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.
Seya
K
K
Department of Pharmacology, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562, Japan.
Furukawa
K I
KI
Motomura
S
S
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