S.A. Child et al.
Archives of Biochemistry and Biophysics 694 (2020) 108596
3
. Experimental procedures
linear gradient was: 0–1 min, 50% A (v/v); 5.5 min, 0% A (v/v); 9.5 min,
0
% A (v/v); 9.75–11 min, 50% A (v/v). The retention times of vitamin
3
3
.1. Catalytic assays
D
3
and the 25-hydroxy product were 8.9 and 5.3 min, respectively. The
peak area was quantified at a wavelength of 265 nm, based on a standard
.1.1. Bovine AdR
curve using an authentic standard of 25-hydroxyvitamin D
ments were performed in duplicate.
3
. Experi-
The Adx concentration-dependence of the catalytic activity of bovine
AdR was tested with both human and bovine Adx. Reaction solutions
were prepared in the wells of a polystyrene 96-well microplate (Greiner
3.1.4. Human P450 27C1
Bio-One North America, Monroe, NC). A 200
each well including 0.015 M AdR, 100 M horse heart cytochrome c,
00 mM potassium phosphate buffer (pH 7.4), and various concentra-
tions of either human or bovine Adx varying from 0.1 to 150 M. NADPH
1.1 mM) was added to initiate the reaction, providing reduction
μ
L solution was prepared in
The activity of human P450 27C1 was tested in a similar way to P450
11B2 described above and as described previously [32,33]. The con-
centration of P450 27C1 was 20 nM, and the concentration of
μ
μ
1
μ
L-
α
-dilauroyl-sn-glycero-3-phosphocholine was 16
μ
M. The reaction
(
tested was the oxidation of all-trans retinol to 3,4-dehydroretinol, and
the initial concentration of all-trans retinol used was 500 nM. Reactions
were initiated with the addition of NADPH (1.5 mM) but quenched by
equivalents to AdR, which can then reduce Adx. Reduced Adx is able to
reduce cytochrome c. The increase in cytochrome c absorbance at 550
nm in this system indirectly reports on the reduction of Adx by AdR.
Under these conditions, the reduction of Adx by AdR is the rate-limiting
step in this series of reactions [18]. After initiating reactions by addition
of NADPH, the absorbance at 550 nm was recorded every 3 s for 4 min
using a Synergy H1 microplate reader (BioTek, Winooski, VT). A linear
fit to the absorbance over the initial 90 s of the experiment was used to
estimate the initial rate of reduction; during this observation period less
than 15% of total cytochrome c was reduced. The absorbance changes
were converted to reduced cytochrome c concentration using an
the addition of 1 mL tert-butyl methyl ether containing 20 M butylated
μ
hydroxytoluene after 1 min (to avoid autoxidation artifacts). After
drying under a nitrogen stream, samples were dissolved in 50% ethanol
(v/v).
Reaction samples were processed in a similar manner as for the P450
11B2 reactions. The chromatography instrumentation was the same
with changes made to the method to optimize for this reaction. The
mobile phases used were A (95% H
and B (95% CH CN, 4.9% H O, 0.1% HCO
2
2
O, 4.9% CH
3 2
CN, 0.1% HCO H (v/v))
3
2
H (v/v)) at a flow rate of 0.5
ꢀ 1
ꢀ 1
extinction coefficient of 21,100 M cm [31]. Reactions were per-
formed in duplicate.
mL/min. The mobile phase linear gradient was: 0–0.1 min, 40% A (v/v);
5–6 min, 25% A (v/v); 6.5–8 min, 0% A (v/v); 8.5–10 min, 40% A (v/v).
The retention times of all-trans retinol and the 3,4-dehydroretinol
product were 6.6 and 5.2 min, respectively. 3,4-Dehydroretinol forma-
tion was identified by co-elution with a commercial standard (Santa
Cruz, Dallas, TX) and quantification was based on the A350 peak areas.
Concentrations of retinoids were determined spectrophotometrically
and samples were kept in amber glass vials at all times. Experiments
were performed in duplicate.
3
.1.2. Human P450 11B2 assays
The activity of human P450 11B2 with both human and bovine Adx
was tested using 11-deoxycorticosterone as the substrate. These assays
were performed based on previous work in this laboratory using P450
1
1B2 [25]. The conditions used for this assay allow for the assay to
primarily report on the production of corticosterone because aldoste-
rone and 18-OH corticosterone production is low. Levels of 18-OH
corticosterone and aldosterone are below the detection limit of the
experiment (2 pmol); whereas, corticosterone production levels are 2.5-
3.2. Microscale thermophoresis (MST)
to 12-fold higher than the detection limit [25]. Briefly, 500
final volume) including 1 nM P450 11B2, 0.5 M AdR, 50
-dilauroyl-sn-glycero-3-phosphocholine (added as lipid vesicles after
sonication of a 1 mg/mL stock), 4 M 11-deoxycorticosterone, and
M) of either human or bovine Adx were
μ
L samples
Binding affinity of Adx forms for bovine AdR and human P450s was
studied using a Monolith NT.115 MST system (NanoTemper Technolo-
gies GmbH, Munich, Germany) in the Vanderbilt Structural Biology Core
Facility. Either human or bovine Adx was labeled using a NanoTemper
Monolith Protein Labeling Kit RED NHS 2nd Generation, which reacts
with amine groups in a protein sample to label the protein with a fluo-
rescent dye (RED) (proprietary). The extent of labeling was determined
by a modification of the manufacturer instructions. Briefly, the Adx
concentration was initially determined using the extinction coefficient
(
μ
μ
M
L-
α
μ
various concentrations (0–100
μ
◦
incubated for 5 min at 37 C (shaking water bath, final concentrations).
NADPH was then added to the mixtures at a final concentration of 1.5
◦
mM to initiate the reactions. Reactions were allowed to proceed at 37 C
for 7.5 min before quenching with the addition of 2 mL of ethyl acetate.
Steroids in the sample were extracted into the organic solvent and an
ꢀ
1
ꢀ 1
ε414 = 9800 M cm [34]. Then, dilution of the Adx from the dye
addition procedure was determined using the ratio of 280 nm absor-
bance before and after adding the dye. The 414 nm Adx absorbance band
was too faint to be measured with the small reaction volume. The dye
concentration in the final reaction volume, after unreacted dye was
removed using a desalting spin-column, was determined using the
aliquot was removed, dried under a N
2
stream, and then dissolved in a
CH CN:H O mixture (1:1, v/v). The resulting samples were analyzed
3
2
using a Waters Acquity UPLC system (Milford, MA), separated with an
Acquity BEH C18 UPLC octadecylsilane column (2.1 mm × 100 mm, 1.7
μ
m, solvents and gradient conditions previously described [25]), with
ꢀ 1
ꢀ 1
detection using a photodiode array detector, and quantified using
external standards. The production of corticosterone from 11-deoxycor-
ticosterone was analyzed in this study. Assays were done in duplicate.
manufacturer-provided extinction coefficient of 195,000 M cm . A
ratio of [dye]/[Adx] in the final purified sample was used to determine
extent of labeling. The kit labeling instructions were modified because
initial attempts at labeling resulted in a degree of labeling of ~10%. By
increasing the dye-protein incubation time from 30 min to 2 h and
increasing the dye:protein ratio from 3.3 to 10, the extent of labeling
was increased to ~50%, as determined by UV–visible absorbance mea-
surements at 650 and 280 nm.
3
.1.3. Human P450 27A1
The activity of human P450 27A1 was assayed in a similar way to
that of P450 11B2 described above. The concentration of P450 27A1 was
.2 M, and the reaction tested was the oxidation of vitamin D
cholecalciferol) to 25-hydroxyvitamin D . The initial concentration of
vitamin D used was 20 M, and the reaction proceeded for 10 min after
0
μ
3
(
3
MST samples contained 20 nM either RED-labeled bovine Adx or
RED-labeled human Adx, 100 mM potassium phosphate buffer (pH 7.4),
0.05% Tween 20 (v/v), and various concentrations of either bovine AdR
3
μ
the addition of NADPH. The reaction samples were processed in a
similar way as for the P450 11B2 reactions. The chromatography
instrumentation was the same, with changes made for this reaction. The
or the human P450s (100 pM–39
μ
M). Samples were loaded into the
standard Monolith NT.115 capillaries and tested by MST analysis at
◦
2
mobile phases used were A (95% H O, 5% CH
3
CN, v/v) and B (95%
25 C. The results were analyzed by plotting the baseline-corrected
CH CN, 5% H O, v/v) at a flow rate of 0.3 mL/min. The mobile phase
3
2
normalized fluorescence (ΔFnorm [%]) versus enzyme concentration.
3