The GAF-Tandem Domain of Phosphodiesterase 5 as a Potential Drug Target - Phosphodiesterases as Drug Targets - page 156

experimental results and previously published data (Bruder et al. 2006 ), the K m of
PDE5-GAF-CyaB1 for ATP is 15 m M. The assay was performed at 75 m MATP,
which is close to substrate saturation. A negative protein control was prepared
from E. coli that was transformed with an empty vector (Fig. 2b ). This protein was
inactive when assayed in parallel to PDE5-GAF-CyaB1. Obviously, it had no
ATPase activity, which otherwise would have released phosphate from substrate
ATP. The activity of PDE5-GAF-CyaB1 was dependent on Mg 2+ ions, and
10 mM Mg 2+ was used in the assay. cGMP concentration/activation curves
were carried out from 0 to 50 m M cGMP using reaction times of 30, 60, and
90 min (Fig. 3a ). For all tested reaction times, half-maximal activation was
observed with approximately 13 m M cGMP in accordance with earlier data
(Bruder et al. 2006 ). These concentrations for half-maximal activation are signifi-
cantly higher than the K d values reported for cGMP-binding (0.027-1.9 m M),
which remains an unexplained discrepancy (Liu et al. 2002 ; Zoraghi et al. 2005 ).
A cGMP concentration of 10 m M cGMP was chosen for the high-throughput
assay. While PDE5-GAF-CyaB1 was not enzymatically active at room tempera-
ture,thereactionrateincreasedintheexpectedfashionfrom30to45 C, reflect-
ing the temperature optimum of the cyclase (Kanacher et al. 2002 ). The assay was
conducted at 37 C in order to provide a physiological temperature for the mam-
malian PDE5 GAF domain. Assay solutions without ATP were preincubated for
15 min at 37 C prior to substrate addition to minimize temperature changes
during the reaction. Furthermore, a concentration-response curve was established

a b

0.8

1.0

0.6 0.8

0.6

0.4

0.4

0.2

0.2

0

0.0

0123456

0 30 60 90

nmol phosphate

Time (min)

Fig. 2 Determination of adenylyl cyclase activity by measurement of released phosphate. (a)Phos-
phate calibration curve of the malachite green assay. 40 m l of malachite reagent was added to 40 m l
of phosphate in assay buffer containing 75 m MATP.OD 650 was measured after 10 min using
a Powerwave HT reader (Biotek). (b) PDE5-GAF-CyaB1 and a negative protein control were
assayed under conditions of the malachite assay used in the high-throughput screen ( 10 m McGMP).
Each point represents the average of quadruplicates. Square PDE5-GAF-CyaB1, circle PDE5-
GAF-CyaB1 + 10 m McGMP, triangle protein negative control 10 m M cGMP (signals cGMP

were identical). Error bars when exceeding the size of the symbol are SD ( n ¼ 4)