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Escherichia coli cells were labeled with (33)PO(4) for several generations and then (32)PO(4) was added, samples were taken at various times, and adenosine triphosphate (ATP) and guanosine triphosphate (GTP) were purified from an acid extract. The ratio of (32)P/(33)P in each of the six phosphate species was determined and compared to the ratio expected at isotopic steady state. The rate constants of the simplest network of pools which could explain the data, and were consistent with metabolic pathways, were then calculated. In the range of times studied (30 to 3,000 s at 21.5 C) the following generalizations can be made. (i) The gamma-phosphate at ATP requires 2,200 s to achieve 50% of its final specific activity and behaves as if it were exchanging with another species 4.4 times as large, with this other species turning over in 540 s. (ii) The beta-phosphate of ATP and the gamma-phosphate of GTP are in equilibrium with the gamma-phosphate of ATP. (iii) The specific activity of the gamma-phosphate of GTP lags 66 s behind the gamma-phosphate of ATP. (iv) The alpha-phosphates of ATP and GTP have equal specific activities which lag far behind the other four phosphates. A single precursor pool with a turnover time of 416s fits the data best, although a fair fit can be obtained assuming several sequential precursor pools with much shorter turnover times. These findings are consistent with known biosynthetic pathways and the probable flows through them, including the relationship of nucleotide biosynthesis to ribonucleic acid synthesis and turnover.

Radioactive ATP and ADP were found in platelets after incubation of human platelet-rich plasma with either [8-(14)C]adenosine or [8-(14)C]ADP. Treatment of the labelled and washed platelets with thrombin indicated that, though considerable amounts of ATP and ADP were released to the supernatant, radioactive ATP and ADP remained predominantly in the cellular fraction. Breakdown of radioactive ATP took place to form mainly IMP and hypoxanthine, the latter compound appearing in the supernatant. The results indicate the presence of at least two pools of nucleotide in platelets. Evidence is given that the two pools contain approximately the same amounts of ATP plus ADP, and that the ratio of ATP to ADP in the pool released to the supernatant by the action of thrombin is about 0.7-0.8.

1. Ox-brain microsomes were incubated with [gamma-(32)P]ATP under various conditions. After the reaction, which was stopped with trichloroacetic acid, a small amount of phosphate remained bound to the washed precipitate. 2. Properties of the bound phosphate were studied by treatment with buffers and solvents. 3. The Na(+)-dependent increment in bound phosphate, predominant at low ATP concentration and features of which suggest involvement in the concomitant adenosine-triphosphatase activity, was rapidly released in both circumstances. 4. In aqueous media the labile phosphate was released entirely as inorganic phosphate at faster rates with increasing alkalinity. 5. In acidified chloroform-alcohol mixtures the released phosphate appeared both as inorganic phosphate and different single (32)P-labelled organic phosphates, which were tentatively identified as the relevant mono-alkyl phosphates, presumably derived by acid-catalysed alcoholysis of a labelled microsomal component, or components. 6. The labile phosphate corresponded to the P exchangeable with non-radioactive ATP added during the enzyme reaction. 7. The possible molecular nature of the labile fraction of the bound phosphate is discussed.

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.