Measurement and significance of ATP and adenine nucleotide pool turnover in microbial cells and environmental samples

Abstract

A method was devised to measure the turnover rates of adenosine 5′-triphosphate (ATP) and total adenine nucleotide (AN) pools in cultures and natural populations of microorganisms. The method relies on 32PO 4 as a tracer for P-flux through the α-, β- and γ-positions of cellular ATP. Following a timed incubation, cellular ATP is extracted and purified. The isolated AT 32P is subjected to enzymatic hydrolysis designed to selectively hydrolyze ATP to either ADP + Pi (ATPase reaction) or to AMP + 2 Pi (apyrase reaction). The released 32Pi is separated from the parent nucleotides (ADP and AMP) and measured by liquid scintillation counting procedures. From time course information on the change in specific activity (i.e., nCi pmol −1) of the γ-P and α-P positions of ATP, the turnover time of the total ATP and AN pools, respectively, can be determined. This kinetic information can be used to estimate total cellular energy flux (derived from the ATP turnover rate) and specific growth rate (derived from the AN turnover rate). The method is relatively simple, reproducible and accurate, and has the sensitivity required for successful application in even the most oligotrophic oceanic habitats.