A real-time fluorescent assay of the purified nitric oxide receptor, guanylyl cyclase.
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.
Newton
Michael
M
Babraham Institute, Cambridge, UK.
Niewczas
Izabella
I
Clark
Jonathan
J
Bellamy
Tomas C
TC
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*
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