Expression of 11/?-Hydroxysteroid
Dehydrogenase Using Recombinant
Vaccinia Virus
Anil K. Agarwal, Maria-Teresa Tusie-Luna, Carl Monder, and
Perrin C. White
Division of Pediatric Endocrinology
Cornell University Medical College
New York, New York 10021
Population Council (CM.)
New York, New York 10021
Ligand specificity of the type I steroid receptor is
apparently conferred by the activity of 11/3-hydroxysteroid dehydrogenase. To determine the kinetic
properties of this enzyme, rat liver cDNA was expressed in cultured cells using recombinant vaccinia
virus. Although this enzyme catalyzes only dehydrogenation when purified from rat liver, the recombinant enzyme obtained from cell lysates catalyzed
both 11/9-dehydrogenation of corticosterone to 11dehydrocorticosterone and the reverse 11-oxoreduction reaction. At pH 8.5, the first order rate constant Kc.,/Km for dehydrogenase activity exceeded
that for reductase (63 vs. 38 min~1 x 10~4), whereas
the rate constants for the two reactions were nearly
equal (48 vs. 47 min"1 x 10~4) at pH 7.0. These
results are consistent with the previously determined pH optima for these activities in liver microsomes. Removal (with glucose-6-phosphate dehydrogenase) of NADP* produced by the reductase
reaction significantly increased reductase activity.
Glycyrrhetinic acid, a known inhibitor of the dehydrogenase reaction, also inhibited the reductase
reaction at slightly higher concentrations (50% inhibitory concentration, <5 nM for dehydrogenase, 1020 nM for reductase). Partial inhibition of glycosylation with Artunicamycin decreased dehydrogenase
activity 50% without affecting reductase activity.
The data demonstrate that a single polypeptide catalyzes both dehydrogenation and reduction, although the presence of additional enzyme forms
catalyzing one or the other activity has not been
ruled out. (Molecular Endocrinology 4: 1827-1832,
1990)
lyzed by 11/3-hydroxysteroid dehydrogenase (11-HSD).
The 11 ^-dehydrogenase (11-DH) activity of 11-HSD is
found in most mammalian tissues (1). In tissues such
as the kidney that express both 11 -HSD and the type I
(mineralocorticoid) receptor, 11 -HSD may act to confer
ligand specificity on the receptor (2,3). Type I receptors
expressed in cells transfected with cloned receptor
cDNA have equal affinities for cortisol and aldosterone
(4), whereas aldosterone is a normally a much more
potent mineralocorticoid in vivo. Cortisol, which is normally present in blood at much higher concentrations
than aldosterone, acts as a mineralocorticoid if 11 -DH
activity is pharmacologically suppressed by carbenoxolone or glycyrrhetinic acid (2); individuals treated with
these compounds develop hypertension. Severe hypertension is also observed in patients with the rare disorder apparent mineralocorticoid excess (5). These individuals lack 11-DH activity, presumably because of a
genetic defect.
Reduction of cortisone to cortisol (and 11-dehydrocorticosterone to corticosterone) also occurs in vivo,
and patients have been described who are deficient in
11-oxoreductase (11-OR) activity but whose 11-DH
activity is unaffected (6).
There is conflicting evidence for the existence of
distinct enzymes (11-DH and 11-OR) catalyzing the
interconversion of the 110-hydroxy and 11 -oxo groups
and for a single 11 -HSD polypeptide expressing both
11-DH and 11-OR activities. Carbenoxolone and glycyrrhetinic acid preferentially inhibit dehydrogenation in
vitro (7), and patients with apparent mineralocorticoid
excess do not have a demonstrable defect in 11 -OR
activity. Furthermore, purified rat liver 11-HSD has only
11-DH and no 11-OR activity (8).
However, cloned cDNA encoding rat liver 11-HSD
was recently isolated and expressed in cultured
Chinese hamster ovary cells by transfection with a clone
containing an SV40 promoter (9). Transfected cells
acquired both 11-DH and 11-OR activities, suggesting
that both activities are carried within the same polypeptide. Nevertheless, these activities were expressed at
levels that were too low to perform kinetic or physico-
INTRODUCTION
The conversion of cortisol to cortisone (in the rat,
corticosterone to 11-dehydrocorticosterone) is cata0888-8809/90/1827-1832S02.00/0
Molecular Endocrinology
Copyright © 1990 by The Endocrine Society
1827
Vol4No. 12
MOL ENDO-1990
1828
chemical studies, so that it was not clear if the reductase activity of this enzyme was physiologically significant.
To address this problem, we have now expressed
11-HSD at relatively high levels in cultured cells using
recombinant vaccinia virus and confirm that this enzyme
catalyzes both 11/3-dehydrogenation and 11-oxoreduction in a manner indistinguishable from the activities
in rat liver.
RESULTS
Immunoprecipitation of Radiolabeled Proteins
Rabbit antirat 11 -HSD serum precipitated a major labeled polypeptide of 34 kDa from cells infected with
vTF7 and v11-HSD, the same size as 11-HSD from rat
liver (Fig. 1). Two smaller and fainter bands migrating
at about 32 and 31 kDa were also seen. When cells
were treated with 50 ng/ml of the An homolog of tunicamycin (an inhibitor of glycosylation), the 34-kDa band
MW
xio 3
97.4-
decreased in intensity and the 31-kDa band became
more prominent. One and 10 ng/ml tunicamycin had no
effect.
The proportion of total labeled protein represented
by recombinant 11-HSD could not be quantitated in
unfractionated cell lysates due to comigrating polypeptides that interfered with densitometry (not shown).
Conditions Affecting Enzymatic Activity
Initial rate studies were performed on 11-HSD in the
oxidative and reductive directions. Figure 2 shows that
11-DH activity was stable for at least 2 h at pH 8.5 or
7.0. In contrast, 11-OR activity was linear with time for
30 min and rapidly lost activity at both pH values when
incubated for longer times. This pattern is consistent
with that shown for rat liver 11-HSD. The decrease in
11 -OR activity was not modified by the addition of 1030% glycerol, 0.1-5 ITIM dithiothreitol, 0.1-1.0 trypsin
units of aprotinin, 0.1-1.0 HIM phenylmethylsulfonyl fluoride, 1-10 mM EDTA, or combinations of these substances (data not shown). Nevertheless, 0.1 mM phenylmethylsulfonylfluoride and 10 mM EDTA were routinely added to the homogenizing and assay buffers.
To determine whether the apparent decrease in 11 OR activity over time represented instability of the
enzyme, cell lysates were preincubated at 37 C in assay
buffer without added substrate or cofactors for varying
66.242.734.0-
31.0-
21.54
5
35
Fig. 1. Immunoprecipitation of S-Labeled Cell Lysates with
Antiserumto 11-HSD
Cells were infected with v11 -HSD and vTF7 together (lane
1), with vTF7 alone (lane 2), or with both viruses in the
presence of A^tunicamycin at 1, 10, or 50 ng/ml (lanes 3-5,
respectively). Immunoprecipitates were resolved on a 12.5%
polyacrylamide gel and autoradiographed. The positions of
size standards are marked.
Fig. 2. Time Curves for 11-DH (O) and 11-OR (•) activities,
represented as percent conversion of 2 ^M substrate. Top, pH
7.0; bottom, pH 8.5.
Expression of 11-HSD
1829
periods of time, followed by determination of 11-DH
and 11-OR activities. Preincubation for up to 1 h had
no effect on either activity (data not shown).
Reductase activity was also measured at pH 8.5 in
the presence of an excess of glucose-6-phosphate
dehydrogenase and glucose-6-phosphate. This reaction consumes any NADP+ generated by the 11-OR
activity of 11-HSD, thus blocking expression of its 11DH activity. The 11-OR activity was significantly increased under these conditions, and the rate of conversion of substrate to product was linear for at least 2
h (Fig. 3). Dehydrogenase activity was completely inhibited.
It has previously been noted that treatment of rat
liver microsomes with various detergents affected 11 DH and 11 -OR activities (10). Consistent with previous
observations, treatment of cell lysates with 3-[(3-cholamidopropyl)-dimethylammonio]-1 -propane sulfonate
decreased 11-DH activity, with no effect on 11-OR
activity, whereas a small increase in both activities was
observed after treatment with Tritons X-100 and DF18 (data not shown). The latter detergent was routinely
added to cell lysates before assay.
Dehydrogenase activity of cells treated with 50 ng/
ml Ai-tunicamycin was decreased by about 50% compared with that of untreated cells; 11 -OR activity was
unaffected (Fig. 4).
Kinetic Measurements (Table 1)
Increased velocity of 11 /3-dehydrogenation at pH 8.5,
expressed as the first order rate constant, Kcat/Km, is
consistent with a pH optimum of 8.5-9.5, which has
been reported with this enzyme from a variety of
sources (1). The first order rate constant of 11-oxored-
ATunicamycIn
(ng/ml)
0
50
50
Fig. 4. Effect of 50 ng/ml ATTunicamycin Added to Culture
Medium on Subsequent Enzymatic Activity in Whole Cells (1
or 10 ng/ml Had no Effect)
The number of determinations is in parentheses. Limit bars
show the SD. **, P < 0.001.
Table 1. Kinetic Parameters of 11/3-Hydroxysteroid
Dehydrogenase
Enzyme
Activity
pH
DH
O-
8.5
8.5
137 + 20
110 20
2.14 ± 0.46
2.83 ± 0.65
63
7.0
7.0
53 ± 03
68 ± 10
1.10 ± 0.10
1.44 ± 0.31
48
Kcat
(104 x
(104/min)
38
R
DH
OR
47
Values for Kcat and apparent Km are the mean ± SD; substrate
is corticosterone for dehydrogenase reactions and 11 -dehydrocorticosterone for reductase reactions.
uction increased at the lower pH in accord with its
reported pH optimum range of 5.5-6.5. The apparent
Km values for dehydrogenation and reduction did not
differ significantly. They were somewhat increased at
the higher pH value, but this difference did not achieve
statistical significance (P > 0.05).
Transforming the data obtained at pH 7.0 according
to the Hill equation produced Hill coefficients of 1 for
both 11-DH and 11-OR reactions, consistent with a
single active site for each reaction.
Inhibition by Glycyrrhetinic Acid
30
60
90
150
TIME (mln)
Fig. 3. Time Curve for 11 -OR Activity (Represented as Percent
Conversion of 2 ^ M 11-DH) at pH 8.5 in the Presence (•) or
Absence (•) of Glucose-6-Phosphate Dehydrogenase and Glucose-6-Phosphate
No 11 -DH activity was observed in the presence of glucose6-phosphate dehydrogenase.
When enzymatic activity was measured in intact infected cells, 500 nM glycyrrhetinic acid inhibited 11-DH
activity by 50% (32% conversion of corticosterone to
dehydrocorticosterone decreased to 16%), but had a
minimal effect on 11 -OR activity (20% conversion decreased to 17%) over 2.5 h. This conforms with our
previous observations made in Chinese hamster ovary
Vol4No. 12
MOL ENDO-1990
1830
cells transfected with a plasmid containing 11-HSD
cDNA under the control of an SV40 promoter (10) and
with the behavior of 11-HSD in rat kidney.
Activities in cell lysates were affected at much lower
concentrations of glycyrrhetinic acid. Dehydrogenase
activity at pH 7.0 was inhibited 50% by less than 5 nwi
glycyrrhetinic acid, and 11-OR activity was inhibited
50% by about 20 nM (Fig. 5).
DISCUSSION
Recombinant 11 -HSD appears to be identical to purified
rat liver 11-HSD in the size of the polypeptide and in
kinetic properties of its 11-DH activity (Km at pH 8.5 of
2.14 MM for recombinant 11-HSD vs. 1.83 M M for the
liver enzyme). The 11-OR activity of rat liver 11-HSD is
lost during purification, and the recombinant enzyme
also has relatively low 11-OR activity at pH 8.5, which
appears to be reduced further during prolonged incubation with substrate and cofactor. This may be due in
part to dehydrogenation of product by the much more
active 11 -DH activity of the enzyme at this pH, because
removal of all NADP+ (the cofactor for the 11-DH reaction) significantly increases 11-OR activity (Fig. 3). Alternatively, binding of NADP+ under the assay conditions may irreversibly inactivate 11-OR. In support of
the latter explanation, it was not possible to reactivate
11 -OR activity of the rat liver enzyme once it had been
lost even when NADP+ was removed using the same
methods as those employed in the present study
(Lakshmi, V., and C. M., unpublished observations).
At pH 7.0, closer to the optimum for rat liver 11-OR
activity, the 11-OR and 11-DH activities of recombinant
11-HSD have identical first order rate constants (Kcat/
Km) and similar Km values. Both activities are inhibited
in vitro by relatively low concentrations of glycyrrhetinic
acid, although 11-DH is more sensitive to it than 11-
OR. Hill coefficients of 1 for both reactions permit us to
conclude that for each substrate the enzyme contains
no noncatalytic regulatory sites, contains a single independent binding site, and undergoes no measurable
conformational change that affects the velocity constants. Thus, the similarities in the kinetic properties of
11-DH and 11-OR are notable. The data are consistent
with either topographically distinct 11 -OR and 11 -DH
sites on a single polypeptide or a single site that undergoes conformational reordering to favor dehydrogenation or reduction. Further studies will be required to
distinguish between these possibilities. The data are
not consistent with the hypothesis that 11 -DH and 11OR are independent enzymes.
There is at present no satisfactory explanation for
the instability of 11-OR activity or for why, if it is indeed
the reciprocal property of a unique active site, its stability is not equal to that of 11-DH activity. However,
other examples exist of enzymes with multiple activities
that may be differentially affected. For example, purified
bovine or porcine steroid 11/3-hydroxylase (P450c11)
also has 18-hydroxylase and 18-oxidase activities; the
last activity is specifically inhibited by an unknown
mechanism in the zona fasciculata of the adrenal cortex,
but not in the zona glomerulosa (11).
These studies do not rule out the existence of additional 11 -DH or 11 -OR isozymes. It is also possible that
additional isoforms of 11-HSD are generated from the
same gene. Because vaccinia replicates in the cytoplasm, only cDNA can be expressed, and so alternatively spliced mRNA species could not be identified in
these experiments. However, 31- and 32-kDa polypeptides were observed in immunoprecipitates in addition
to the major 34-kDa band. These might be unglycosylated or partially glycosylated products; 11-HSD is a
glycoprotein, and the predicted size of the polypeptide
alone is 31.7 kDa. The increased relative intensity of
the 31-kDa band in lysates from cells treated with A t tunicamycin is consistent with this possibility. Inspection of the cDNA sequence suggests an alternative
possibility: residue 27 is an additional in-frame methionine in a good context (GAAATGC) for initiation of
translation (12), and if ribosomes occasionally initiated
at this internal methionine, the resulting polypeptide
would be about 3 kDa smaller than the full-length
product. This methionine occurs before the predicted
NADP+/NADPH-binding site, and so it is possible that
such a truncated enzyme would still be active (Fig. 6).
11-HSD
17-HSD
5
10
15
20
100
GE(nM)
Fig. 5. Inhibition of 11-DH (O) and 11-OR (•) Activities by
Glycyrrhetinic Acid (GE)
Activities are measured as the percent conversion of 2 HM
substrate.
MKKYLLPVLVLCLGYYYSTNEEFRPE1ILQGKKVIVTGASKGIG
MARTWLITGCSSGIG
+
*++** * ***
Fig. 6. Comparison between the Amino-Terminal Sequence of
11-HSD (9) and That of a Related Enzyme, 17/8-Hydroxysteroid Dehydrogenase (17-HSD) (21). Single letter amino acid
codes are used. Amino acid residues that are identical in these
two enzymes are indicated by asterisks, and functionally conserved residues by crosses. The second methionine residue
in 11 -HSD is underlined; note that if translation were initiated
at this methionine, the two enzymes would have very similar
amino-termini.
1831
Expression of 11-HSD
Tunicamycin-treated cells had a relative decrease in
11-DH activity, suggesting that glycosylation might be
important for full 11 -DH activity. A more definitive way
to determine whether unglycosylated or truncated enzymes differ from the normal enzyme in relative 11 -DH
and 11 -OR activities is to study recombinant enzymes
that have had potential glycosylation sites and/or the
initial methionine modified by in vitro mutagenesis of
the cDNA.
MATERIALS AND METHODS
Construction of Recombinant Vaccinia Virus
A 1.2-kb EcoRI fragment was isolated containing a nearly fulllength cDNA for rat 11 -HSD, including the entire coding region.
The shuttle vector pTF7 (13) (obtained from B. Moss) was
digested with BamH\. The vector and fragment were rendered
blunt-ended with the Klenow fragment of DNA polymerase
and ligated. E. coli transformants carrying the insert were
selected by colony hybridization, and a clone with the insert in
the correct orientation relative to the vector's T7 promoter
was identified by restriction mapping.
TK~143B human osteosarcoma cells were obtained from
American Type Culture Collection and grown in Dulbecco's
Modified Eagle Medium supplemented with 10% fetal calf
serum and 25 (tg/m\ 5-bromodeoxyuridine. Cells were transfected with the pTF7/11-HSD construct and infected with wildtype vaccinia virus strain WR, as previously described (14).
Recombinant viruses lacked thymidine kinase (i.e. they were
tk~) and could form plaques in the presence of bromodeoxyuridine. The presence of 11-HSD cDNA in the viral genome
was verified by dot blot hybridization. One clone was termed
v11-HSD.
Infection of Cells
TK~143B cells were infected simultaneously with v11-HSD
and with vTF7, a recombinant vaccinia virus expressing T7
RNA polymerase under the control of a vaccinia promoter. T7
polymerase synthesized under the direction of vTF7 transcribed 11-HSD cDNA from the T7 promoter included in the
pTF7 shuttle vector used to generate v11-HSD.
Viruses (10:10 multiplicities of infection of for vTF7 and v11 HSD) were adsorbed to cells at 37 C for 60 min in a minimal
volume of serum-free medium with occasional rocking. A,Tunicamycin homolog(Boehringer-Mannheim, Indianapolis, IN)
was added to some wells at 1, 10, or 50 ng/ml immediately
after adsorption of virus. Control plates were infected with
vTF7 alone. Cells were then fed with medium supplemented
with 10% serum. Activity was assayed in intact cells or cell
lysates 24 h after infection.
Preparation of Cell Lysates
Cells were washed once with PBS and once with ice-cold
homogenizing buffer (0.1 M Tris-HCI, pH 8.5-0.1 mM phenylmethylsulfonylfluoride-10 mM EDTA). Cells were scraped off
the culture dishes and homogenized in the same buffer using
a Dounce homogenizer (Kontes Co., Vineland, NJ). The lysate
was centrifuged at 1000 x g for 5 min. The supernatant was
used directly for enzyme assays or frozen in aliquots at - 2 0
C and used within 1 week of preparation. Protein concentrations were determined according to the method of Bradford
(15), using reagents from Bio-Rad (Richmond, CA).
Assays of Enzymatic Activity in Cell Lysates
Except where noted, lysates were treated with Triton DF-18
at a ratio of 0.15 mg detergent/mg protein for 60 min at 4 C
immediately before being assayed. Dehydrogenase activity
was determined by measuring the conversion of corticosterone to 11-dehydrocorticosterone in the presence of NADP+.
Assays were performed in 1 ml buffer containing 0.1 M TrisHCI (pH 8.5), 0.1 mM phenylmethylsulfonylfluoride, 10 mM
EDTA, 250 fiM NADP\ approximately 20,000 cpm [1,2-3H]
corticosterone (SA, 60 Ci/mmol), and 2 ^M unlabeled corticosterone (in kinetic studies 0.125-4.0 MM corticosterone was
used). In some experiments 0.1 M Na2HPO4, pH 7.0, was
substituted for 0.1 M Tris-HCI, pH 8.5. After 10 min of preincubation at 37 C, 50 M9 cell lysate protein were added, and
incubation was continued for various times. Steroids were
extracted into ethyl acetate, and unlabeled corticosterone and
11-dehydrocorticosterone were added as markers. Extracts
were concentrated, and the steroids were separated by TLC
on silica support in chloroform-methanol (95:5, vol/vol) (16).
Spots corresponding to steroids were located under UV light,
cut out, and counted by scintillation spectrophotometry.
Reductase activity was assayed in a similar manner by
measuring the conversion of 11 -dehydro-[1,2-3H]corticosterone [prepared as described previously (16)] to corticosterone
in the presence of NADPH. Assays performed at pH 8.5 used
200 ng protein/reaction.
Reductase activity was also determined at pH 8.5 in the
presence of 2 U glucose-6-phosphate dehydrogenase purified
from L. mesenteroides (Sigma, St. Louis, MO) and 1 mM
glucose-6-phosphate.
Kinetic measurements were performed using a 60-min incubation for 11-DH activity and a 30-min incubation for 11-OR
activity. Reaction rates were linear with time within these
intervals, and less than 25% of substrate was converted under
these reaction conditions. Each experimental point was determined in duplicate, and the data were analyzed using double
reciprocal plots. Data were also analyzed using an adaptation
of the Hill equation (17).
Inhibition of enzyme activity by glycyrrhetinic acid was determined at pH 7.0 using the assay conditions described above
in the presence of 2 ^M steroid substrate. Reductase activity
was assayed during a 15-min incubation using 300 ng protein/
reaction.
All determinations were repeated using lysates prepared
from cells infected with vTF7 helper virus alone. Background
values were subtracted from experimental values before further analysis. Typical background conversions observed during 60-min incubations of lysates from vTF7-infected cells were
approximately 2% for dehydrogenation of corticosterone to
dehydrocorticosterone and approximately 8% for reduction of
dehydrocorticosterone to corticosterone.
Statistical Analysis
Experimental points were plotted using the Enzfit program.
Enzyme kinetic data were fitted to K^/Km as proposed previously (18) for the two-step reaction:
Ki
Ko
K_i
K_2
E + S ^ E S ^ E + P,
where E is 11-DH or 11-OR, and S is the appropriate steroid
substrate. It is assumed that the enzyme is saturated with
cofactor (NADP+ or NADPH > 10 Km) and that the pyridine
nucleotides are not dead-end inhibitors (Monder, C , and V.
Lakshmi, unpublished observations). The Michaelis-Menten
equation may be rewritten as: v = (Km x S x Kca^K^Kn, +
S), where K^t/Kn, = k^zAkz + k_,).
The data were analyzed by nonlinear regression. Parametric
analysis was performed using a weighted least squares curvefitting program with iterative reweighting (19). Significance of
differences between means was determined using two-tailed
Student's f test for the appropriate degrees of freedom.
Vol4No. 12
MOL ENDO-1990
1832
Radiolabeling and Immunoprecipitation
TK~143B cells were seeded in six-well plates and grown to
80% confluency. Cells were infected with vTF7 alone or vTF7
and v11-HSD together. At-Tunicamycin homolog (1-50 ng/ml)
was added to some wells. After 24 h, the medium was changed
to 2 ml medium (including tunicamycin if previously used)
containing 150 fid [35S]methionine/cysteine (1000 Ci/mmol)
without additional methionine and cysteine. After 1 h, cells in
each well were washed once in PBS, collected, and lysed in
250 n\ 0.01 M Tris-HCI, pH 8.0-1% Triton X-100-0.1% sodium
dodecyl sulfate. To 200 n\ lysate were added 300 n\ dilution
buffer (0.1 M Tris-HCI, pH 8.0-0.14 M NaCI-0.1% Triton X100). Lysates were precleared by successive 1 -h incubations
on ice with a 1:100 dilution of normal rabbit serum and a 1:10
volume of protein-A-Sepharose. After centrifugation, a 1:100
dilution of rabbit antirat 11-HSD serum was added to the
supernatant and incubated for 2 h at 4 C. Antigen-antibody
complexes were adsorbed to protein-A-Sepharose and
washed twice in dilution buffer, once in 0.1 M Tris-HCI, pH
8.0-0.14 M NaCI, and once in 0.05 M Tris-HCI, pH 6.8 (20).
Samples were boiled in Laemmli sample buffer and analyzed
by electrophoresis in a 12.5% polyacrylamide gel. The gel was
dried and exposed to Kodak XAR film (Eastman Kodak, Rochester, NY) for 4 days.
Acknowledgments
Received August 27, 1990. Revision received September 20,
1990. Accepted September 20, 1990.
Address requests for reprints to: Dr. Perrin C. White, Division of Pediatric Endocrinology, Cornell University Medical
College, New York, New York 10021.
This work was supported by NIH Grants DK-37094 and DK37867.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
REFERENCES
1. Monder C, Shackleton CHL 1984 110-Hydroxysteroid
dehydrogenase: fact or fancy? Steroids 44:383-417
2. Funder JW, Pearce PT, Smith R, Smith Al 1988 Mineralcorticoid action: target tissue specificity is enzyme, not
receptor, mediated. Science 242:583-585
3. Edwards CRW, Burt D, Mclntyre MA, deKloet ER, Stewart
PM, Brett L, Sutanto WS, Monder C 1988 Localization of
11/3-hydroxysteroid dehdrogenase-tissue specific protector of the mineralocorticoid receptor. Lancet 2:986-989
4. Arriza JL, Weinberger C, Cerelli G, Glaser TM, Handelin
BL, Housman DE, Evans RM 1987 Cloning of human
mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor. Science 237:268-275
5. Ulick S, Levine LS, Gunczler P, Zanconato G, Ramirez
17.
18.
19.
20.
21.
LC, Rauh W, Rosier A, Bradlow HL, New Ml 1979 A
syndrome of apparent mineralocorticoid excess associated with defects in the peripheral metabolism of cortisol.
J Clin Endocrinol Metab 49:757-764
Phillipou G, Higgins BA 1985 A new defect in the peripheral conversion of cortisone to cortisol. J Steroid Biochem
22:435-437
Monder C, Stewart PM, Lakshmi V, Valentino R, Burt D,
Edwards CRW 1989 Licorice inhibits corticosterond 110dehydrogenase of rat kidney and liver: in vivo and in vitro
studies. Endocrinology 125:1046-1053
Lakshmi V, Monder C 1988 Purification and characterisation of the corticosteroid 110-dehydrogenase component of the rat liver 11 /3-hydroxysteroid dehydrogenase
complex. Endocrinology 123:2390-2398
Agarwal AK, Monder C, Eckstein B, White PC 1989
Cloning and expression of rat cDNA encoding corticosteroid 11/S-dehydrogenase. J Biol Chem 264:1893918943
Lakshmi V, Monder C 1985 Extraction of 11 /3-hyroxysteroid dehydrogenase from rat liver microsomes by detergents. J Steroid Biochem 22:331 -340
Yanagibashi K, Haniu M, Shively JE, Shen WH, Hall P
1986 The synthesis of aldosterone by the adrenal cortex.
J Biol Chem 261:3556-3562
Kozak M 1986 Point mutations define a sequence flanking
the AUG initiator codon that modulates translation by
eukaryotic ribosomes. Cell 44:283-292
Fuerst TR, Earl PL, Moss B 1987 Use of a hybrid vaccinia
virsu-T7 RNA polymerase system for expresssion of target genes. Mol Cell Biol 7:2538-2544
Fuerst TR, Niles EG, Studier FW, Moss B1986 Eukaryotic
transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci USA 83:8122-8126
Bradford MM 1976 A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing
the principle of protein-dye binding. Anal Biochem
72:248-254
Lakshmi V, Monder C 1985 Evidence for independent 11 oxidase and 11 -reductase activities of 11 /3-hydroxysteroid
dehydrogenase: enzyme latency, phase transition, and
lipid requirements. Endocrinology 116:552-560
Segal IH 1975 Enzyme Kinetics. Wiley and Sons, New
York, pp 346-385
Northrop DB 1983 Fitting enzyme-kinetic data to V/K.
Anal Biochem 132:457-461
Bevington P 1969 Data Reduction and Error Analysis for
the Physical Sciences. MacGraw-Hill, New York
Kessler SW 1975 Rapid isolation of antigens from cells
with a staphylococcal protein A-antibody absorbent: parameters of the interaction of antibody-antigen complexes
with protein A. J Immunol 115:1617-1624
The VL, Labrie C, Zhao HF, Couet J, Lachance Y, Simard
J, Leblanc G, Cote J, Berube D, Gagne R, Labrie F 1989
Characterization of cDNAs for human estradiol 17/3-dehydrogenase and assignment of the gene to chromosome
17: evidence of two mRNA species with distinct 5'-termini
in human placenta. Mol Endocrinol 3:1301-1309