A Fluorometric Assay for Cyclic Guanosine 3′,5′-Monophosphate Incorporating a Sep-Pak Cartridge and Enzymatic Cycling
✍ Scribed by Kazuhiko Seya; Ken-Ichi Furukawa; Shigeru Motomura
- Publisher
- Elsevier Science
- Year
- 1999
- Tongue
- English
- Weight
- 85 KB
- Volume
- 272
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
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 M) 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.