A fluorometric assay for cyclic guanosine 3',5'-monophosphate incorporating a Sep-Pak cartridge and enzymatic cycling.
We developed a sensitive and nonradioactive fluorometric assay for cyclic guanosine 3',5'-monophosphate (cGMP). Guanine nucleotides except cGMP were enzymatically phosphorylated to GTP. cGMP, absorbed into a Sep-Pak amino propyl cartridge, was eluted separately from GTP. Purified cGMP was enzymatically converted to GTP, which was applied to the GTP-GDP cycle using succinic thiokinase and pyruvate kinase. When pyruvic acid produced by the GTP-GDP cycle was reduced by lactate dehydrogenase, a reduced form of nicotinamide adenine dinucleotide (NADH) was equivalently oxidized to NAD(+). NAD(+) was further converted into fluorescent compound, which was excited at 370 nm and emitted fluorescence at 460 nm, by a strong alkali. When 20 nmol NADH was used for this assay, the calibration curve over 50 to 500 fmol cGMP became sufficiently linear. The detection limit for cGMP was ca. 5 fmol (signal to noise ratio >3). Using this assay, we confirmed that the cGMP content in the left atrial strip of dog was changed from 11.4 +/- 3.8 to 19.3 +/- 2.6 fmol/mg wet wt of tissue (mean +/- SE, n = 6) by electrical driving at 1 Hz. Carbachol (1 microM) further increased the cGMP to 45.6 +/- 9.2 fmol/mg wet wt of tissue. From these results, it is suggested that this novel assay for cGMP is highly sensitive and can be applied to various biological samples.
Department of Pharmacology, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562, Japan.
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Seya
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Furukawa
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Motomura
D000818:Animals; D016279:Atrial Function, Left; D002138:Calibration; D002217:Carbachol / Q000494:pharmacology; D003066:Coenzyme A Ligases; D006152:Cyclic GMP / Q000032:analysis*; D004285:Dogs; D005260:Female; D006160:Guanosine Triphosphate / Q000032:analysis / Q000302:isolation & purification; D006321:Heart / Q000187:drug effects / Q000502:physiology*; D007202:Indicators and Reagents; D007770:L-Lactate Dehydrogenase; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D009243:NAD; D010766:Phosphorylation; D011770:Pyruvate Kinase; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods
D007202:Indicators and Reagents; D009243:NAD; D006160:Guanosine Triphosphate; D002217:Carbachol; D007770:L-Lactate Dehydrogenase; D011770:Pyruvate Kinase; D003066:Coenzyme A Ligases; D006152:Cyclic GMP