Abstract
A method was devised for the quantitative determination of guanine ribonucleotides (GTP, GDP, and GMP) in extracts of biological materlals. The technique is based upon selective enzymatic hydrolysis of UTP and ATP contained within the cell extracts, followed by a quantitative determination of GTP. GTP is measured using a nucleoside diphosphate kinase-firefly luciferase coupled bioluminescent reaction, during which the GTP is enzymatically coupled to ATP production, resulting in ATP-dependent light emission. The methods are simple and reproducible and extremely sensitive (≤ 10 −9 m GTP), and require no preparatory chromatographic separation procedures. Methods are also presented for the enzymatic conversions of GDP and GMP to GTP in addition to the determination of GTP.
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