactions in mammalian cells, and hypoxia provokes acute as well as long term physiological responses. The expression of many genes is known to be induced by hypoxia, including the genes encoding tyrosine hydroxylase(2) , endothelin(3) , glycolytic enzymes (4, 5) and growth factors such as platelet-derived growth factor B(6) , vascular endothelial growth factor(7) , and erythropoietin (Epo)(^1)(8, 9) . The molecular mechanisms that underlie hypoxia signal transduction in mammalian cells, however, remain undefined.

Epo is a glycoprotein growth factor that stimulates proliferation and differentiation of erythroid progenitor cells. Tissue hypoxia resulting from reduced ambient O(2) levels or reduced blood O(2) carrying capacity (anemia) activates EPO gene expression, which is regulated primarily at the level of transcription (10, 11) . A hypoxia-inducible enhancer was defined in the 3`-flanking sequence of the EPO gene(12, 13, 14, 15) . The hypoxia-inducible enhancer is functionally tripartite, with the first two sites essential for hypoxia inducibility and a third site functioning to amplify the induction signal(14, 15, 16, 17, 18) . A hypoxia-induced DNA-binding protein (HIF-1) that binds specifically to site 1 of the hypoxia-inducible enhancer was identified(15) . The induction of HIF-1 DNA binding activity in hypoxic cells requires on-going protein and RNA synthesis (15, 19) . Protein phosphorylation is also required for hypoxia signal transduction and HIF-1 DNA binding activity(19) . HIF-1 recognizes an 8-base pair DNA sequence 5`-TACGTGCT-3` in the EPO enhancer and contacts both DNA strands in the major groove(19) . UV cross-linking analysis indicated that HIF-1 contains a DNA-binding subunit of 120 kDa as well as a subunit of 94 kDa that was cross-linked less efficiently(20) . The divalent cation Co and the iron chelator desferrioxamine also induce EPO gene expression (10, 21, 22) as well as HIF-1 DNA binding activity(20, 23) .

The potential importance of HIF-1 as a transcriptional activator was underscored by the finding that it was induced by hypoxia in all cell types tested (20) and that the EPO enhancer can mediate hypoxia-inducible reporter gene expression in non-Epo-producing cells (20, 24) . Furthermore, HIF-1 binding sites are present in several hypoxia-inducible genes encoding glycolytic enzymes, and DNA sequences encompassing these sites can mediate hypoxia-inducible reporter gene transcription(5) . These results suggest the existence of a common hypoxia signal transduction pathway and a central role for HIF-1 in the transcriptional regulation of hypoxia-responsive genes. Here we describe the biochemical purification of human HIF-1 and the characterization of its DNA binding activity and molecular composition.


MATERIALS AND METHODS

Cell Culture and Nuclear Extract Preparation

Human Hep3B and HeLa cells were maintained and treated with 1% O(2) and CoCl(2)(20) , and nuclear extracts were prepared as described previously(15, 25) . HeLa S3 cells, obtained from American Type Culture Collection, were adapted to suspension growth in Spinner's minimum essential medium supplemented with 5% (v/v) horse serum (Quality Biological, Gaithersburg, MD). The cells were grown to a density of 8 times 10^5 cells/ml and maintained by dilution to 2 times 10^5 cells/ml with fresh complete medium every 2 days. For induction of HIF-1 DNA binding activity, HeLa S3 cells were treated with 125 µM cobalt chloride for 4 h at 37 °C before harvesting by centrifugation for 10 min at 2,500 times g. Cell pellets were washed twice with ice-cold phosphate-buffered saline and resuspended in 5 packed cell volumes of buffer A (10 mM Tris-HCl (pH 7.6), 1.5 mM MgCl(2), 10 mM KCl) supplemented with 2 mM dithiothreitol (DTT), 0.4 mM phenylmethylsulfonyl fluoride and 1 mM Na(3)VO(4). After incubation on ice for 10 min, cells were pelleted at 2,500 times g for 5 min, resuspended in 2 packed cell volumes of buffer A, and lysed by 20 strokes in a glass Dounce homogenizer with type B pestle. Nuclei were pelleted at 10,000 times g for 10 min and resuspended in 3.5 packed nuclear volumes of buffer C (0.42 M KCl, 20 mM Tris-HCl (pH 7.6), 20% glycerol, 1.5 mM MgCl(2)) supplemented with 2 mM DTT, 0.4 mM phenylmethylsulfonyl fluoride, and 1 mM Na(3)VO(4). Nuclear proteins were extracted by stirring at 4 °C for 30 min. After centrifugation at 15,000 times g for 30 min, the supernatant was dialyzed against buffer Z-100 (25 mM Tris-HCl (pH 7.6), 0.2 mM EDTA, 20% glycerol, 2 mM DTT, 0.4 mM phenylmethylsulfonyl fluoride, and 1 mM Na(3)VO(4) and 100 mM KCl) at 4 °C. The dialysate was clarified by ultracentrifugation at 100,000 times g for 60 min at 4 °C and designated as crude nuclear extract. The nuclear extracts were aliquoted, frozen in liquid N(2), and stored at -80 °C. Protein concentration was determined by the method of Bradford (26) with a commercial kit (Bio-Rad) using bovine serum albumin (BSA) as standard.

Gel Shift Assays

Gel shift assays were performed as described previously (15) except that the binding reaction was in buffer Z-100. For gel shift assays with partially purified and affinity-purified HIF-1 preparations, 0.25 mg/ml of BSA and 0.05% Nonidet P-40 were included in the binding reaction. Nonspecific competitor calf thymus DNA (Sigma) was used in reduced amounts for partially purified fractions, and no calf thymus DNA was used for affinity-purified HIF-1 fractions. For competition experiments, unlabeled oligonucleotide DNA was incubated with DEAE-Sepharose column fractions for 5 min on ice before probe DNA was added.

Methylation Interference Analysis

Methylation interference analysis was performed as described previously(19) , except 100 µg of nuclear extract prepared from CoCl(2)-treated HeLa cells were used in the binding reactions.

Preparation of DNA Affinity Columns

DNA affinity columns were prepared by coupling multimerized double-stranded oligonucleotides to CNBr-activated Sepharose(27) . The wild-type and the mutant column contained multimerized oligonucleotide W18 () and M18 (, mutation underlined), respectively.

Equal amounts of complementary oligonucleotides were annealed, phosphorylated, and ligated. Ligated oligonucleotides (60-500 base pairs) were extracted with phenol/chloroform, ethanol precipitated, resuspended in deionized water, and coupled to CNBr-activated Sepharose 4B as instructed by the manufacturer (Pharmacia Biotech Inc.). Approximately 50 µg of ligated double-stranded oligonucleotides were coupled per ml of Sepharose.

Purification of HIF-1

Crude nuclear extracts from 120 liters of CoCl(2)-treated HeLa S3 cells (435 ml, 3,040 mg) were thawed on ice and clarified by centrifugation at 15,000 times g for 10 min. Extracts were fractionated as three batches over a 36-ml DEAE-Sepharose CL-6B column (Pharmacia) in buffer Z-100 with a step gradient of increasing KCl. Fractions containing peak activity were pooled and dialyzed against buffer Z-100. The dialysate from DEAE-Sepharose columns was incubated with calf thymus DNA (Sigma) at a concentration of 4.4 µg/ml for 15 min on ice. After centrifugation at 15,000 times g for 10 min, the supernatant (240 ml; 2.3 mg/ml) was applied to a 6-ml DNA affinity column prepared with concatenated W18 oligonucleotide. The fractions containing HIF-1 activity were pooled and dialyzed against buffer Z-100. The dialysate from the first DNA-affinity column was mixed with calf thymus DNA at a concentration of 2.5 µg/ml and incubated on ice for 15 min. After centrifugation (as described above), the supernatant was applied to a 1.5-ml M18 DNA-Sepharose column. The flowthrough from the M18 column was collected and reapplied to a second 2-ml W18 column. All buffers used for DNA affinity chromatography were supplemented with 0.05% Nonidet P-40 and 5 mM DTT. The amount of protein in affinity column fractions was quantitated by silver staining of SDS-polyacrylamide gels or by Amido Black (Sigma) staining of nitrocellulose membranes (Schleicher & Schuell) spotted with protein samples and compared against known amounts of protein standards (Bio-Rad).

For purification of HIF-1 from hypoxia-treated Hep3B cells, nuclear extracts (95 mg) were fractionated by the use of a 4-ml DEAESepharose CL-6B column as described above. 0.25 M KCl elute fractions were dialyzed against buffer Z-100 and applied onto a Sephacryl S-300 gel filtration column (50 ml, 1.5 times 30 cm). The fractions containing HIF-1 activity were pooled and applied to a 2-ml calf thymus DNA column (0.8 mg of calf thymus DNA/ml of Sepharose) prepared by coupling single-stranded calf thymus DNA to CNBr-activated Sepharose 4B. The flowthrough was collected and applied to a 0.4-ml W18 column as described above after incubation with calf thymus DNA (2.2 µg/ml) for 10 min followed by another 0.2-ml W18 column after dialysis against buffer Z-100.

SDS-PAGE and Silver Staining

SDS-PAGE was carried out as described by Laemmli(28) . The gels were calibrated with high range molecular weight standards or prestained molecular weight markers (Bio-Rad). Electrophoresis was performed at 30 mA. Silver staining was performed with silver nitrate as described(29) . Molecular weight estimation for HIF-1 polypeptides was based on SDS-polyacrylamide gels with 3.2% cross-linking (acrylamide/bisacrylamide ratio of 30:1).

Analysis of HIF-1 Subunits

Preparative gel shift assays were performed with 30 µl of affinity-purified HIF-1 and probe W18. Gel slices containing HIF-1 and surrounding areas were isolated after autoradiography of a wet gel. Gel slices were placed on the stacking gel of a 6% SDS-polyacrylamide gel and incubated with Laemmli buffer in situ for 15 min, and electrophoresis was performed in parallel with 30 µl of affinity-purified HIF-1 and molecular weight markers. For two-dimensional denaturing gel electrophoresis, two aliquots of affinity-purified HIF-1 were resolved on a 6% SDS-polyacrylamide gel with 5% cross-linking (acrylamide/bisacrylamide ratio of 19:1). One lane was stained with silver nitrate. The gel slices corresponding to regions of interest were isolated from the unstained lane. The isolated gel slices were placed directly on the stacking gel of the second dimension 6% SDS-polyacrylamide gel with 3.2% cross-linking, and electrophoresis was performed in parallel with 30 µl of affinity purified HIF-1.

Peptide Mapping of HIF-1 Subunits

2 ml of the affinity-purified HIF-1 were dialyzed against 10 mM ammonium bicarbonate, 0.05% SDS and lyophilized. After resuspension in a solubilizing solution (100 mM sucrose, 3% SDS, 31.25 mM Tris-HCl (pH 6.9), 1 mM EDTA, 5% beta-mercaptoethanol, 0.005% bromphenol blue), the protein samples were heated to 37 °C for 15 min and resolved on a 6% polyacrylamide gel containing 0.2% SDS. Polypeptides were transferred electrophoretically at 4 °C to a polyvinylidene difluoride membrane (Bio-Rad) in 0.5 times Towbin buffer (30) (96 mM glycine, 12.5 mM Tris-HCl (pH 8.3)) with 10% methanol at 250 mA for 12 h. After transfer, the polyvinylidene difluoride membrane was stained for 1 min with 1% Amido Black in 10% acetic acid, destained with 5% acetic acid and rinsed with Milli-Q water. Membrane slices containing the HIF-1 polypeptides of 120, 94/93, and 91 kDa were excised and subjected to peptide mapping(31) . In situ tryptic digestion and reverse phase HPLC were performed by the Wistar Protein Microchemistry Laboratory.

UV Cross-linking Analysis

UV cross-linking was carried out as described previously (20) except that 30 µl of affinity-purified HIF-1 were used in the binding reaction. Affinity-purified HIF-1 was incubated with W18 probe in the absence or presence of unlabeled W18 or M18 oligonucleotide. After incubation for 15 min at 4 °C, the reaction mixtures were irradiated with UV light (312 nm; Fisher Scientific) for 30 min and resolved by 6% SDS-PAGE with pre-stained molecular weight markers and visualized by autoradiography.

Glycerol Gradient Sedimentation

Linear gradients of 12 ml, 10-30% glycerol in a buffer containing 100 mM KCl, 25 mM Tris-HCl (pH 7.6), 0.2 mM EDTA, 5 mM DTT, and 0.4 mM phenylmethylsulfonyl fluoride, were prepared for centrifugation in a Beckman SW40 rotor for 48 h at 4 °C. Nuclear extract prepared from hypoxic Hep3B cells (100 µl, 5 mg/ml) was mixed with an equal volume of glycerol gradient buffer containing 10% glycerol and layered on the top of the gradient. A marker gradient was sedimented in parallel and contained 50 µg each of thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), and BSA (67 kDa) (Pharmacia). Markers were adjusted to the same volume and glycerol concentration as the sample. Fractions (0.5 ml) were collected from the top of the tubes, and DNA binding activity was measured by the gel shift assay. Markers were assayed by SDS-PAGE and silver staining.


RESULTS

Induction of HIF-1 by Cobalt Chloride Treatment of HeLa Cells

HIF-1 was previously found to be induced by CoCl(2) treatment of Hep3B cells and HeLa cells(20) . To determine the optimal concentration of CoCl(2) for induction of HIF-1 DNA binding activity, HeLa cells were treated for 4 h with 5 µM to 1 mM CoCl(2). Nuclear extracts were prepared, and 5-µg aliquots were analyzed by gel shift assay with oligonucleotide W18 as probe (Fig. 1A). Induction of HIF-1 DNA binding activity by CoCl(2) was dose-dependent. HIF-1 activity in nuclear extracts was detected at 25 µM CoCl(2) and reached a peak activity at 250 µM (Fig. 1A, lanes 8 and 10). Significant cell death, however, was observed at CoCl(2) concentrations geq 250 µM, resulting in decreased yield of nuclear proteins. For this reason 125 µM CoCl(2) was chosen for subsequent large scale nuclear extract preparation. Constitutive DNA binding activities (C), which also bind W18 probe sequence specifically(15, 20) , remained relatively unchanged in cells treated with 0-100 µM CoCl(2) (Fig. 1A, lanes 1-7), and decreased at CoCl(2) concentration greater than 250 µM (Fig. 1A, lanes 10-12), suggesting an adverse effect of high CoCl(2) concentration on the cells. Nonspecific DNA binding activities (NS) were barely detectable in this particular gel shift assay and vary with cell type and the relative amount of nonspecific competitor DNA used(15, 20) .


Figure 1: Analysis of HIF-1 induced by CoCl(2) treatment of HeLa cells. A, dose-dependent induction of HIF-1 DNA binding activity by CoCl(2) treatment. Nuclear extracts, prepared from HeLa cells cultured in the presence of the indicated concentration of CoCl(2) for 4 h at 37 °C, were incubated with W18 probe and analyzed by gel shift assay. Lanes 1-8 and 9-12 represent extracts prepared in two separate experiments. Arrows indicate HIF-1, constitutive DNA binding activity (C), nonspecific activity (NS), and free probe (F). B, methylation interference analysis with nuclear extracts from CoCl(2)-treated HeLa cells. W18 was 5`-end labeled on the coding or noncoding strand, partially methylated, and incubated with nuclear extracts. DNA-protein complexes corresponding to HIF-1, constitutive DNA binding activities (C1 and C2), and nonspecific binding activity (NS) were isolated from a preparative gel shift assay (lower) in addition to free probe (F) (not shown). DNA was purified, cleaved with piperidine, and analyzed on a 15% denaturing polyacrylamide gel (upper). Results are summarized at left for coding strand and at right for noncoding strand. The guanine residues are numbered according to their locations in the W18 probe. The HIF-1 binding site is boxed. Complete methylation interference with HIF-1 binding is indicated by closed circles. Partial and complete methylation interference with constitutive DNA binding activity are indicated by open and closed squares, respectively.



To provide evidence that HIF-1 DNA binding activity in crude nuclear extracts from hypoxic Hep3B cells and CoCl(2)-treated HeLa cells has the same DNA binding properties, methylation interference analysis was performed. Crude nuclear extract from CoCl(2)-treated HeLa cells was incubated with partially methylated W18 probe, labeled at the 5` end of either coding or noncoding strand, and the reaction mixtures were resolved on a native polyacrylamide gel (Fig. 1B, lower). Probe DNA from the HIF-1, constitutive, and nonspecific bands as well as free probe was isolated, cleaved with piperidine, and analyzed by denaturing polyacrylamide gel electrophoresis (Fig. 1B, upper). On the coding strand, methylation of G(8) or G eliminated or greatly reduced HIF-1 binding, respectively (Fig. 1B, lane 2). Methylation of G only partially interfered with the binding of constitutive factors (Fig. 1B, lanes 3 and 4). On the noncoding strand, methylation of G(7) or G blocked HIF-1 binding to the probe (Fig. 1B, lane 8). Only the methylation of G(7) interfered with binding of constitutive factors (Fig. 1B, lanes 9 and 10). The nonspecific binding activity was unaffected by DNA methylation on either strand (Fig. 1B, lanes 5 and 11). These results are identical to those obtained with crude nuclear extract from hypoxic Hep3B cells(19) . In both cases, the results indicate that (i) HIF-1 closely contacts G(8) and G on the coding strand and G(7) and G on the noncoding strand through the major groove of the DNA helix and (ii) HIF-1 and the constitutive DNA binding factors can be distinguished by the nature of their DNA binding site contacts.

Biochemical Purification of HIF-1

HIF-1 was initially detected in hypoxic Hep3B cells(15) , but it is difficult to obtain large amounts of Hep3B nuclear extract because these cells grow as a monolayer. Since HIF-1 DNA binding activity from hypoxic Hep3B cells and CoCl(2)-treated HeLa cells are indistinguishable, HeLa S3 cells treated with 125 µM CoCl(2) were used as starting material for the large scale purification of HIF-1. To purify HIF-1 by DNA affinity chromatography, the constitutive DNA binding activity must first be separated from HIF-1 since both bind specifically to the W18 DNA sequence(15) . Various ion-exchange resins and gel filtration matrices were examined. HIF-1 was retained on DEAE anion-exchange resins in buffer Z-100, whereas constitutive DNA binding activity was found in the flowthrough (data not shown). HIF-1 DNA binding activity was eluted with 250 mM KCl in buffer Z. DEAE-Sepharose chromatography effectively removed constitutive DNA binding activity and resulted in a 4-fold purification of HIF-1 (Fig. 2A, lanes 1 and 2). This step, however, appeared to destabilize the HIF-1 protein complex and resulted in a faster migrating form of HIF-1 (Fig. 2A, lane 2, second arrow), which was also occasionally seen in crude nuclear extract preparations. This faster migrating form could be converted to the slower migrating HIF-1 band at higher salt concentrations (data not shown), and HIF-1 appeared predominantly as the slower migrating form again after the first round of DNA affinity column chromatography (Fig. 2A, lanes 10-12), suggesting that no HIF-1 component was lost during the DEAE-Sepharose chromatography step. Probe binding of both HIF-1 forms could be competed by unlabeled W18 (Fig. 2B, lanes 2-4) but not M18 oligonucleotide (Fig. 2B, lanes 5-7), which contained a three-base pair substitution (see ``Materials and Methods'') that abolished the ability of the EPO enhancer to mediate hypoxia-inducible transcription (15) .


Figure 2: Purification of HIF-1 from CoCl(2)-treated HeLa S3 cells. A, gel shift assay of column fractions for HIF-1 DNA binding activity. Nuclear extracts were fractionated by DEAE-Sepharose chromatography, and fractions containing HIF-1 activity were applied to a W18 DNA affinity column. 5 µg of protein were incubated with 0.1 µg of calf thymus DNA for gel shift analysis of crude nuclear extract (Crude NE, lane 1) and HIF-1 active fractions from DEAE-Sepharose columns (DEAE, lane 2). For fractions from the W18 column (lanes 3-13), 1-µl aliquots were incubated with 5 ng of calf thymus DNA. The positions of the two HIF-1 bands, constitutive activity (C), nonspecific activity (NS), and free probe (F) are indicated. FT, flowthrough. 0.25 M, 0.5 M, 1 M, and 2 M are fractions eluted with indicated concentration of KCl in buffer Z. B, sequence-specific DNA binding of the partially purified fractions. Aliquots (5 µg) of fractions from the DEAE-Sepharose column were incubated with W18 probe in the presence of no competitor (lane 1), 10-fold (lanes 2 and 5), 50-fold (lanes 3 and 6), or 250-fold (lanes 4 and 7) molar excess of unlabeled W18 (W; lanes 2-4) or M18 (M; lanes 5-7) oligonucleotide.



Partially purified HIF-1 fractions were then incubated with nonspecific competitor calf thymus DNA at concentrations that allowed optimal detection of HIF-1 DNA binding activity by gel shift assays and applied to a W18 DNA affinity column. HIF-1 was eluted in 0.5 M and 1 M KCl (Fig. 2A, lanes 10 and 11). Fractions containing HIF-1 were pooled and dialyzed against buffer Z-100. To eliminate nonspecific DNA-binding proteins that were not removed by calf thymus DNA competitor, the dialysate was applied to an M18 DNA column. HIF-1 DNA binding activity was detected in the flowthrough, which was then applied directly onto second W18 column. HIF-1 activity was detected exclusively in 0.5 M KCl fractions (data not shown). Two rounds of W18 and one round of M18 column chromatography resulted in a purification of 2,800-fold.

The results of the final large scale purification are summarized in Table 1. From 120 liters of HeLa cells, approximately 60 µg of highly purified HIF-1 were obtained (Table 1). The total purification was 11,250-fold and yielded approximately 22% of the starting of HIF-1 DNA binding activity (Table 1). Our objective was to identify HIF-1 subunits and isolate HIF-1 components for the purpose of peptide mapping and protein microsequencing analysis. Since additional steps of purification resulted in markedly lower yield, we did not purify HIF-1 further to homogeneity. Aliquots from flowthrough of the M18 column (Fig. 3A, Load) as well as the 0.25 M KCl wash and 0.5 M KCl elute fractions of the second W18 column were analyzed by 6% SDS-PAGE and silver staining. Four polypeptides of 90-120 kDa were highly enriched in the 0.5 M KCl fraction (lane 3, arrows), which had high HIF-1 DNA binding activity compared with the 0.25 M KCl fraction (lane 2), which had very little HIF-1 activity. The 0.5 M KCl fraction, however, still had many of the contaminant proteins found in the 0.25 M KCl fraction.




Figure 3: Analysis of affinity-purified HIF-1 by SDS-PAGE. A, purification of HIF-1 from CoCl(2)-treated HeLa S3 cells. Flowthrough fraction from the M18 DNA column (Load, lane 1) and 0.25 M KCl and 0.5 M KCl fractions from the second W18 DNA affinity column (lanes 2 and 3) were analyzed. An aliquot of each fraction (5 µg of load or 1 µg of affinity column fractions) was resolved by 6% SDS-PAGE and silver stained. B, purification of HIF-1 from hypoxic Hep3B cells. HIF-1 fractions from the first W18 column (Load, lane 1) and 0.25 M KCl and 0.5 M KCl fractions from the second W18 column (lanes 2 and 3) were analyzed. An aliquot of each fraction (50 µl) was resolved by 7% SDS-PAGE and silver stained. Molecular mass markers are myosin (200 kDa), beta-galactosidase (116 kDa), phosphorylase (97 kDa), BSA (66 kDa), and ovalbumin (45 kDa). HIF-1 polypeptides in lanes3 are indicated by arrows at right of each figure.



In an initial pilot purification of HIF-1 from hypoxia-induced Hep3B cells, a different purification protocol was used. Gel filtration over a Sephacryl S-300 column was also found to be effective in separating HIF-1 from constitutive DNA binding activity (data not shown). In addition, a calf thymus DNA column was used to remove nonspecific DNA-binding proteins prior to two rounds of W18 DNA affinity chromatography. HIF-1 activity was detected in 0.5 M KCl fractions from both DNA affinity columns. An aliquot from the 0.5 M KCl elute fraction of the first W18 column (Fig. 3B, Load) as well as the 0.25 M KCl wash and 0.5 M KCl elute fractions of the second W18 column were analyzed by 7% SDS-PAGE and silver staining. Four polypeptides of similar molecular mass to those that co-purified with HIF-1 DNA binding activity in CoCl(2)-treated HeLa cells were present in the affinity-purified preparation from hypoxic Hep3B cells (lane 3, arrows), indicating that HIF-1 from the two different cell types is composed of the same polypeptide subunits. Affinity-purified HIF-1 from both CoCl(2)-treated HeLa cells and hypoxic Hep3B cells bound specifically to the W18 probe in gel shift assays (data not shown).

HIF-1 Contains Four Polypeptides of Apparent Molecular Mass 120, 94, 93, and 91 kDa

To identify polypeptides that are part of the HIF-1bulletDNA binding complex, preparative gel shift assays were performed with affinity-purified HIF-1 and W18 probe. Gel slices containing the HIF-1bulletDNA complex were isolated, inserted directly into the wells of an SDS-polyacrylamide gel, and analyzed by electrophoresis in parallel with an aliquot of affinity-purified HIF-1 (Fig. 4A). Four polypeptides present in the HIF-1 complex migrated with an apparent molecular weight of 120, 94, 93, and 91 kDa, respectively (Fig. 4A, HIF-1). None of these peptides were detected in gel slices isolated from other regions of the same lane. These four polypeptides migrated at the same positions as the polypeptides that co-purified with HIF-1 DNA binding activity by DNA affinity chromatography (Fig. 4A, lane A). The 120-kDa polypeptide and the 91-94-kDa polypeptides appear to be present in an equimolar ratio, suggesting that the 120-kDa polypeptide forms complexes with any one of the 91-, 93-, and 94-kDa polypeptides.


Figure 4: Identification of HIF-1 polypeptides. A, identification of HIF-1 components on a 6% SDS-polyacrylamide gel with 3.2% cross-linking. An aliquot of affinity-purified HIF-1 (A) was resolved on a 6% SDS-polyacrylamide gel along with the HIF-1 protein complex isolated by a preparative native gel shift assay (HIF-1). MW, molecular mass markers with size (kDa) indicated at left. Numbers to the right indicate the apparent molecular mass (in kDa) of HIF-1 polypeptides. B, identification of HIF-1 components on a 6% SDS-polyacrylamide gel with 5% cross-linking. An aliquot of affinity-purified HIF-1 was resolved on a 6% SDS-polyacrylamide gel along with the HIF-1 protein complex isolated by a preparative gel shift assay. The 120-kDa polypeptide, 94/93/91-kDa polypeptides, and two contaminant proteins (1 and 2) are indicated. C, alignment of HIF-1 components identified on two gel systems with different degrees of cross-linking. Gel slices isolated from a 6% SDS-polyacrylamide gel with 5% cross-linking corresponding to 120-kDa HIF-1 polypeptide (120), 94/93/91-kDa HIF-1 polypeptides (94/93/91), and two contaminant proteins (1 and 2) were resolved on a 6% SDS-polyacrylamide gel with 3.2% cross-linking in parallel with an aliquot (30 µl) of affinity-purified HIF-1 (A).



On a 6% SDS-polyacrylamide gel with 3.2% cross-linking, the 120 kDa HIF-1 polypeptide migrated very close to a contaminant polypeptide of slightly greater apparent molecular weight (Fig. 4A, lane A), making isolation of the 120 kDa polypeptide difficult. This problem was resolved by separating the HIF-1 polypeptides on a 6% SDS-polyacrylamide gel with 5% cross-linking. The 120-kDa polypeptide migrated much faster on the more highly cross-linked gel relative to the migration of the 116-kDa molecular mass marker, whereas migration of the contaminant band (1) was unchanged (Fig. 4B, lane A). Under these conditions, however, the 91-kDa polypeptide ran very close to another contaminant band (2) below it. Two polyacrylamide gel systems with different degrees of cross-linking were therefore required for the isolation of the 91-94-kDa and the 120-kDa HIF-1 polypeptides, respectively.

To confirm that the HIF-1 polypeptides identified by the two gel systems were identical, two dimensional denaturing gel electrophoresis was performed. Affinity-purified HIF-1 was first resolved on a 6% SDS-polyacrylamide gel with 5% cross-linking (as in Fig. 4B, lane A). Regions of the gel containing the 120-kDa, 94/93/91-kDa HIF-1 polypeptides, as well as the two contaminant bands, were isolated and analyzed by electrophoresis on a 6% SDS-polyacrylamide gel with 3.2% cross-linking in parallel with an aliquot of the affinity-purified HIF-1. As shown in Fig. 4C, the isolated HIF-1 and contaminant polypeptides co-migrate with the corresponding bands in the control sample, indicating that the differences in their migration were due to different degrees of cross-linking of the SDS-polyacrylamide gels.

HIF-1 Is Composed of Two Subunits, HIF-1alpha and HIF-1beta

To determine whether the four polypeptides from the HIF-1 complex represent distinct protein species, tryptic peptide mapping was performed. The 91 kDa band was isolated individually while the 93 and 94 kDa bands were excised together after electrophoretic separation and transfer to a polyvinylidene difluoride membrane. Proteins were digested with trypsin in situ, and the tryptic peptides were separated by reverse phase HPLC (Fig. 5A). The elution profiles of tryptic peptides derived from 91-kDa protein and 93/94-kDa proteins were nearly superimposable (Fig. 5A), suggesting that they were derived from similar polypeptides. Another aliquot of HIF-1 was resolved on a 6% polyacrylamide gel of 5% cross-linking for isolation of the 120-kDa HIF-1 polypeptide. The tryptic peptide elution profile derived from the 120-kDa polypeptide was distinct from those of the 91-94-kDa polypeptides (data not shown). These results suggest that HIF-1 is composed of two different subunits, 120-kDa HIF-1alpha and 91/93/94-kDa HIF-1beta.


Figure 5: Analysis of HIF-1 subunits. A, reverse phase HPLC of tryptic peptides derived from 91- and 93/94-kDa polypeptides of HIF-1. Absorbance profile at 215 nm of tryptic peptides derived from 91-kDa HIF-1 polypeptide (top), 93/94-kDa polypeptides (middle), and trypsin (bottom). B, HIF-1alpha and HIF-1beta contact DNA directly. UV cross-linking analysis with affinity-purified HIF-1 and probe W18 in the absence (lane 1) or presence of 250-fold molar excess of unlabeled W18 (lane 2) or M18 (lane 3) oligonucleotide. The binding reaction mixtures were UV-irradiated and analyzed on a 6% SDS-polyacrylamide gel. Molecular mass standards are indicated at left.



HIF-1alpha and HIF-1beta Both Contact DNA Directly

To identify the DNA-binding subunit(s), affinity-purified HIF-1 was incubated with W18 probe. After UV irradiation to cross-link the DNA-binding proteins to nucleotide residues at the binding site, the reaction mixtures were boiled in Laemmli buffer and resolved by SDS-PAGE, and cross-linked proteins were visualized by autoradiography. Two DNA-binding proteins were detected (Fig. 5B, lane 1). Their molecular masses were estimated to be approximately 120 and 92 kDa (after the 16-kDa molecular mass contributed by probe DNA was subtracted), similar to those of HIF-1alpha and HIF-1beta. The binding of both proteins to the probe was sequence-specific since it could be competed by unlabeled wild-type W18 (lane 2) but not mutant M18 (lane 3) oligonucleotide. These results suggest that both HIF-1alpha and HIF-1beta contact DNA directly. HIF-1alpha was cross-linked to DNA much more strongly than HIF-1beta (lanes 1 and 3). Similar results were obtained with cross-linked HIF-1bulletDNA complex isolated from a gel shift assay with crude nuclear extract prepared from hypoxic Hep3B cells(20) . These data provided further evidence that the four polypeptides purified by DNA affinity chromatography are bona fide components of HIF-1 DNA binding activity.

HIF-1 Exists Predominantly in a Dimeric Form in Solution

To estimate the native size of HIF-1, glycerol gradient sedimentation analysis was performed with crude nuclear extract prepared from hypoxic Hep3B cells. HIF-1 and the constitutive DNA binding activity were monitored by gel shift assays. In hypoxic Hep3B nuclear extracts, HIF-1bulletDNA complexes are present in two forms, whereas in CoCl(2)-treated HeLa extracts, the faster migrating form predominates(20, 23) . The results shown in Fig. 6demonstrate that the two bands of the HIF-1 doublet are separable by sedimentation. The faster migrating form was estimated to have a molecular mass of approximately 200-220 kDa. Longer exposure of the autoradiograph (not shown) revealed that the slower migrating band comigrated with ferritin, which has a molecular mass of 440 kDa. Assuming a globular conformation for both protein complexes, these results are consistent with the hypothesis that the faster migrating form represents a heterodimeric complex, consisting of a 120-kDa HIF-1alpha subunit and a 91-94-kDa HIF-1beta subunit, whereas the slower migrating form may represent a heterotetramer. The exact nature and stoichiometry of these HIF-1 complexes, however, remains to be determined. The constitutive DNA binding activity has a molecular mass less than the 67-kDa BSA protein. Since UV cross-linking analysis indicated that the constitutive factor has a DNA-binding subunit of approximately 40-50 kDa(20) , it is most likely that the constitutive factor binds DNA as a monomer. Consistent with the results of glycerol gradient sedimentation analysis, HIF-1 eluted from a Sephacryl S-300 gel filtration column before the constitutive binding activity, and the slower migrating HIF-1 gel shift activity eluted before the faster migrating form (data not shown). These results suggest that HIF-1 exists predominantly as a heterodimer in solution and to a lesser extent as a higher order complex and that these complexes contain at least one HIF-1alpha and one HIF-1beta subunit.


Figure 6: Glycerol gradient sedimentation analysis. Nuclear extract prepared from Hep3B cells exposed to 1% O(2) for 4 h (Load) was sedimented through a 10-30% linear glycerol gradient. Aliquots (10 µl) from each fraction were analyzed by gel shift assay. Arrows at top indicate the peak migration for ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), and BSA (67 kDa).




DISCUSSION

HIF-1 was identified as a nuclear protein that binds to the hypoxia-inducible enhancer in the 3`-flanking region of the EPO gene(15) . Several lines of evidence support the proposed role of HIF-1 as a physiological regulator of EPO transcription in response to hypoxia: (i) induction of both HIF-1 activity (15) and EPO transcription in hypoxic cells (22) requires de novo protein synthesis; (ii) treatment of hypoxic cells with the protein kinase inhibitor 2-aminopurine inhibits induction of both HIF-1 DNA binding activity and EPO RNA (19) ; (iii) the kinetics of HIF-1 induction by hypoxia and of HIF-1 decay after return to normoxia parallel EPO transcriptional activity(19) ; (iv) CoCl(2) and desferrioxamine induce EPO expression and HIF-1 activity(19, 20) . In addition to regulating EPO transcription, HIF-1 appears to play a more general role in regulating other hypoxia-inducible genes as well(5, 20) .

In order to investigate the biochemical properties of HIF-1 and the role of HIF-1 in the hypoxia signal transduction pathway, it is necessary to clone the genes encoding HIF-1 subunits. Our initial attempt to clone the DNA-binding subunit of HIF-1 by screening cDNA expression libraries with oligonucleotide W18 was unsuccessful. This may be in part due to the fact that HIF-1 binds DNA as a heterodimer and that the DNA binding activity of HIF-1 requires protein phosphorylation(19) . Many transcription factors have been purified biochemically by DNA affinity chromatography(27, 32) . However, HIF-1 has a very rapid dissociation rate from DNA with a half-time of less than 1 min(19) , a factor that probably reduced the yield obtained by affinity chromatography. The binding of HIF-1 to DNA may have been stabilized by using concatenated oligonucleotides containing the HIF-1 binding site (but note HIF-1 in flowthrough fractions of the affinity column in Fig. 2A). For the purpose of large scale protein purification, CoCl(2)-treated HeLa S3 cells were used. The mechanism of HIF-1 induction by CoCl(2) is unknown. Co may substitute for ferrous iron in the heme moiety of a putative hemoprotein oxygen sensor, resulting in a fixed deoxy conformation(22) . Induction of HIF-1 and EPO RNA in Hep3B cells by hypoxia and CoCl(2) have similar kinetics(10, 11, 20) , and the effects of these stimuli are not additive(23) , suggesting a common mechanism of HIF-1 induction by these two stimuli. HIF-1 activity induced by hypoxia and CoCl(2) are indistinguishable with respect to DNA binding specificity and contacts with target DNA sequences. Furthermore, the four polypeptides identified as HIF-1 components in CoCl(2)-treated HeLa cells also co-purified with HIF-1 DNA binding activity in hypoxic Hep3B cells (Fig. 3).

Purification of HIF-1 required separation of HIF-1 DNA binding activity from a constitutive activity since both factors specifically bind to the EPO enhancer. Several lines of evidence suggested that the constitutive DNA binding activity is not part of the HIF-1 complex: (i) UV cross-linking analysis identified DNA-binding subunits of different molecular weights in the HIF-1 and constitutive activities isolated from a preparative gel shift assay(20) ; (ii) HIF-1 is highly sensitive to heat treatment, whereas the constitutive activity remained mostly intact when extracts were incubated at 65 °C for 15 min or at 80 °C for 2 min (data not shown); (iii) HIF-1 and the constitutive activity bind an overlapping DNA sequence but have distinct methylation interference patterns ((19) ; Fig. 1B); (iv) HIF-1 and constitutive activity can be separated according to their biochemical properties by ion-exchange, glycerol gradient sedimentation, and gel filtration chromatography; (v) they have a different sensitivity to the alkylation reagent N-ethylmaleimide (data not shown).

In this report we provide evidence to support the conclusion that the 120-, 94-, 93-, and 91-kDa polypeptides are components of HIF-1. First, all four polypeptides co-purified with HIF-1 DNA binding activity in both Epo-producing Hep3B cells and non-Epo-producing HeLa cells, in which HIF-1 was induced by the physiological stimulus hypoxia and the chemical inducer CoCl(2), respectively (Fig. 3). Second, the HIF-1bulletDNA complex isolated from native polyacrylamide gels contained these four polypeptides (Fig. 4). Finally, the molecular sizes of these polypeptides are consistent with the size of DNA-binding proteins detected by UV cross-linking analysis with either crude nuclear extracts or affinity-purified HIF-1 ((20) , Fig. 5B). The 94-, 93-, and 91-kDa polypeptides have a similar profile of tryptic peptides (Fig. 4A), suggesting that they represent related sequences. The difference in their apparent molecular masses could be due to differences in post-translational modification. To determine whether HIF-1 is a glycoprotein as is the case for several other transcription factors(33, 34) , we applied nuclear extracts from CoCl(2)-treated HeLa cells onto a wheat germ agglutinin affinity column. HIF-1 was found in the flowthrough (data not shown), suggesting that HIF-1 is unlikely to be modified by glycosylation. The possibility of protein phosphorylation can be investigated when antibodies against HIF-1 are available. Alternatively, the 91-94-kDa species could represent proteolytic products generated during purification or the differential usage of small exons not detected by tryptic peptide mapping.

Glycerol gradient sedimentation analysis indicated that HIF-1 is present in solution as stable protein complexes. The estimation of native sizes of the HIF-1 complexes suggested that HIF-1 exists either as a heterodimer, consisting of one 120-kDa HIF-1alpha subunit and one of the 91-94-kDa HIF-1beta subunits or as a heterotetramer, which corresponds to the lower and upper bands of the HIF-1 doublet detected by gel shift assay, respectively. The proteins in each band of the HIF-1 doublet have same DNA binding specificity (15) and make identical binding site contacts(19) . At least one 120-kDa HIF-1alpha and one 91-94-kDa HIF-1beta are also present in the larger protein complex since UV cross-linking analysis of the isolated slower migrating band identified both subunits(20) . The exact stoichiometry of the larger form of HIF-1, however, is unknown. It is possible that protein component(s) other than HIF-1 subunits are also part of the complex or contribute to the stabilization of the complex. The larger form of HIF-1 is more abundant in nuclear extracts of hypoxic Hep3B cells than in nuclear extracts of CoCl(2)-treated Hep3B cells, hypoxic HeLa cells, or CoCl(2)-treated HeLa cells(20) .

To study the role of HIF-1 in the hypoxia signal transduction pathway, it is necessary to clone the genes encoding HIF-1 subunits in order to express recombinant HIF-1 proteins and to generate antibodies against HIF-1 subunits. As a first step, we have biochemically purified HIF-1 from HeLa cells. Since the hypoxic induction of HIF-1 DNA binding activity requires on-going transcription and translation, it is possible that genes encoding HIF-1 subunits are hypoxia-inducible. Alternatively, the newly synthesized component could be a modifying enzyme of HIF-1, such as a protein kinase or phosphatase. Phosphorylation is known to be involved in the hypoxic induction of HIF-1 DNA binding activity (19) and EPO gene expression(35, 36) . Protein microsequence analysis of purified HIF-1 subunits will allow the isolation of cDNA sequences necessary to establish the precise mechanisms of HIF-1 induction.


FOOTNOTES

*
This work was supported in part by grants from the Council for Tobacco Research, the Lucille P. Markey Charitable Trust, and the NIDDK, National Institutes of Health (Grant DK 39869). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
A Lucille P. Markey Scholar. To whom correspondence should be addressed: Johns Hopkins Hospital, CMSC-1004, 600 N. Wolfe St., Baltimore, MD 21287-3914. Tel: 410-955-1619; Fax: 410-955-0484.

(^1)
The abbreviations used are: Epo, erythropoietin; HIF-1, hypoxia-inducible factor 1; DTT, dithiothreitol; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

We thank Dr. Thomas Kelly, Jr., in whose laboratory the large scale cell culture and initial purification were performed. We thank Drs. Dan Herendeen, George Brush, and other Kelly lab members for providing assistance and valuable advice; Drs. Chi Dang, and Gary Pasternack for helpful suggestions; Dr. Dan Herendeen for critical reading of the manuscript; and Drs. David Speicher and David Reim of the Wistar Protein Microchemistry Laboratory for expertise in tryptic peptide mapping.


REFERENCES

  1. Maniatis, T., Goodbourn, S., and Fischer, J. A. (1987) Science 236, 1237-1244 [Medline] [Order article via Infotrieve]
  2. Czyzyk-Krzeska, M. F., Furnari, B. A., Lawson, E. E., and Millhorn, D. E. (1994) J. Biol. Chem. 269, 760-764 [Abstract/Free Full Text]
  3. Kourembanas, S., Marsden, P. A., McQuillan, L. P., and Faller, D. V. (1991) J. Clin. Invest. 88, 1054-1057 [Medline] [Order article via Infotrieve]
  4. Webster, K. A., Gunning, P., Hardeman, E., Wallace, D. C., and Kedes, L. (1990) J. Cell. Physiol. 142, 566-573 [Medline] [Order article via Infotrieve]
  5. Semenza, G. L., Roth, P. H., Fang, H.-M., and Wang, G. L. (1994) J. Biol. Chem. 269, 23757-23763 [Abstract/Free Full Text]
  6. Kourembanas, S., Hannan, R. L., and Faller, D. V. (1990) J. Clin. Invest. 86, 670-674 [Medline] [Order article via Infotrieve]
  7. Shweiki, D., Itin, A., Soffer, D., and Keshet, E. (1992) Nature 359, 843-845 [CrossRef][Medline] [Order article via Infotrieve]
  8. Krantz, S. B. (1991) Blood 77, 419-434 [Medline] [Order article via Infotrieve]
  9. Jelkmann, W. (1992) Physiol. Rev. 72, 449-149 [Free Full Text]
  10. Schuster, S. J., Badiavas, E. V., Costa-Giomi, P., Weinmann, R., Erslev, A. J., and Caro, J. (1989) Blood 73, 13-16 [Abstract]
  11. Goldberg, M. A., Gaut, C. C., and Bunn, H. F. (1991) Blood 77, 271-277 [Abstract]
  12. Beck, I., Ramirez, S., Weinmann, R., and Caro, J. (1991) J. Biol. Chem. 266, 15563-15566 [Abstract/Free Full Text]
  13. Pugh, C. W., Tan, C. C., Jones, R. W., and Ratcliffe, P. J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10553-10557 [Abstract]
  14. Semenza, G. L., Nejfelt, M. K., Chi, S. M., and Antonarakis, S. E. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5680-5684 [Abstract]
  15. Semenza, G. L., and Wang, G. L. (1992) Mol. Cell. Biol. 12, 5447-5454 [Abstract]
  16. Beck, I., Weinmann, R., and Caro, J. (1993) Blood 82, 704-711 [Abstract]
  17. Blanchard, K. L., Acquaviva, A. M., Galson, D. L., and Bunn, H. F. (1992) Mol. Cell. Biol. 12, 5373-5385 [Abstract]
  18. Madan, A., and Curtin, P. T. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3928-3932 [Abstract]
  19. Wang, G. L., and Semenza, G. L. (1993) J. Biol. Chem. 268, 21513-21518 [Abstract/Free Full Text]
  20. Wang, G. L., and Semenza, G. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4304-4308 [Abstract]
  21. Beru, N., McDonald, J., Lacombe, C., and Goldwasser, E. (1986) Mol. Cell. Biol. 6, 2571-2575 [Medline] [Order article via Infotrieve]
  22. Goldberg, M. A., Dunning, S. P., and Bunn, H. F. (1988) Science 242, 1412-1415 [Medline] [Order article via Infotrieve]
  23. Wang, G. L., and Semenza, G. L. (1993) Blood 82, 3610-3615 [Abstract]
  24. Maxwell, P. H., Pugh, C. W., and Ratcliffe, P. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2423-2427 [Abstract]
  25. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11, 1474-1489
  26. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  27. Kadonaga, J. T., and Tjian, R. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5889-5893 [Abstract]
  28. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  29. Switzer, R. C., Merril, C. R., and Shifrin, S. (1979) Anal. Biochem. 98, 231-237 [Medline] [Order article via Infotrieve]
  30. Towbin, H., Stachelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354 [Abstract]
  31. Best, S., Reim, D. F., Mozdzanowski, J., and Speicher, D. W. (1994) in Techniques in Protein Chemistry V (Crabb, J. W., ed) pp. 205-213, Academic Press, San Diego
  32. Rosenfeld, P. J., and Kelly, T. J. (1986) J. Biol. Chem. 261, 1398-1408 [Abstract/Free Full Text]
  33. Jackson, S. P., and Tjian, R. (1989) Proc. Natl. Acad. Sci. U. S. A. 80, 1781-1785
  34. Lichtsteiner, S., and Schibler, N. (1989) Cell 57, 1179-1187 [Medline] [Order article via Infotrieve]
  35. Jelkmann, W., Huwiler, A., Fandrey, J., and Pfeilschifter, J. (1991) Biochem. Biophys. Res. Commun. 179, 1441-1448 [Medline] [Order article via Infotrieve]
  36. Kurtz, A., Eckardt, K.-U., Pugh, C., Corvol, P., Fabbro, D., and Ratcliffe, P. (1992) Am. J. Physiol. 262, C1204-C1210

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.