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INTRODUCTION |
Glucocorticoid hormones regulate a multitude of
physiological processes including carbohydrate and lipid metabolism,
development, blood pressure, and immunity. Like other steroid hormones,
glucocorticoids act through binding to specific nuclear receptors,
thereby activating or repressing gene transcription (1).
In analogy to other steroid hormones, glucocorticoids are submitted to
prereceptor control through metabolic conversion by enzymes acting as
metabolic "switches," producing "active" (i.e. binding to the receptor) or "inactive" (i.e. not binding
to the receptor) hormone (2-6). The glucocorticoid "shuttle"
mechanism consists of 11
-hydroxysteroid dehydrogenases
(11
-HSDs),1 mediating the
reversible oxoreduction/hydroxydehydrogenation at position C11 of
cortisone and cortisol, respectively. At present, two 11
-HSD
isozymes, the type 1 and 2 enzymes, have been characterized, and both
mediate activation or inactivation of the hormone in a tissue-specific
manner (7, 8). Oxoreduction of 11-oxo-corticosteroids (cortisone in
humans, dehydrocorticosterone in rodents) is mediated by the
NADPH-dependent type 1 11
-HSD (11
-HSD1), thus
performing activation to the receptor ligand cortisol
(corticosterone in rodents). The
NAD+-dependent type 2 enzyme (11
-HSD2)
catalyzes 11
-OH dehydrogenation of cortisol (corticosterone), which
leads to the inactivation of glucocorticoid receptor ligands.
The two 11
-HSD isozymes of this enzymatic shuttle constitute
important components within the glucocorticoid endocrine system. The
type 2 enzyme has been shown to be involved in the "protection" of
the mineralocorticoid receptor in peripheral tissues (such as distal
renal tubules) against occupation by cortisol, exemplified by
11
-HSD2 gene defects leading to the "apparent mineralocorticoid excess syndrome" (9, 10). The physiological role of the type 1 form
is less clear, but several studies established a critical role of
11
-HSD1 in the tissue-specific activation of corticosteroid ligands
(11-14). This physiological role is established in hepatic and adipose
tissue where the inhibition of enzyme activity might be beneficial in
diseases such as obesity and non-insulin-dependent diabetes
mellitus (12-15). However, the wide tissue distribution suggests that
the enzyme has further specific functions.
In this study, we investigated enzymological properties of 11
-HSD1
from human and guinea pig. The latter species displays high circulating
levels of glucocorticoids with lowered affinities of the glucocorticoid
receptor for its ligand cortisol (16). The data obtained suggest that
11
-HSD1 is not a critical determinant of peripheral glucocorticoid
resistance in guinea pig. Furthermore, we established an enzymological
profile of recombinant human and guinea pig 11
-HSD1 and show for the
first time high levels of reductive and oxidative activities of
purified recombinant enzyme, demonstrating the suitability of this
system for systematic investigations.
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EXPERIMENTAL PROCEDURES |
Cloning of 11
-Hydroxysteroid Dehydrogenases--
Guinea pig
11
-HSD1 was cloned by reverse transcriptase-PCR from a liver
sample (Hartley strain). Total liver RNA was prepared by acid
guanidinium-thiocyanate/phenol extraction, and reverse transcriptase-PCR was carried out using sequence-specific primers (17).
After digestion of the purified PCR product with nucleases SnabI and NotI, the resulting cDNA was
subcloned into pPIC3.5 (Invitrogen). The sequences of the constructs
were verified by DNA sequencing. All clones obtained from guinea pig
liver display identical sequences but show two differences from the
published data (17). These exchanges comprise protein sequence
position 22 (Pro
Ser) and position 103 (Ala
Val). All of the
11
-HSD1 sequences known to date show the same residues as the guinea
pig sequence determined in this study. These structural details were reported to the Swiss Protein Data Bank. Human 11
-HSD1 was prepared as described previously (18).
Yeast Transformation, Selection, and Overexpression--
After
linearization of the plasmid DNA with endonucleases BglII or
SacI, yeast strains KM71 or GS115 were transformed using the
spheroplast method. Clones obtained after homologous recombination of
the 11
-HSD-1 DNA into the Pichia alcohol oxidase locus
were selected, and phenotyping for His+Mut+ and
His+Muts was carried out on appropriate
media. Recombinant strains were confirmed by PCR of genomic DNA
with gene-specific primers. Positive clones for each phenotype were
selected, and expression trials were performed. Finally, a recombinant
strain (His+MutS) for all further experiments
was chosen. To exclude intrinsic activities, mock-transformed strains
(vector alone, no insert) were used as background controls. Yeast
clones were grown at 30 °C for 3-5 days in buffered complex media
(1% yeast extract, 2% peptone, 100 mM potassium
phosphate, pH 6.0, 1.34% yeast nitrogen base, 4 × 10
5% biotin) containing 0.5% (v/v) methanol for
induction of recombinant proteins.
Preparation of Yeast Fractions--
Yeast subcellular fractions
were prepared by mechanically disrupting the washed cells with glass
beads (245-400 µm, Sigma) in 20 mM sodium
phosphate buffer, 5% glycerol, 1 mM EDTA, pH 7.0, containing 1 mM PMSF. After the removal of cell debris by
low speed centrifugation, an S9 fraction was prepared by a 9,000 × g centrifugation step for 10 min.
Cell Culture and Transient Transfection of 11
-HSD1--
COS7
cells were transfected with 1.4 µg of guinea pig or human 11
-HSD1
plasmid DNA subcloned into the mammalian expression vector pcDNA3.1
(Invitrogen) using the FuGENE 6 transfection reagent (Roche Molecular
Biochemicals). Cells (0.5 × 106 in 6-well plates)
were grown in 3 ml of medium/well, and 11
-HSD activities were
analyzed 24 h after transfection. At this point, cells reached a
confluency of ~80-90%. Cells were incubated for 1-3 h in the
presence of unlabeled cortisone or cortisol and appropriate glucocorticoid tracer (200,000 cpm, 3H-labeled).
Supernatants were extracted on solid phase (Oasis, Waters) and further
analyzed by thin layer chromatography and liquid scintillation counter
(14) to determine the fractional conversion of substrate.
3H-Labeled cortisol (specific activity 64.0 Ci/mmol) was
obtained from Amersham Biosciences, and tritiated cortisone was
synthesized from cortisol using recombinant 11
-HSD1 and was further
purified chromatographically (14). Background activities (ranging from 0.9 to 2% of total recovered tracer radioactivity) were obtained in
mock transfection experiments and were subtracted from the respective measurements.
Heterologous Expression and Purification of Soluble
11
-HSD1--
Expression constructs without the
NH2-terminal transmembrane domain were made for
human 11
-HSD1 according to Walker et al. (19) and in an
analogous manner for guinea pig 11
-HSD1 in the pET28a (Novagen)
vector, which introduces a His6 tag at the amino terminus.
BL21(DE3) cells were transformed and grown in LB or Terrific Broth
medium containing 50 µg/ml kanamycin until
A600 reached 0.5-1.0. Induction was
achieved by the addition of 0.2 mM
isopropyl-1-thio-
-D-galactopyranoside, and incubation
was continued for 12-16 h at room temperature. Cells were harvested and lysed by sonication, and purification of soluble 11
-HSD1 protein
was achieved by His-bind affinity chromatography on a HiTrap Chelating
HP column using an ÄKTA protein purifier system (Amersham
Biosciences) with a stepwise gradient of imidazole. Active fractions
were pooled and dialyzed against 20 mM Tris-HCl, pH 8.0, 2 mM tris-(2-carboxyethyl)phosphine (Sigma), 5% glycerol, and concentrated using Amicon or Millipore concentration devices.
Enzymatic Assays and Data Analysis--
S9 fractions were
incubated in a reaction mixture containing 0.5 mM of
appropriate coenzyme (NADP+ or NADPH or NADPH-regenerating
system), steroid substrate concentrations ranging from 0.1 to 120 µM, and 30-40 µg of protein in 50 mM
Tris-Cl, pH 7.4, at 37 °C for 10-60 min. Initial reaction
velocities were measured, and conditions were chosen with no >20% of
substrate conversion, and measurements were carried out in the linear
range of product formation versus reaction time and enzyme
concentration. At substrate concentrations over 5 µM,
reactions were stopped by a 3-fold volume of ice-cold acetonitrile.
After a brief centrifugation at 20,000 × g,
supernatants were directly analyzed on reverse phase-high pressure
liquid chromatography consisting of a C18 stationary phase and an
eluent of 30% acetonitrile in 0.1% ammonium acetate, pH 7.0. UV
detection of metabolites was achieved at 240 nm. Below 5 µM of substrate concentrations, reactions were stopped by
the addition of 40 µl of phosphoric acid/ml, and samples were extracted by liquid phase extraction in ethyl acetate or by solid phase
extraction using Oasis columns. After drying under nitrogen, samples
were reconstituted in high pressure liquid chromatography eluent and
injected into the system. Using a series of potassium phosphate
buffers, pH optima were determined for the purified enzymes, and
kinetic measurements were subsequently carried out at pH 6.0 (reductase) and at pH 9.2 (dehydrogenase) in 50 mM
potassium phosphate.
Kinetic constants were calculated using Prism (GraphPad, San Diego,
CA) software by linear or non-linear regression analysis and by fitting
to different models describing either the Michaelis-Menten kinetics
(V = Vmax × Sh/(Kmh + Sh);
h = 1) or a cooperative kinetic behavior (h
1), respectively.
Active Site Titration--
Fractional velocities (dehydrogenase
reaction, 50 µM cortisol, 100 µM
NADP+) were measured in the presence of increasing amounts
of the arylsulfonamidothiazole inhibitor compound BVT.24829 (Biovitrum)
(15) at enzyme concentrations between 0.5 and 1 µM. Data
obtained were fitted by non-linear regression to Equation 1
(20),
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(Eq. 1)
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where vi is the observed velocity in the presence of
varied inhibitor concentrations, vo is the uninhibited velocity, E is the concentration of active enzyme,
K
is the apparent inhibition
constant, and I is the experimental inhibitor concentration.
Protein Analysis--
Protein concentrations were determined
using the Lowry method with bovine serum albumin as standard.
Concentrations of purified samples were determined by compositional
analysis on an Biochrom 20 Plus system (Amersham Biosciences) after
hydrolysis of samples in 6 M HCl, 0.1% phenol.
Immunohistochemistry--
Liver and adrenal glands from guinea
pig (Hartley strain) were fixed in 1% buffered formaldehyde,
dehydrated, and embedded into paraplast. Sections (about 5 µM) were processed for immunohistochemistry using
established protocols. Primary antibodies used were anti-mouse liver
11
-HSD1 affinity-purified through absorption on purified immobilized
11
-HSD1 (21) and corresponding preimmunization serum at 1:100
dilution in PBS. Western blot analysis of the antiserum revealed a
single band at 32 kDa in guinea pig tissues (21) and non-reactivity
with preimmunization serum. Detection was achieved using alkaline
phosphatase-labeled secondary antibodies and nitro blue
tetrazolium/X-phosphate. Control sections were stained with hematoxylin-eosin.
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RESULTS |
Kinetic Analysis of Recombinant Full-length 11
-HSD1 from Guinea
Pig and Human--
Comparative kinetic analyses of recombinant human
and guinea pig 11
-HSD1 were performed in homogenates prepared from
Pichia pastoris strains and from COS7 cells (Table
I and Fig. 1, A and B). Furthermore, kinetic
constants were determined from intact monolayers of transiently transfected COS7 cells by analyzing supernatants of conditioned medium. These experiments yield an apparent Km in the low micromolar region measured in homogenates, similar for both guinea pig and human. The values range
from 2.2 to 8.2 µM for the guinea pig enzyme in P. pastoris and COS7 homogenates, respectively, whereas the human
forms display Km values of 1.9 and 8.9 µM for P. pastoris and COS7 homogenates,
respectively. These values are in agreement with data obtained from
previous studies (18, 22) using guinea pig and human liver microsomes
(Table I). Specific activities obtained range from 30.3 (P. pastoris) to 150 (COS7) pmol/min × mg protein for the human
enzyme, whereas guinea pig extracts have activities ranging from 42.0 (P. pastoris) to 221 (COS7) pmol/min × mg
protein. In contrast, intact COS7 cells transiently transfected with
plasmids expressing human or guinea pig 11
-HSD1 show a markedly
higher affinity (approximately one order of magnitude) for cortisone
reduction over homogenates with an apparent Km of
0.28 µM for the guinea pig and 0.33 µM for
the human enzyme. Specific activities obtained in this system are
similar and range from 173 pmol/106 cells × h to 191 pmol/106 cells × h for the guinea pig and human
forms, respectively.
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Table I
Comparative kinetic analysis for cortisone reduction of 11 -HSD1
expressed in different hosts
Experiments were carried out as described under "Experimental
Procedures." Km is in µM,
Vmax is in pmol × min 1 × mg 1, and for intact cells, units are in pmol × (106 cells) × h 1. n = 3-5. P. pastoris, transiently transfected COS7 cells; COS
intact, measured in intact monolayer cultures; COS extract, measured in
homogenates.
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Fig. 1.
Enzyme kinetic analysis of cortisone
reduction in intact COS7 cell monolayers transiently transfected with
plasmids expressing guinea pig 11 -HSD1
(A) or the human isoform (B).
Insets show linear regression analysis by Eadie-Hofstee
plots.
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An analysis of COS7 extracts expressing human or guinea pig 11
-HSD1
using a wide range of cortisone substrate concentrations reveals in
each case a hyperbolic character of the graph, indicating the
Michaelis-Menten kinetics of the enzyme. In particular, no indication
for a cooperative behavior was obtained (Fig. 1, A and
B).
Kinetic Analysis of Soluble and Purified 11
-HSD1
from Human and Guinea Pig--
Heterologous expression experiments
with transmembrane-deleted soluble 11
-HSD1 enzymes carrying an
NH2-terminal His6 tag (19) were carried out in
E. coli. Both proteins could be produced to a level of 1-5
mg/l culture and were purified to homogeneity (Fig.
2) and analyzed by steady-state kinetic
measurements (Table II, Fig.
3). Using the arylsulfonamidothiazole
inhibitor BVT.24829, active site titration was carried out with the
human form showing 40% of active sites/mol purified protein (Fig.
4). Corresponding experiments with the
guinea pig enzyme were not successful as revealed by the lack of
inhibition (80% residual activity at 25 µM) (data not
shown). Analyzing the 11-oxo-reductase activity with cortisone as
substrate, the Km values determined are 0.8 µM for human and 0.6 µM for guinea pig. The
kcat/Km values obtained are
1.5 (human) and 1.3 (guinea pig) M
1
min
1 × 106 with both enzymes displaying
hyperbolic kinetic behavior (Fig. 4). With cortisol as substrate,
Km values are 2.9 µM (human) and 3.1 µM (guinea pig), whereas
kcat/Km values range from 3.8 (human) to 0.6 M
1 min
1 × 106 (guinea pig).

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Fig. 2.
SDS-PAGE analysis of purified recombinant
human and guinea pig 11 -HSD1,
respectively. Coomassie Blue-stained NuPAGE 10% bis-Tris gel
(Invitrogen) is shown. Both orthologs lack the NH2-terminal
transmembrane domain of native 11 -HSD1 and carry a His6
tag at the amino terminus to assist purification by His-bind affinity
chromatography. Lane 1, SeeBlue Plus2 Pre-Stained Protein
Standard (Invitrogen) (molecular masses (in kDa) are indicated on the
left); lane 2, recombinant human 11 -HSD1;
lane 3, recombinant guinea pig 11 -HSD1
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Table II
Kinetic constants for purified soluble 11 -HSD1 from human and guinea
pig
Data are given as the mean ± S.D. and are from n = 3-6 experiments. Experiments were carried out at saturating cofactor
(100 µM NADP+, 250 µM NADPH) and
varied cortisone/cortisol concentrations at pH 6.0 for reduction and pH
9.2 for oxidation reactions. Values in italics for cited references
were calculated based on molecular masses of the proteins under study
using the Vmax data given. s, soluble; tm,
deleted transmembrane domain; h, human; r, rat; rec, recombinant; fl,
full-length; n, native; l, liver; na, not active. Km
is in µM; Vmax is in nmol × min 1 × mg 1; kcat is in
min 1; kcat/Km is in
106 × M 1 × min 1.
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Fig. 3.
Kinetic analysis of soluble human and guinea
pig 11 -HSD1. A, human.
B, guinea pig. Inserts show linear regression
analysis through Eadie-Hofstee plots.
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Fig. 4.
Active site titration of soluble human
11 -HSD1. Fractional velocities
(vi/vo) of purified soluble human 11 -HSD1 were
determined in the presence of increasing amounts of the inhibitor
BVT.24829. Values obtained were fitted to the equation as defined by
Morrison (20), yielding a value for the enzyme active site
concentration of 0.21 ± 0.07 µM and a
K of 0.09 ± 0.03 µM.
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Immunohistochemistry of Guinea Pig Liver and Adrenal
Gland--
Histochemical localization studies of 11
-HSD1 were
carried out in guinea pig tissues. In liver, intense immunoreactivity around the central vein is noted (Fig.
5A), in agreement with histochemical data from the human (23). In the adrenal gland, strong
staining is observed in subcapsular areas corresponding to the Zona
glomerulosa, whereas decreasing intensities are noted in areas
corresponding to the Zona fasciculata and Zona reticularis (Fig.
5B). No staining is observed in the adrenal medulla (data not shown).

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Fig. 5.
Expression of
11 -HSD1 in guinea pig liver and adrenal
gland. A, section of guinea pig liver stained with
affinity-purified primary antibody (a), preimmunization
serum (b), and hematoxylin-eosin (c).
B, section of guinea pig adrenal gland stained with
affinity-purified primary antibody (a and b),
preimmunization serum (c), and hematoxylin-eosin
(d). Immunoreactive staining is clearly prominent in
subcapsular (sc) areas.
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DISCUSSION |
The enzyme 11
-HSD1 has been suggested as a critical determinant
protecting against hypercortisolism in the guinea pig model of
glucocorticoid resistance (17). However, a comparison of the kinetic
data obtained from the guinea pig and the human enzymes (Tables I and
II) indicates no significant differences in substrate affinities.
Combined with the observation of higher expression levels in guinea pig
liver versus human liver, these data argue against a role of
11
-HSD1 as a crucial determinant protecting against hypercortisolism
as suggested previously (17) in the guinea pig model of glucocorticoid
resistance (16, 24-27). Thus, neither in the squirrel monkey (another
natural model to study cortisol resistance) (28) nor in the guinea pig
does 11
-HSD1 seem to contribute to the observed peripheral
resistance to corticosteroids (28). Rather, altered glucocorticoid
receptor affinities, ACTH-mediated cortisol secretion, lowered plasma
protein binding, and other interactions (16, 24-27) but not 11
-HSD1
are concluded to be the factors mediating resistance to
glucocorticoids. Consequently, the importance of 11
-HSD1 in
mammalian physiology is underscored by the lack of excess states such
as apparent mineralocorticoid excess or Cushing syndrome in species
displaying high levels of circulating glucocorticoids. However, species
differences in tissue-specific expression patterns of 11
-HSD
isozymes are apparent (17, 28, 29). In contrast to humans and rodents,
absent or low levels of 11
-HSD1 in the central nervous system are
found in the guinea pig and squirrel monkey (17, 28), indicating a
possible role in central feedback control. Notably, we detected the
expression of 11
-HSD1 in the Zona glomerulosa of guinea pig adrenal
gland and lower levels in Zona fasciculata and Zona reticularis. This is in contrast to the expression of 11
-HSD1 in human adrenal gland
in which higher levels in segments close to the medulla are found,
suggesting involvement in the regulation of catecholamine synthesis
(23, 30, 31). Consequently, the expression of 11
-HSD1 in the
aldosterone-synthesizing compartment suggests regulatory roles of
glucocorticoids in mineralocorticoid secretion or synthesis in guinea
pig, similar to data obtained in rat (32).
Interpretation of kinetic data obtained for 11
-HSD1 has been
complicated by the occurrence of bidirectional activity
(i.e. displaying both dehydrogenase and labile reductase
activities) in vitro in homogenized or purified material,
whereas intact cells (transfected or primary culture) display reductase
activity with little or no dehydrogenase activity (14, 28, 33-39).
This effect partially reflects intracellular cofactor availability,
because 11
-HSD1 is clearly a NADPH-dependent reductase
and localized to the lumen of the endoplasmic reticulum (40). In most
studies, the kinetic analysis of homogenates reveals an apparent
Km value for cortisone/dehydrocorticosterone in the
low micromolar range (7, 17, 18, 22), whereas some researchers report higher Km values (41, 42) for recombinant
homogenates or purified material. These latter in vitro data
are clearly inconsistent with the circulating glucocorticoid levels,
which are in the nanomolar concentration range. Therefore, cooperative
kinetic behavior has been suggested recently as an explanation of how a
micromolar Km is compatible with the in
vivo function at nanomolar substrate concentrations in humans
(42). A Hill coefficient of 1.6 obtained through regression analysis
was reported, but allosteric behavior was not further corroborated.
However, a K0.5 of 10 µM obtained
in the study reported by Maser et al. (42) is still ~100
times higher than the physiological glucocorticoid levels, thus making
cooperative kinetics an unlikely explanation with these data obtained.
Furthermore, as observed in our study and by others (14, 28, 33-39),
intact cells, transfected or as primary culture, display an apparent
Km clearly around the physiological level,
i.e. between 100 and 400 nM. The observed affinity difference to homogenate material therefore points to further
explanations. These explanations could be that the intact intracellular
environment, e.g. lipids, stabilizes the reductive enzyme
activity or that protein modification occurs, that the glucocorticoid
substrate is concentrated at the intracellular expression site,
i.e. the lumen of the endoplasmic reticulum where the
catalytic center is located (40), or that cellular factors inhibit
activity upon disruption of cell material. Finally, cooperative kinetic
behavior is not observed in intact cells or homogenates neither in our
work nor other studies (14, 28, 33-39). To investigate the possibility
of cooperative kinetics, we performed incubations with homogenates or
purified enzyme at substrate concentrations spanning several orders of
magnitude, indicating in no case cooperative kinetic characteristics.
Importantly, the purified recombinant soluble 11
-HSD1 material
employed in our study displays no sign of sigmoidal kinetics.
Therefore, we believe that the most probable explanation for the
postulated allosteric behavior (42) is the occurrence of partial
inactivation of the purified native enzyme, thus suggesting cooperative
behavior deduced from non-linear regression analysis. This is in line
with the 150-380-fold lower
kcat/Km values of material
prepared from human liver versus our purified 11
-HSD1
forms (Table II, compare references and data). To our knowledge, active
site titration of purified 11
-HSD1 has never been performed earlier.
In this study, we show that ~40-50% active sites/mol enzyme can be
obtained by the protocol used, suggesting that all earlier purification
attempts reached lower ratios of active molecules. Apparent
cooperativity is frequently observed at low substrate concentrations
with labile enzymes (43), and instability of 11
-HSD1 reductase
activity upon purification has been noted already previously (44) and
has consistently been confirmed in all purification studies using
native material. However, here we demonstrate that purified recombinant
11
-HSD1 from human and guinea pig display Km
values close to those observed in intact mammalian cells. This
indicates that during purification of native enzymes, either the
conditions or intrinsic factors are employed that destabilize the
enzyme or that unidentified cellular factors inhibiting activity
copurify. Importantly, we demonstrate high level expression and
maintenance of kinetic characteristics compared with that of the enzyme
from its native cellular environment upon purification. Consequently,
heterologous expression in E. coli of engineered constructs
lacking the transmembrane domain is a promising route to derive at
structural and functional details of 11
-HSD1.