Adenine Nucleotide Levels in Neurospora , as Influenced by Conditions of Growth and by Metabolic Inhibitors

Abstract

By a combination of luciferase and fluorescence methods adenine nucleotide pools in Neurospora crassa have been examined under various conditions of growth and metabolic inhibition. During sustained exponential growth (25 C, shaking liquid cultures), the intracellular adenosine 5′-triphosphate (ATP) concentration, [ATP]_i, rises slowly from the conidial level near 1 mM (1 mmol/kg of cell water) to a maximum of 2.0 to 2.5 mM at 14 h, after which it slowly declines. The adenosine 5′-diphosphate and adenosine 5′-monophosphate (AMP) curves show two peaks, at 8 and 20 h, with a minimum at 16 h. The “energy charge” function varies around a mean of 0.72 throughout the period of exponential growth. Transferral of growing cells to buffer lacking a nitrogen source stabilizes the [ATP]_i near 2.5 mM, apparently independent of the cell age, and most studies of metabolic inhibitors were carried out on cells grown 14 to 16 h and then shifted to N-free buffer. Under these conditions sudden respiratory blockade (cyanide) produces exponential decay of ATP with a time constant of about 5.7 s (half-time of 3.9 s), and at a rate which implies a minimal ATP turnover of 0.44 mM/min. This figure is about one-third the rate (1.17 mM/min) which would be calculated from steady-state respiration, a discrepancy which may partly be accounted for by transphosphorylation from appreciable amounts of non-adenine nucleoside di- and triphosphates present in Neurospora. For all three adenine nucleotides, the transients associated with sudden respiratory blockade include overshoots or undershoots of several minutes duration, which are consistent with feedback regulation of glycolysis by the AMP/ATP ratio.