Adenosine Triphosphate and Guanosine Triphosphate Determinations in Intertidal Sediments

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Abstract
Measurements were made of the spatial distributions of adenosine triphosphate (ATP) on a variety of sandy beaches. Samples were collected during ebb flow and extracted in the field using cold sulfuric acid and ethylenediamine tetraacetate (EDTA); the neutralized extracts were immediately frozen for subsequent analyses. ATP was measured using the firefly bioluminescent reaction; however, during these investigations, a disparity was observed between the concentrations of ATP that were calculated from peak height and integrated light flux determinations. Integral measurements were consistently higher. Examination of the kinetics of the light emission reaction revealed an altered pattern of reactivity (relative to ATP standards) for nearly all of the sediment extracts. Thin-layer chromatographic separation procedures indicated that the sediment extracts contained additional nucleotide triphosphates (that is, GTP, UTP) at levels high enough to significantly interfere with quantitative ATP determinations. A technique was devised to eliminate this altered kinetic pattern in order to calculate true ATP levels. Additional methods were developed for the quantitative determination of guanosine triphosphate (GTP) using a nucleoside diphosphate kinase-firefly luciferase coupled reaction. The role of GTP in microbial metabolism, especially in the process of protein synthesis, results in an increase in the [GTP]/[ATP] ratio as a function of increasing growth rate. The measurement of [GTP]/[ATP] ratios in cell extracts may be useful for determining the metabolic states and rates of growth of microbial populations in nature.