Fig. 5.
Generation and characterization of adenylyl cyclases. a Sequence alignment of adenylyl cyclases (ACs) and guanylyl cyclases (GCs) from various organisms (full alignment and accession numbers in Supplementary Figure 6) shows key residues (*), involved in nucleotide binding, which differ between ACs and GCs. Insertion of the double mutation E497K, C566D-generated Ca/BeRhACs. Four additional mutations (564-QYDIW-568) were inserted in Ca/BeRhACs-6×. Enzymatic specificities of the RhACs were determined via ELISA-based quantification of cAMP (b) and cGMP (c) within oocytes, expressing the N-terminal YFP-tagged constructs as indicated. Oocytes were kept in the dark or illuminated (light 4 min, 532 nm, 0.3 mW mm−2) immediately before lysis. Bar graphs show data as means ± s.d., n = 3 samples of 5 oocytes each, ***p = 8 × 10−5, 9 × 10−4, **p = 0.01, unpaired t-test light vs dark; ###p < 0.0001, ##p = 0.001, #p = 0.02, ns = not significant from control one-way ANOVA (dark conditions p < 0.0001) followed by Tukey’s multiple comparisons, for clarity not all comparisions are shown. Control = non-injected oocytes