HPLC and 32P‐radiolabeling method for quantification of microbial adenylate concentrations and turnover rates in seawater

Abstract

We optimized a high throughput method to quantify turnover rates of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) in marine microbes from simultaneous measures of the respective stocks and phosphorylation rates. We combined a microbial adenylate extraction method using boiling 20 mM Tris buffer with purification and analysis by high pressure liquid chromatography optimized to quantify these intracellular adenylate concentrations in marine microbes. Additionally, we incorporated radiolabeled phosphate (32Pi) incubations to quantify phosphorus (P) uptake rates and the phosphorylation rates for these adenylate compounds in microbial cells. With this method, we can directly assess the variations in microbial growth rates, metabolic turnover rates, energy charge, and adenylate storage. We applied and validated this method application with environmental samples from Biscayne Bay, Florida, and quantified adenylate turnover times of 12, 15, and 73 min, for ATP, ADP, and AMP, respectively. Future incorporation of this method into experiments and geographic surveys across marine environments will allow for direct assessments of changes in microbial metabolic activity in relation to other ecological variables.