Nucleotidyl cyclase activity of recombinant soluble guanylyl cyclase

Background The ubiquitously expressed soluble guanylyl cyclase (sGC) converts guanosine 5'-triphosphate (GTP) to guanosine 3':5'-cyclic monophosphate (cGMP). The heterodimeric protein is activated by nitric oxide (NO). sGC plays a key role in the regulation of vascular tone and neurotransmission. Hence, sGC is an important target for the treatment of cardiovascular diseases e.g. pulmonary hypertension, heart failure and coronary heart disease. In addition to the biosynthesis of cGMP, we had previously shown by radiochemical analysis that sGC also generates the cyclic pyrimidine nucleotide uridine 3':5'-cyclic monophosphate (cUMP)[1]. However, cUMP could only be described as putative product but an exact identification by structure analysis was still missing. Therefore, we have established a new analytical method based on high performance liquid chromatography/mass spectrometry (HPLC-MS/MS) [see poster: CM Spangler et al.]. In comparison with classic radiochemical assays an analysis by HPLC-MS/MS allows the detection of non-radioactive compounds. Accordingly, a broader spectrum of substrates that is not available for radioactive assays can be used. Additionally, different nucleotides can be detected at the same time and the detection is directly structurebased. Making use of the new HPLC-MS/MS method we systematically characterised the substrate specificity of sGC.