کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10532858 | 961756 | 2010 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
A real-time fluorescent assay of the purified nitric oxide receptor, guanylyl cyclase
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موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
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چکیده انگلیسی
Nitric oxide (NO) mediates intercellular signaling through activation of its receptor, soluble guanylyl cyclase (sGC), leading to elevation of intracellular guanosine 3â²,5â²-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 (2â²-O-(N-methylanthraniloyl) guanosine 5â²-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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 402, Issue 2, 15 July 2010, Pages 129-136
Journal: Analytical Biochemistry - Volume 402, Issue 2, 15 July 2010, Pages 129-136
نویسندگان
Michael Newton, Izabella Niewczas, Jonathan Clark, Tomas C. Bellamy,