A real-time fluorescent assay of the purified nitric oxide receptor, guanylyl cyclase.
Michael Newton, Izabella Niewczas, Jonathan Clark, Tomas C Bellamy
文献索引:Anal. Biochem. 402 , 129-136, (2010)
全文:HTML全文
摘要
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.2010 Elsevier Inc. All rights reserved.
相关化合物
相关文献:
2010-05-01
[Endocrinology 151 , 2151-2161, (2010)]
2009-12-15
[Neuroscience 164 , 1244-1251, (2009)]
1980-01-01
[J. Cyclic Nucleotide Res. 6(6) , 461-8, (1980)]
2009-04-01
[Biochim. Biophys. Acta 1794(4) , 642-54, (2009)]
1985-03-01
[Endocrinology 116(3) , 935-44, (1985)]