31P NMR magnetization transfer study of the control of ATP turnover in Saccharomyces cerevisiae.

Abstract

ABSTRACT31P NMR magnetization transfer measurements have been used to measure the steady state flux between P1 and ATP in yeast cells genetically modified to overexpress an adenine nucleotide translocase isoform. An increase in Pi -> ATP flux and apparent ratio of moles of ATP synthesized/ atoms of oxygen consumed (P:O ratio), when these cells were incubated with glucose, demonstrated that the reactions catalyzed by the translocase and F1Fo ATP synthase were readily reversible in vivo. However, when the same cells were incubated with ethanol alone, translocase overexpression had no effect on the measured Pi -> ATP flux or apparent P:O ratio, suggesting that the synthase was now operating irreversibly. 31P NMR magnetization transfer techniques have been used to measure directly the rate of ATP turnover in a variety of systems, including Escherichia coli (1, 2), yeast (3-8), rat brain (9), and the perfused rat heart (10-12). A primary objective of these studies, particularly those on the heart, has been to determine the extent to which the F1Fo ATP synthase is displaced from equilibrium and, thus, to assess its potential for exerting kinetic control over flux in mitochondrial oxidative phosphorylation (13). These measurements have also been used to determine the degree of mitochondrial coupling, as reflected by the apparent ratio of moles of ATP synthesized/ atoms of oxygen consumed (P:O ratio) (14).Previous magnetization transfer measurements of Pi --ATP flux in vivo, in which the contribution of the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) and phosphoglycerate kinase (PGK; EC 2.7.2.3) to this measured flux has been removed (see Fig. 1), have produced values for the apparent P:O ratio of between 2 and 3 (6, 12). The similarity of these values to those measured with isolated mitochondria catalyzing net ATP synthesis in vitro was taken to indicate that the reaction catalyzed by the mitochondrial ATP synthase is effectively irreversible in vivo and that the magnetization transfer experiment is measuring net ATP turnover. However, isotope exchange measurements on isolated rat heart and liver mitochondria have shown that the unidirectionality of ATP synthesis depends on the rate of respiration (15). At high rates of respiration, the reaction was unidirectional, whereas at low rates, it was near-toequilibrium. This study suggested that an exchange reaction between P1 and ATP should be observable in NMR magnetization transfer measurements in the perfused rat heart at low workloads (15). Determination of P:O ratios in isolated mitochondria and rat hepatocytes have shown that the P:O ratio also depends on the rate of respiration, being close to zero at low rates of respiration and approaching its maximal value at high rates of respiration (16-18). These latter studies undermine the value of the P:O ratio as an indicator of the irreversibility of the ATP synthase reaction in NMR magnetization transfer measurements. Both studies suggest that an ATP < Pi exchange reaction, catalyzed by...

Comparison of in vivo postexercise phosphocreatine recovery and resting ATP synthesis flux for the assessment of skeletal muscle mitochondrial function

“…Up to this point, it has been assumed that the mitochondrial F 1 F 0 -ATP synthase flux, which constitutes only a small part of the total V ATP measured, is unidirectional. However, it has been shown that at low rates of respiration (e.g., resting muscle) the F 1 F 0 -ATP synthase reaction is near to equilibrium and that mitochondrial exchange between P i and ATP can make a significant contribution to the measured V ATP (50). Taken together, if there would have been an effect of DPI on the rate of net resting mitochondrial ATP production, this effect might not be measurable due to the large contribution of glycolytic exchange flux and possibly also a significant contribution of mitochondrial exchange flux to the total measured V ATP .…”

Comparison of measuring energy metabolism by different 31P‐magnetic resonance spectroscopy techniques in resting, ischemic, and exercising muscle

“…There is evidence from yeast that glycolytic reactions contribute strongly to fATP measured by 31 P-magnetic resonance spectroscopy (32,33), indicating that reversible glycolytic reactions might explain the higher ATP synthetic rates. Finally, mitochondrial ATP formation is not strictly irreversible (34). As a consequence, the apparent forward flux (fATP) exceeds the net production many times (26).…”

The significance and mechanism of mitochondrial proton conductance

“…18,19 If the substrate remains the same, a drop in PaO ratio below the theoretical or control value is caused by diversion of the proton¯ux into the leak pathway.…”