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Biocenter Oulu and Department of Clinical Chemistry (T.P.,
M.P., P.V., R.V.) University of Oulu FIN-90220 Oulu,
Finland
Hauptman-Woodward Medical Research Institute, Inc.
(D.G.) Buffalo, New York 14203
Roswell Park Cancer
Institute (D.G.) Buffalo, New York 14263
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Human 17-HSD type 1 mainly catalyzes the reduction of estrone to a biologically more active estrogen, estradiol (9, 10), both in steroidogenic tissues (11, 12, 13) and in certain estrogen target tissues, such as healthy and malignant breast and endometrial tissues (14, 15, 16, 17). It is therefore suggested that the enzyme might have a significant role in the regulation of estrogen exposure and estrogen-dependent growth of breast cancer tissue. Hence, 17-HSD type 1 inhibitors could be a new potential approach in blocking estradiol biosynthesis, both in the gonads and in target tissues.
It has been shown that human 17-HSD type 1 protein is a dimer
consisting of identical subunits of 35 kDa in size (18, 19, 20, 21). The
protein has been purified in several laboratories (11, 18, 20, 22), but
the microheterogeneity (23) and the possible role of phosphorylation in
regulating enzyme activity have not been studied in detail. Based on
the x-ray structure of the enzyme (21), a model for the reaction
mechanism of human 17-HSD type 1 has been recently proposed. According
to this model, the side chains of Ser142,
Tyr155, and Lys159 residues are involved in
hydride transfer by the enzyme. These amino acids are strictly
conserved in the SDR family members, and results of several studies
indicate that the tyrosine residue corresponding to Tyr155
in human 17-HSD type 1 is directly involved in the catalytic action of
SDR members (24, 25, 26, 27, 28). Similarly, substituting arginine, isoleucine,
glutamine, or leucine in place of the largely conserved lysine residue
always abolishes the enzymatic activity totally (25, 26, 27, 29). The role
of the conserved serine residue (Ser142 in human 17-HSD
type 1) in the catalytic activity of any of the enzyme family members
has not been studied so far. The x-ray structure of the bacterial
3
,20ß-hydroxysteroid dehydrogenase-carbenoxolone complex, however,
indicates that Ser139 forms a hydrogen bond with the
hydroxyl group of strictly conserved Tyr152 (30), and
could, therefore, directly facilitate proton transfer (21, 31).
Structural data have also suggested that in human 17-HSD type 1, the
His221 residue could have an important role in substrate
recognition. The data, furthermore, indicate that the side chain of
Glu282 forms a salt bridge with the side chain of
His221, which could further interact with the 3-hydroxyl
group of the substrate (21). In the present study, several central
aspects concerning the biochemical and catalytical properties of human
17-HSD type 1, including dimerization, microheterogeneity, and
phosphorylation, were characterized. In addition, site-directed
substitutions were constructed to evaluate the current model of hydride
transfer and substrate recognition by human 17-HSD type 1, based on the
x-ray structure of the enzyme.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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), native PAGE (Fig. 1B), and Superdex 75 gel
filtration column chromatography (Fig. 4C) confirmed that similar to
human 17-HSD type 1 purified from placental tissue, the resulting
recombinant protein exists as a dimer of 70–78 kDa. The structural
data and the fact that a monomeric stage of the protein was observed in
SDS-PAGE without ß-mercaptoethanol, indicated that the enzyme
monomers were not covalently bound. Hence, to characterize the affinity
between the noncovalently bound enzyme monomers, a series of human type
1 protein dilutions were analyzed by Superdex 75 gel filtration
chromatography. At all the protein concentrations used, the enzyme
migrated in an elution volume corresponding to a homodimer, and
therefore the exact dissociation constant (Kd) of human
17-HSD type 1 could not be measured. The lowest amount of protein
measured in the eluted fractions by immunofluorometric assay was 0.35
ng/ml, indicating that the Kd is less than 5 pmol/liter.
Hence, it is evident that the enzyme exists predominantly as a dimer,
and there is no equilibrium between monomer and dimer in
vitro. The previously resolved x-ray structure of the enzyme dimer
indicated that there are two dimerization helices in each monomer (E
and F), forming a four-helix bundle. In the present study, two
substitutions (Leu111GluVal113Phe and
Ala170Glu+Phe172) were generated to
analyze the role of the dimeric interface in the folding of an active
protein. The substituted proteins eluted in the void volume in Superose
12 gel filtration column chromatography. The data, therefore, indicate
that disrupting the formation of one of the two helices results in an
aggregated protein of more than 300 kDa, while dimeric or monomeric
forms of the enzyme could not be detected. Both of the enzymes
generated were found to be inactive both in vitro (Table 1) and in cultured Sf9 cells (data not shown).
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). The
primary structure of the human 17-HSD type 1 enzyme contains a
potential cAMP-dependent phosphorylation site (Ser134). We
therefore investigated whether phosphorylation could contribute to the
catalytic and/or biochemical properties of human 17-HSD type 1,
including its microheterogeneity. The data indicated that the protein
was efficiently phosphorylated by protein kinase A (PKA) in
vitro (Fig. 2). Furthermore, destroying the
potential phosphorylation site by substituting alanine in place of
Ser134 totally abolished phosphorylation (Fig. 2). The
substitution, however, did not have any significant effects on the
catalytic properties (Table 1) or microheterogeneity (Fig. 1C) of the
purified enzyme. Our studies further indicated that the
Ser134Ala-substituted and the wild type enzymes had
identical enzymatic properties in cultured Sf9 cells (Fig. 3). Identical properties were also observed using MCF-7
human breast cancer cells transiently transfected with the cDNAs for
Ser134Ala-substituted and wild type enzymes (data not
shown). Furthermore, in mass spectrometric (MALDITOF) analysis of the
purified recombinant enzyme, only one fragment with the correct
molecular mass was observed. This indicates that no significant mass
heterogeneity, resulting, for example, from differential
phosphorylation states, was present. In addition, no electron density
appropriate for a phosphate moiety at the Ser134 side chain
was detected in the crystal structure of the enzyme. The results,
therefore, indicate that human 17-HSD type 1 is phosphorylated by PKA
exclusively at Ser134 in vitro, but this
phosphorylation has no effect on the catalytic properties of 17-HSD
type 1, and most likely it does not occur in vivo.
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), and gel filtration analysis revealed that the
truncated enzyme was still present as a dimer (Fig. 4C) of
approximately 54 kDa. The data, furthermore, indicated that deleting
the last 36 amino acids did not have a dramatic effect on the catalytic
properties measured in cultured Sf9 cells (Fig. 3), and that the
truncated enzyme possessed similar microheterogeneity to that detected
in the native type 1 enzyme (Fig. 4B).
Substitutions at the Active Site of Human 17-HSD Type 1
Recently, a model for the catalytic mechanism of 17-HSD type 1 has
been proposed by utilizing the three-dimensional structure of the
enzyme (21). In this model the side chains of Ser142,
Tyr155, and Lys159 are involved in hydride
transfer between the cofactor and 17-keto group of the substrate, and
the His221 side chain is responsible for substrate
recognition by the enzyme. Our previous activity measurements in
vitro showed that a His221Ala substitution resulted in
a marked (11-fold) decrease in reductive enzyme activity (28).
Unexpectedly, our present data indicate that in cultured Sf9 cells a
His221Ala substitution does not result in a decreased
conversion of E1 to E2, compared with the wild type enzyme (Fig. 5). The Glu282 residue is in close proximity
to His221 and forms a salt bridge with the histidine
residue. Hence, we suggested that Glu282 could replace the
function of His221, and additional substitutions at the
putative substrate recognition helices
(His221AlaGlu282Ala,
His221AlaGlu282Gln, Glu282Ala and
Glu282Gln) were constructed. After substituting alanine or
glutamine in place of Glu282, no major effects on the
catalytic properties of the enzyme were observed in vitro or
in intact cultured cells (Table 1 and Fig. 5), and simultaneous
substitutions of His221 and Glu282 resulted in
similar inactivation of the enzyme in vitro (Table 1) as
observed after the substitution of His221 alone (28).
However, in cultured Sf9 insect cells (Fig. 5), all the
His221 and/or Glu282-substituted proteins
catalyzed the reduction of E1 to E2 in a manner similar to that
observed for the wild type enzyme, which indicates that neither
His221 nor Glu282 is critical for substrate
recognition in vivo.
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) and in cultured Sf9 cells (Fig. 5). The catalytic
efficiencies [turnover rate (kcat)/Michaelis-Menten
constant (Km)] of the substituted enzymes were more than
200-fold lower for both reduction and oxidation (E1 
E2) compared
with the values measured for the wild type enzyme. This indicates that
these amino acids have important roles in the catalytic mechanism of
human 17-HSD type 1. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In line with the results of previous studies (20, 35), the human
17-HSD type 1 protein was found to be expressed exclusively as a
homodimer. Our present results further indicate a strong association
between the subunits, resulting in a stable dimerization state of the
enzyme. Structural data (21) and the activity measurements reported in
the present study indicate that the substitutions
Leu111GluVal113Phe and
Ala170Glu+Phe172 disrupt the dimer interface of
human 17-HSD type 1. When these substitutions were introduced in a
three-dimensional structural model of human 17-HSD type 1 (21),
Leu111Glu substitution was seen to result in a position in
which two negatively charged side chains approach each other in close
proximity, and Val113Phe substitution generated steric
conflict with the region between residues Ala91 to
Leu93 of strand ßD, as well as with the adenosine moiety
of the cofactor. Ala170 resides in a hydrophobic pocket
that contains Val276 from a neighboring subunit and the
Leu251 side chain. The pocket is at a region close to the
dimer interface, stabilizing the interior of the molecule. Hence, both
of the substitutions generated are harmful with respect to the activity
and folding of the enzyme. The fact that disrupting the dimerization of
these helices results in totally inactive aggregates and no monomeric
forms were observed is in line with the highly hydrophobic nature of
their outer surfaces. Construction of a monomeric form of the enzyme
might require a change in the character of the outer surfaces of these
helices, which in turn might result in improper folding of the enzyme.
In addition, it has been suggested that the neighboring amino acids of
the conserved tyrosine and lysine residues in the active site of SDR
family members might interact with the hydrophobic outer surface of the
F-helix and hence stabilize the dimer interface of the proteins
(36). The close proximity of these residues to the catalytic site
responsible for hydride transfer in the enzymes, together with our
results, suggests that monomerization of the dimeric or tetrameric
proteins in the SDR family most likely abolishes their enzyme
activity.
Purified recombinant human 17-HSD type 1 yielded a pattern of three
isoforms resolved by IEF. This disproves the assumption that the
microheterogeneity previously also detected in type 1 enzyme purified
from human placenta (23) would be a result of the frequent protein
polymorphism reported in 17-HSD type 1 (37, 38). The primary structures
of human, rat (39), and mouse (40) 17-HSD type 1 proteins contain a
potential cAMP-dependent phosphorylation site (Ser134). The
three-dimensional structure of the human enzyme revealed that
Ser134 is exposed at the turn between helix E and strand
ßE and could be conducive to phosphorylation. The results of the
present study, together with previous findings (41), indicate that
human 17-HSD type 1 is phosphorylated exclusively at Ser134
by PKA in vitro. However, in our study no indication of a
phosphate moiety at Ser134 was detected by x-ray
diffraction. Furthermore, destroying the potential phosphorylation site
by substituting alanine in place of Ser134 totally
abolished phosphorylation but did not affect the enzymatic properties
of the enzyme. These data are in line with the fact that
Ser134 is not near the catalytic site or at the dimer
interface. We therefore conclude that phosphorylation of
Ser134 does not have a critical role in the function of
human 17-HSD type 1. The results also clearly indicate that
microheterogeneity of the enzyme does not result from the different
phosphorylation states of the Ser134 residue. The reason
for the discrepancy between our detailed data and those previously
described by Barbieri et al. (41) is not known. Their data
indicated that treating BeWo cell lysates with alkaline phosphatase
decreased 17-HSD enzyme activity and that, in addition to serine,
threonine residues were also slightly phosphorylated. However, in the
mass-spectrometric analysis carried out in the present study, such
post-translational modifications were not detected. Furthermore, three
isoforms resolved by IEF showed identical densities after silver
staining (Fig. 1C). This indicates a similar concentration of each of
the isoforms, suggesting that they are not a result of weak
phosphorylation.
The last 43 amino acids of the C terminus of human 17-HSD type 1 could not be resolved in the three-dimensional structure of the enzyme (21). We therefore hypothesized that the C terminus of the enzyme is highly flexible and could form several conformations with differential charges at the outer surface, and thereby could be responsible for the microheterogeneity observed. However, the results showed that the last 36 amino acids of the enzyme do not play any role in the microheterogeneity or catalytic activity of human 17-HSD type 1. In addition, the results of the present study exclude the possibility that fast purification procedures could eliminate the microheterogeneity of the protein, as suggested by Yang et al. (42). In addition, the absence of carbohydrates in the enzyme (18) rules out this source of heterogeneity. The reasons for and functional importance, if any, of the charge differences in human 17-HSD type 1 remain to be clarified. Asparagine and glutamine residues may, for example, undergo deamination, which might not be detectable by mass spectrometry.
In the present study we also evaluated the model for substrate recognition and hydride transfer proposed from the x-ray structure of the enzyme. We constructed several site-directed substitutions at the potential substrate recognition helices of the enzyme. Even though the x-ray structure of the enzyme suggests that the side chain of His221 is involved in substrate recognition, our results indicate that the residue does not have a critical role in substrate binding in vivo. Since the three-dimensional structure of 17-HSD type 1 was determined from the solubilized enzyme in vitro, interaction of the 3-hydroxy group of the substrate with the His221 side chain could possibly occur in vitro only, which is in agreement with our activity measurements in vitro. The nature of the substrate-protein interaction may, however, be modified in vivo, by a membrane association near the active site region. The fact that glycerol or other ampholytes are needed to retain the catalytic activity of the enzyme in vitro is an additional indication of the hydrophobic interaction needed for an active enzyme. Based on the structure of the enzyme, one of the most probable candidate residues able to replace the function of His221 was Glu282. However, the present results indicate that there is no significant interaction between Glu282 and the 3-hydroxy group of the substrate either in vitro or in intact cultured cells.
The current data indicate that Ser142, Lys159, and Tyr155 residues are all essential for the activity of human 17-HSD type 1, which perfectly matches the proposed hydride transfer mechanism (21). With respect to the tyrosine and lysine residues, the results of several studies (24, 25, 26, 29), together with our own, indicate that these strictly conserved residues are essential for the activity of all the members of the SDR family. Our present results also show that the highly conserved serine residue (Ser142 in human 17-HSD type 1) has a significant role in the catalytic action of SDR family members. The side chain of Ser142 forms a hydrogen bond with the hydroxyl oxygen of the Tyr155 residue by donating a proton (21, 30, 31). This could lower the pKa of the Tyr155 side chain proton, thereby allowing it to approach the nucleophilic carbonyl oxygen of the substrate. The role of Ser142 could be similar to that suggested for the positively charged side chain of Lys159, whose proximity to Tyr155 could also have a pKa-lowering effect.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-32P]ATP (3000 Ci/mmol),
[2,4,6,7-3H]estradiol (120 Ci/mmol),
[2,4,6,7-3H]estrone (120 Ci/mmol),
[1,2,6,7-3H]testosterone (105 Ci/mmol), and
[1,2,6,7-3H]androst-4-ene-3,17-dione (110 Ci/mmol) were
purchased from Amersham Life Science (Little Chalfont, UK).
17ß-Estradiol, estrone, testosterone, and androstenedione were the
products of Steraloids Inc. (Wilton, NY). Spodoptera
frugiperda insect cell line (Sf9) was obtained from Invitrogen
(San Diego, CA). All the media, buffers, supplements, and reagents for
cell culture were obtained from GIBCO BRL-Life Technologics (Grand
Island, NY) and the Sigma Chemical Co. (St. Louis, MO). Other reagents
not mentioned in the text were obtained from the Sigma Chemical Co.,
Boehringer Mannheim (Mannheim, Germany), New England Biolabs (Beverly,
MA), and Merck A. G. (Darmstadt, Germany) and were of the highest
purity grade available.
Purification of Recombinant Human 17-HSD Type 1 from Sf9 Insect
Cells
Recombinant Autographa californica
nuclear-polyhedrosis viruses (AcNPVs) for human 17-HSD type 1 were
generated using the BaculoGold Transfection System (Pharmingen, San
Diego, CA) as previously described (28). Sf9 cells were grown at a
density of 2.0 x 106 cells per ml in 500- and 1000-ml
spinner flasks (Techne, Cambridge, UK) in complete TNM-FH insect medium
containing 10% FCS. Exponentially growing cells were infected with
recombinant 17-HSD type 1-AcNPVs at a multiplicity of infection of
1–10 (43). An optimal level of expression was reached after about
70 h, and the cells were then harvested by centrifugation at
1000 x g for 10 min, washed once with PBS, and stored
at -70 C before purification of the enzyme.
Harvested cells were suspended in 6 volumes (vol/vol) of 10 mM potassium phosphate buffer, pH 7.5, containing 1 mM EDTA, 0.5 mM phenylmethylsulfonylfluoride, 0.02% NaN3, and 20% glycerol (vol/vol) and disrupted by sonication (4 x 20 sec at 0.5-min intervals) in an ice bath. Disruption of the cells was controlled by microscopic observation. The cell suspension was centrifuged for 1 h at 100,000 x g. Human 17-HSD type 1 protein was then purified using a cofactor analogy affinity chromatography column (Reactive Red-agarose, Sigma) as previously described (11). The bound proteins were eluted with 250 µM NADP+, and the fractions containing 17-HSD type 1 protein were pooled. Thereafter, the protein was dialyzed against 0.02 M sodium acetate buffer, pH 6.3. The dialyzed sample was loaded onto a Mono Q anion-exchange chromatography column (0.5 x 5 cm) connected to an fast protein liquid chromatography system (Pharmacia Biotech, Uppsala, Sweden), and the proteins were eluted with a linear gradient of sodium acetate (0.02–1.35 M, pH 6.3) at a flow rate of 0.5 ml/min. Fractions containing purified human 17-HSD type 1 were pooled and stored at -70 C. Protein concentrations of the purified samples were measured by Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, CA), using BSA as a standard.
Mass Spectrometry
Purified human 17-HSD type 1 (0.6 nmol) was evaporated and
dissolved in 20 µl 70% trifluoroacetic acid. Thereafter, a sample of
1 µl was subjected to MALDITOF-mass spectrometric analysis (KOMPACT
MALDI III, Kratos Analytical, Manchester, UK).
Modeling of Human 17-HSD Type 1 Proteins
Model building was performed on an SGI Elan workstation with the
refined 2.20 Å x-ray structure of human 17-HSD type 1 (21). The
software used for this purpose was CHAIN, a modified version of FRODO
(44). Substitutions in human 17-HSD type 1 were modeled by replacing
side chains of the amino acids with the substituted ones and
orientating them in favorable conformations. The environments of the
newly introduced side chains were examined for possible steric
conflicts and van der Waals and polar/charge interactions.
Site-Directed Mutagenesis of Human 17-HSD Type 1 Proteins
Ser134Ala, Ser142Ala,
Lys159Ala, His221AlaGlu282Ala,
His221AlaGlu282Gln,
Glu282Ala, Glu282Gln, and
Leu111GluVal113Phe substitutions in human
17-HSD type 1 were generated using the overlap-extension method (45).
The method was also used to construct an enzyme that contained an
Ala170Glu substitution and insertion of Phe at position 172
(Ala170Glu+Phe172). Furthermore, the method was
used to generate human 17-HSD type 1 lacking the last 36 C-terminal
amino acids. The modifications in human 17-HSD type 1 cDNA were
constructed by using flanking primers
(5'-TTATATTAGCGGCCGCACCATGGCCCGCACCGTG-3' and
5'-TATATGAATTCAGGAAGCCTTTACTGCGGGGC-3') and the internal primers
presented in Table 2. In PCR reactions, wild type cDNA
was used as a template except in the cases of
His221AlaGlu282Ala and
His221AlaGlu282Gln substitutions, which were
amplified with a template of previously constructed human
His211Ala-substituted 17-HSD type 1 cDNA (28). All the
constructs generated were confirmed by sequencing.
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Gel Electrophoresis, IEF, and Immunoblotting
The purified recombinant 17-HSD type 1 enzymes were analyzed by
SDS-PAGE, native PAGE, and IEF. The electrophoreses were carried out on
a PhastSystem (Pharmacia Biotech, Uppsala, Sweden) using acrylamide
gradient gels (10–15% for SDS-PAGE and 8–25% for native PAGE), and
IEF was performed on a pH gradient of 3–9. After electrophoreses or
IEF, the proteins were detected by silver staining. Properties of the
wild type and deleted human 17-HSD type 1 proteins were characterized
by immunoblotting. Cytosolic proteins from the Sf9 cell homogenates
were separated by SDS-PAGE (Mini-PROTEAN II, Bio-Rad Laboratories) or
IEF (pH gradient of 3–9, PhastSystem). Thereafter, the proteins were
transferred onto a nitrocellulose membrane and immunostained as
previously described by Poutanen et al. (17) using
polyclonal antibodies raised against the wild type human 17-HSD type 1
enzyme.
Gel Filtration Chromatography
The molecular masses of human 17-HSD type 1 proteins were
determined using Superdex 75 (wild type enzyme and enzyme lacking 36
C-terminal amino acids) and Superose 12 (enzymes having substitutions
at the dimerization helices) gel filtration columns connected to a
SMART System (Pharmacia Biotech). The columns were equilibrated with
0.15 M potassium phosphate, pH 7.2. A 50-µl sample was
then applied, the system was operated at a flow rate of 60 µl/min,
and 30-µl fractions were collected. Migration of the purified native
protein was followed by UV spectrometry at 280 nm, whereas migration of
the substituted human 17-HSD type 1 proteins in the cytosolic fractions
of Sf9 cell lysates were followed by measuring 17-HSD type 1
concentrations in the collected fractions using an immunofluorometric
assay (47).
Identical conditions were used to determine the Kd value of the monomers of purified human 17-HSD type 1 using a Superdex 75 gel filtration column. The purified enzyme was applied to the column at different concentrations (10, 5, 1, 0.5, 0.1, and 0.05 µg/ml), and the 17-HSD type 1 concentrations in the collected fractions were measured by immunofluorometric assay (47).
Measurement of 17-HSD Type 1 Activity In Vitro and In
Cultured Cells
The activity of 17-HSD was measured in vitro as
previously described by Puranen et al. (28). Briefly,
samples were diluted in 10 mM potassium phosphate, pH 7.5,
containing 0.01% BSA and were then mixed with
[3H]estradiol or [3H]estrone (0.73–7.3
µmol of substrate/liter). The reactions were started by adding a
cofactor (NAD+/NADH, Boehringer Mannheim) to a final
concentration of 1.3 mmol/liter, and the samples were incubated for 20
sec at 37 C. After incubation, the reactions were stopped by quickly
freezing the reaction mixtures in an ethanol-dry ice bath. The steroids
were extracted into diethyl ether-ethyl acetate (9:1). The substrates
and the products were then separated in a Sephasil C18 reverse-phase
chromatography column connected to a SMART System (Pharmacia Biotech)
using an acetonitrile-water solution as a mobile phase, as previously
described (28, 48). Alternatively we used an acetonitrile/water (48:52,
vol/vol) solution as a mobile phase in a Symmetry C18 reverse-phase
chromatography column (3.9 x 150 mm) connected to a HPLC system
(Waters, Milford, MA). Radioactivity was measured by an on-line
ß-counter (150TR, FLO-ONE Radiomatic, Packard, Meriden, CT) connected
to the HPLC system, using Ecoscint A scintillation solution (National
Diagnostics, Atlanta, GA). Km and kcat values
for the 17-HSD type 1 enzymes were calculated by using a GraFit-program
(Erithacus Software Ltd., Staines, UK). The program fits data to the
Michaelis-Menten equation using nonlinear regression analysis. The
values presented represent the average ± SD of at
least three independent experiments. One micromole of product formed
per minute was defined as one unit of enzyme activity.
Activity measurements in cultured Sf9 insect cells were carried out by plating the cells in six-well plates at a density of 1.2 x 106 cells per well. The cells were allowed to attach for 1 h and were then infected with 17-HSD type 1-AcNPVs at a multiplicity of infection of 2. After 50 h incubation at 27 C, the culture medium was removed, and both reductive (E1 to E2, A-dione to T) and oxidative (E2 to E1, T to A-dione) activities were measured in the intact cells in culture by adding 2 ml serum-free TNM-FH insect medium, containing 10 µM [3H]substrate (20 nmol/well) to each well. To assess the linearity of the reactions, the activities were measured at four different time points (10, 20, 40, 80 min). After incubation, the media were collected, frozen in dry ice, and kept at -20 C until the steroids were extracted, and the amount of substrate converted was measured as described above.
The concentrations of the constructed enzymes in the Sf9 cell homogenates were measured using a immunofluorometric assay (47). Conversion of E1 to E2 was then calculated according to the amount of enzyme expressed.
Phosphorylation of 17-HSD Type 1 Enzymes in Vitro
Phosphorylation of wild type and
Ser134Ala-substituted human 17-HSD type 1 enzymes was
carried out through the use of PKA in vitro in a buffer
containing 20 mM Tris-HCl, pH 7.5, 10 mM
MgCl2, 1 mM dithiothreitol, 10 µCi
[-32P]ATP, and 20 U of the catalytic subunit of PKA in
a final reaction volume of 50 µl. The amount of substrate was 15 µg
of protein per reaction. After 30 min incubation at 30 C, the reactions
were stopped by keeping them on ice for 10 min.
[-32P]ATP was separated from the reaction mixtures by
using Bio-Spin Chromatography Columns (Bio-Rad Laboratories) according
to the manufacturer’s instructions, after which the reaction mixtures
were subjected to electrophoresis (SDS-PAGE, Mini-PROTEAN II, Bio-Rad
Laboratories). The gel was then stained with Coomassie Blue and dried.
Thereafter, phosphorylated proteins were visualized by
autoradiography.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by the Research Council for Health of the Academy of Finland (project numbers 3314 and 30099), the Technology Development Center of Finland (TEKES, project number 4476), and by NIH Grant DK-26546. The Department of Clinical Chemistry, University of Oulu, is a World Health Organization Collaborating Center for Research in Human Reproduction, supported by the Ministries of Education, Social Affairs and Health, and Foreign Affairs, Finland.
Received for publication June 3, 1996. Accepted for publication October 4, 1996.
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 O. O. Oduwole, Y. Li, V. V. Isomaa, A. Mantyniemi, A. E. Pulkka, Y. Soini, and P. T. Vihko 17{beta}-Hydroxysteroid Dehydrogenase Type 1 Is an Independent Prognostic Marker in Breast Cancer Cancer Res., October 15, 2004; 64(20): 7604 - 7609. [Abstract] [Full Text] [PDF]
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 J. L. Thomas, J. I. Mason, S. Brandt, B. R. Spencer Jr., and W. Norris Structure/Function Relationships Responsible for the Kinetic Differences between Human Type 1 and Type 2 3beta -Hydroxysteroid Dehydrogenase and for the Catalysis of the Type 1 Activity J. Biol. Chem., November 1, 2002; 277(45): 42795 - 42801. [Abstract] [Full Text] [PDF]
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F. Beaudoin, K. Gable, O. Sayanova, T. Dunn, and J. A. Napier A Saccharomyces cerevisiae Gene Required for Heterologous Fatty Acid Elongase Activity Encodes a Microsomal beta -Keto-reductase J. Biol. Chem., March 22, 2002; 277(13): 11481 - 11488. [Abstract] [Full Text] [PDF]
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 Y.-W. Huang, I. Pineau, H.-J. Chang, A. Azzi, V. Bellemare, S. Laberge, and S.-X. Lin Critical Residues for the Specificity of Cofactors and Substrates in Human Estrogenic 17{beta}-Hydroxysteroid Dehydrogenase 1: Variants Designed from the Three-Dimensional Structure of the Enzyme Mol. Endocrinol., November 1, 2001; 15(11): 2010 - 2020. [Abstract] [Full Text] [PDF]
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 T. J. Puranen, R. M. Kurkela, J. T. Lakkakorpi, M. H. Poutanen, P. V. Itäranta, J. P. J. Melis, D. Ghosh, R. K. Vihko, and P. T. Vihko Characterization of Molecular and Catalytic Properties of Intact and Truncated Human 17{beta}-Hydroxysteroid Dehydrogenase Type 2 Enzymes: Intracellular Localization of the Wild-Type Enzyme in the Endoplasmic Reticulum Endocrinology, July 1, 1999; 140(7): 3334 - 3341. [Abstract] [Full Text]
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 A. G. Mensah-Nyagan, J.-L. Do-Rego, D. Beaujean, V. Luu-The, G. Pelletier, and H. Vaudry Neurosteroids: Expression of Steroidogenic Enzymes and Regulation of Steroid Biosynthesis in the Central Nervous System Pharmacol. Rev., March 1, 1999; 51(1): 63 - 82. [Abstract] [Full Text] [PDF]
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 M. W. Sawicki, M. Erman, T. Puranen, P. Vihko, and D. Ghosh Structure of the ternary complex of human 17beta -hydroxysteroid dehydrogenase type 1 with 3-hydroxyestra-1,3,5,7-tetraen-17-one (equilin) and NADP+ PNAS, February 2, 1999; 96(3): 840 - 845. [Abstract] [Full Text] [PDF]
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P. Nokelainen, H. Peltoketo, R. Vihko, and P. Vihko Expression Cloning of a Novel Estrogenic Mouse 17{beta}-Hydroxysteroid Dehydrogenase/ 17-Ketosteroid Reductase (m17HSD7), Previously Described as a Prolactin Receptor-Associated Protein (PRAP) in Rat Mol. Endocrinol., July 1, 1998; 12(7): 1048 - 1059. [Abstract] [Full Text]
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T. Puranen, M. Poutanen, D. Ghosh, R. Vihko, and P. Vihko Origin of Substrate Specificity of Human and Rat 17{beta}-Hydroxysteroid Dehydrogenase Type 1, Using Chimeric Enzymes and Site-Directed Substitutions Endocrinology, August 1, 1997; 138(8): 3532 - 3539. [Abstract] [Full Text]
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