Fig. 4. Guanylyl cyclase activity of Gyc-88E and Gyc-89Db. COS-7 cells were transiently transfected with pcDNA3.1 vectors containing the open reading frames of various soluble guanylyl cyclase subunits and the cell extracts assayed for guanylyl cyclases activity under the conditions shown. (A) Gyc-88E exhibits enzyme activity in the absence of additional subunits and has higher levels of activity in the presence of Mn compared with Mg. The Manduca guanylyl cyclase, MsGC-ß3, exhibits similar properties and was included for comparison. The two splice variants of Gyc-88E (Gyc-88E-S and Gyc-88E-L) yielded similar levels of activity as each other. Data shown are the means ± S.E.M. of three determinations. (B) Kinetic analysis of Gyc-88E-S and Gyc-88E-L. Cell extracts were assayed for guanylyl cyclase activity in the presence of 0.1–10 mmol l^-1 GTP in the presence of either 4 mmol l^-1 Mg or Mn. A Michaelis–Menten curve was applied to the resulting data using Graphpad Prism 3.0. No difference in K_m or V_max was observed between the splice variants in the presence of Mg or Mn. (C) The NO donor sodium nitroprusside (SNP) stimulated the activity of both splice variants of Gyc-88E and this stimulation was unaffected by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxaline-1-one (ODQ). By contrast, ODQ virtually eliminated the activation of the Manduca MsGC-1/ß1 heterodimer by SNP. Assays were carried out in the presence of 4 mmol l^-1 Mg. Data shown are the means ± S.E.M. of three determinations. (D) Guanylyl cyclase activity of Gyc-89Db. No enzyme activity was detected when Gyc-89Db was expressed in the absence of additional subunits or when co-expressed with Gyc-99B in either the presence of 4 mmol l^-1 Mg or 4 mmol l^-1 Mn. However, when Gyc-89Db was co-expressed with Gyc-88E, greater basal activity was detected than when Gyc-88E was expressed alone. The basal activity was enhanced in the presence of both Mg and Mn. The data shown represent pooled values for Gyc-88E-S and Gyc-88-L, as no differences were seen between the different splice variants. Data shown represent the means ± S.E.M. of six determinations. (E–G) Guanylyl cyclase activity of the Drosophila soluble guanylyl cyclase subunits in the presence of 100 µmol l^-1 of the NO donors SNP, SIN-1, SNAP, SNOG, DEA-NONOate, NOC-12 and spermine NONOate and the NO-independent activator of soluble guanylyl cyclase YC-1. The subunit combinations shown are Gyc-88E (E), Gyc-88E co-expressed with Gyc-89Db (F) and Gyc-99B/Gycß-100B (G). The data shown represent pooled values for Gyc-88E-S and Gyc-88-L, as no differences were seen between the different splice variants. Data shown represent the means ± S.E.M. of at least four determinations. For all graphs, the data were analyzed using one-way ANOVA: `ns' represents P>0.05, ^**P<0.01 and ^***P<0.001. For the data shown in A, C and D, Tukey–Kramer post-hoc test was used and, for E–G, Dunnett's multiple comparison test was used.