Figure 1.
Activation pattern of sGC with tetracysteine (TC) motif.
Concentration response curves of WT sGC (A) and sGC with an intramolecular tetracysteine motif without (B) or with FlAsH labelling (C) are shown. cGMP reporter cells were transiently cotransfected with WT α1 subunit and WT or a TC motif carrying β1 subunit as indicated in the respective figures. Cells were incubated with increasing concentrations of BAY 58-2667 or BAY 41-2272 alone or in combination with 10 µM ODQ or 10 nM DEA/NO (NO), respectively. sGC activity is represented as x-fold stimulation compared to transfected control cells. Data are means ± S.E.M. from 5–14 independent experiments, performed in duplicate. Following basal activities were measured: (A) 10732 relative light units (RLUs), (B) 9720 RLUs, (C) 3541 RLUs.
Figure 2.
Fluorescence of FlAsH-labelled TC4-WT sGC.
cGMP reporter cells were cotransfected with WT α1 sGC and TC4-WT β1 sGC and labelled with FlAsH. The cells were then incubated with 0.1, 1 or 10 µM BAY 58-2667 (A) 1, 30 or 100 µM rotenone (B) or 1, 10 or 100 µM NS 2028 (C) for 90 min. Fluorescence intensity of single cells was monitored in a time series of 120 laser scans and is expressed as % of start value which was obtained before application of the test compounds. Data are means ± S.E.M. from 9–28 single cells assayed on different days. *p<0.05; ***p<0.005: Student's t-test compared to control. Representative traces of the fluorescence measurement as % of start value are given in the right part of the figure.
Table 1.
Effects of 100 µM NS 2028 on fluorescence intensity of FlAsH labelled haem- free TC4-Y135A/R139A sGC (A) and ReAsH labelled TC4-WT sGC (B) after 90 min incubation.
Figure 3.
Effect of 90 min incubation with NS 2028 and rotenone on sGC activity.
cGMP reporter cells expressing TC4-WT sGC were pre-incubated with 100 µM NS 2028 or rotenone and then incubated with increasing concentrations of BAY 58-2667, BAY 41-2272 alone or in combination with 10 µM ODQ or 10 nM DEA/NO (NO), respectively. Enzyme activity is expressed as x-fold stimulation compared to pre-treated but not stimulated control. Data are means ± S.E.M. from 3–10 independent experiments, performed in duplicate. A basal activity of 7747 relative light units was measured. A) is identical to Fig. 1B.
Table 2.
Effects of 90 min pre-treatment with 100 µM NS 2028 or rotenone on TC4-WT sGC protein levels.
Figure 4.
Principle of the fluorescence dequenching method.
A) The green fluorescent biarsenical dye FlAsH binds to the tetracysteine motif CCPGCC in sGC. In holo-sGC, the fluorescence of FlAsH is quenched by the haem group as energy from FlAsH can be transferred to the haem group due to the overlap in the respective absorption and emission spectra. Haem oxidation leads to loss of the heam group and dequenching of FlAsH fluorescence. Hence, apo-sGC shows full FlAsH fluorescence. B) Replacement of the haem anchoring residues Y135 and R139 with alanine results in a constitutive haem-free sGC. As FlAsH fluorescence cannot be quenched by the haem group FlAsH shows full fluorescence. This sGC form was therefore used as a negative control to test if changes in FlAsH fluorescence are actually due to haem loss. C) Like FlAsH, the red fluorescent biarsenical dye ReAsH binds to the CCPGCC motif. But compared to FlAsH the emission spectrum of ReAsH overlaps with the absorption spectrum of the haem group to a much lesser extent. Consequently, the haem group does not quench the ReAsH fluorescence. ReAsH could therefore be used as a negative control to test if changes in FlAsH fluorescence are due to the overlapping spectra of haem and FlAsH.