title: A real-time fluorescent assay of the purified nitric oxide receptor, guanylyl cyclase.
abstract: Nitric oxide (NO) mediates intercellular signaling through activation of its receptor, soluble guanylyl cyclase (sGC), leading to elevation of intracellular guanosine 3',5'-cyclic monophosphate (cGMP) levels. Through this signal transduction pathway, NO regulates a diverse range of physiological effects, from vasodilatation and platelet disaggregation to synaptic plasticity. Measurement of sGC activity has traditionally been carried out using end-point assays of cGMP accumulation or by transfection of cells with "detector" proteins such as fluorescent proteins coupled to cGMP binding domains or cyclic nucleotide gated channels. Here we report a simpler approach: the use of a fluorescently labeled substrate analog, mant-GTP (2'-O-(N-methylanthraniloyl) guanosine 5'-triphosphate), which gives an increase in emission intensity after enzymatic cyclization to mant-cGMP. Activation of purified recombinant sGC by NO led to a rapid rise in fluorescence intensity within seconds, reaching a maximal 1.6- to 1.8-fold increase above basal levels. The sGC inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), eliminated the fluorescence increase due to NO, and the synergistic activator of sGC, BAY 41-2272 (3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine), increased the rate at which the maximal fluorescence increase was attained. High-performance liquid chromatography (HPLC) confirmed the formation of mant-cGMP product. This real-time assay allows the progress of purified sGC activation to be quantified precisely and, with refinement, could be optimized for use in a cellular environment.
authors.affiliation: Babraham Institute, Cambridge, UK.
authors.forename: Michael
authors.initials: M
authors.lastname: Newton
authors.forename: Izabella
authors.initials: I
authors.lastname: Niewczas
authors.forename: Jonathan
authors.initials: J
authors.lastname: Clark
authors.forename: Tomas C
authors.initials: TC
authors.lastname: Bellamy
mesh_terms: D002851:Chromatography, High Pressure Liquid; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000737:chemistry*; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D012680:Sensitivity and Specificity; D000071756:Soluble Guanylyl Cyclase; D013050:Spectrometry, Fluorescence / Q000379:methods; D062367:ortho-Aminobenzoates / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D062367:ortho-Aminobenzoates; D009569:Nitric Oxide; C052862:2'-methylanthraniloyl guanosine cyclic monophosphate; C063981:2'(3')-O-(N-methyl)anthraniloylguanosine 5'-triphosphate; D006160:Guanosine Triphosphate; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D006152:Cyclic GMP