FIG 5 .

CNP, BNP, and NPR agonist and isatin affinities for AmiC in vitro. (A) Recombinant AmiC was fluorescently labeled and incubated with various concentrations of natriuretic peptide receptor agonists and isatin. These mixtures were then analyzed using by microscale thermophoresis. The results were normalized to a fitted zero for unbound AmiC, and the thermophoresis signal is shown, in parts per thousand. Addition of cANF^4-23 caused AmiC fluorescence to increase in a dose-dependent fashion; this increase was used as the signal rather than thermophoresis, as the effect confounds the thermophoresis signal. (B) The results were fitted to the dissociation constant formula from the law of mass action. The fitted values for K_D are shown for isatin, BNP, sANP, cANF^4-23, and osteocrin. We showed that these compounds either bound very weakly (sANP), bound more tightly (osteocrin), or exhibited no interaction (BNP). The data are the means from three independent experiments.