Biology Reference
In-Depth Information
a
0.0
12 h
-0.5
-1.0
15 min
PD
E
5
Δ
1-419
C Domain
B
-1.5
0
10
20
30
Minutes
b
0.0
12 h
-0.5
-1.0
15 min
PDE5
Δ
1-465
B
C Domain
-1.5
0
10
20
30
Minutes
Fig. 5 Exchange-dissociation of [
3
H]tadalafil from PDE5
D
1-419 but not PDE5
D
1-465 is affected
by time of preincubation with the inhibitor. PDE5
D
1-419 (
top
) or PDE5
D
1-465 (
bottom
) (80
m
l;
0.035 nM final) was added to 4.5 ml of binding reaction mixture containing [
3
H]tadalafil (30 nM
final concentration) after being preincubated for either 15 min or 12 h at 4
C. The zero time point
(B
o
) was determined as described in Blount et al. (
2007
). Following 15-min or 12-h preincubation
of the respective constructs with [
3
H]tadalafil, the stoichiometry of inhibitor binding (B
o
) was not
significantly different. Approximately 30
m
l of a 1 mM tadalafil solution was then added to the
remaining incubating binding reaction mixture at 4
C. Aliquots were removed at various times
(B
t
) and filtered by the same procedure at the indicated time points
characteristics of apparently different conformations of the same catalytic site;
however, results of recent X-ray crystal structures of various constructs of PDE4
isoforms in complex with certain inhibitors (described above) reveal that the high-
affinity interaction of some inhibitors results from direct contacts with the catalytic
pocket as well as with UCR2 (Burgin et al.
2010
; Houslay and Adams
2010
).
The molecular factors that determine whether the HARBS or LARBS form
predominates for a particular inhibitor have not been unequivocally identified, nor
has it been established whether these states are interconvertible, as occurs when
inhibitor binding to the PDE5 catalytic site shifts the low-affinity site to the high-
affinity site. The chemical characteristics of the inhibitor, the contacts that it forms
with the catalytic site and/or other regions of the PDE, as well as the oligomerization
state, degree of phosphorylation, or metal-ion occupancy of the catalytic center
