Charges of nicotinamide adenine nucleotides and adenylate energy charge as regulatory parameters of the metabolism in Escherichia coli.

Methods for measurements of catabolic reduction charge (defined as NADH/(NADH+NAD+)) and anabolic reduction charge (defined as NADPH/(NADPH + NADP+)) are described using [14C]nicotinamide labeling of Escherichia coli cultures. Together with these parameters the adenylate energy charge (ATP + 1/2ADP)/(ATP + ADP + AMP) was measured using labeling with [2-3H]adenine. These three charges were found under different exponential growth conditions to have values independent of the growth conditions: catabolic reduction charge, 0.05; anabolic reduction charge, 0.45; and adenylate energy charge, 0.9. The charges were examined during interruption of growth primarily affecting catabolism, respiration, or anabolism, leading to changes of the charges. The changes of charges are evaluated as a possible regulation of the metabolic rates utilizing or producing the nucleotides by their respective charges.

Methods for measurements of catabolic reduction charge (defined as NADH/(NADH + NAD+)) and anabolic reduction charge (defined as NADPH/(NADPH + NADP+)) are described using ['Wnicotinamide labeling of Escherichia coli cultures. Together with these parameters the adenylate energy charge (ATP + VzADP)I(ATP + ADP + AMP) was measured using labeling with [2-"Hladenine. These three charges were found under different exponential growth conditions to have values independent of the growth conditions: catabolic reduction charge, 0.05; anabolic reduction charge, 0.45; and adenylate energy charge, 0.9. The charges were examined during interruption of growth primarily affecting catabolism, respiration, or anabolism, leading to changes of the charges. The changes of charges are evaluated as a possible regulation of the metabolic rates utilizing or producing the nucleotides by their respective charges.  (21, or from the energy-dependent transhydrogenase (3) to anabolism.
The turnover rates of the three nucleotide systems are all very rapid; 1 to 10 s-l (4,5). The internal concentrations of these different compounds are in the order of millimolar; thus, it is possible that they are saturating their respective enzyme systems (6). An  were extracted and separated as the adenine nucleotides were, except that after the rinse with methanol and three runs in water, the plates were run in two steps: to 5 cm above origin in 0.1 M LiCl, and rest (15 cm) in 0.8 M LiCl. A typical chromatogram is shown in Fig. B. The catabolic reduction charge is calculated as NADH/(NADH + NAD+) and the anabolic reduction charge as NADPH/(NADPH+ NADP+). Autoradiograms were regularly performed of the chromatograms.

Charge
Measurements -Nicotinamide is taken up by Escherichia coli and incorporated into the nicotinamide nucleotides (11). The K,, for uptake of nicotinamide was estimated to 0.2 KM (results not shown) (5). Total incorporation, determined after filtration of the cells, showed a biphasic uptake ( Fig. 2A), indicating a rapid equilibration of the total nicotinamide pool. Virtually all of the labeled material in the cells was acid soluble, indicating that it consisted of small molecules. The sum of the four nicotinamide nucleotides measured by the chromatographic method was equal to the total incorporation measured by filtration ( Fig. 2B), showing that almost all radioactive material in the cell had been incorcpm 3000 porated exclusively into the four nicotinamide nucleotides, as reported previously (12).
NADH and NADPH are labile in acid, which makes acid extraction impossible for measurements by fluorescence and enzymatic methods. The reaction of hydrogen ions with the reduced nicotinamide nucleotides forms products (13) which are not fluorescent.
However, acid-treated NADH and NADPH are found at the same positions as the untreated compounds in the chromatographic system, determined by ultraviolet detection of the spots of pure markers (5). The result of this method for determination of nicotinamide charges are in general agreement with enzymatic measurements (14) on cell extracts as indicated in Table I. Charges during Exponential Growth-E.
coli was grown with different carbon sources, which gave growth rates ranging from 0.2 to 2.4, doubling per h. As shown in Table II   tially and then rose during the starvation to 0.45 by 130 min, possibly because the cells were adapting to acetate growth. Thus, addition of glucose at this time did not change the anabolic reduction charge. A down shift of a culture growing on glucose was induced by addition of cY-methylglucoside, which has been shown to inhibit glucose uptake competitively (22,23). Fig. 5A shows the charges during adaption to this new growth condition. Energy charge and anabolic reduction charge fell initially from 0.9 to 0.8 and 0.5 to 0.3, respectively, and rose again while the growth rate was changing during the lag phase to the new exponential rate. Catabolic reduction charge stayed constant, with the value 0.05.
A shift from glucose to mannose (Fig. 5B) did not cause any growth lag; a steady state growth on mannose was achieved immediately after the end of glucose utilization. All charges remained constant during this shift. This is remarkable since the specific rate of oxygen consumption fell abruptly during this transition, and similarly to the cr-methylglucoside-induced down shift described above (5 shown in Fig. 6A. Since succinate cannot be fermented this shift leads to a total termination of the ATP production and NADH utilization. was here constant, with the value 0.05. During the oxygen starvation the anabolic reduction charge slowly decreased from 0.55 to 0.2. This result is difficult to interpret since the rate of NADPH production is possible both coupled to the respiration via the energy-dependent transdehydrogenase (3) and to the catabolism via the NADP-dependent isocitrate dehydrogenase (21. In case of oxygen starvation during growth on glucose fermentation will result. Energy charge in this case has been reported to drop from 0.8 to 0.7 and to rise slowly to 0.8 during the starvation (16). The latter can be seen as a result of the adaption to fermentation.
The catabolic reduction charge stayed constant, at a value of 0.05; the anabolic reduction charge dropped transiently from 0.5 to 0.35 upon the oxygen starvation, as shown in Fig. 6B. The catabolic reduction charge has been reported to rise from 0.2 to 0.6 during anaerobiosis of,!?. coli grown in rich media (181, measured enzymatitally, and the sum of reduced nicotinamide adenine nucleotides (NADH, NADPH) has been shown by the irz viuo fluorescence technique to rise during anaerobiosis (17,20).  Fig. 3). Intermediate values of the rate of NADPH utilization (proportional to the growth rate) and anabolic reduction charge are seen during the glucose starvation and a-methylglucosideinduced shift (Fig. 4, Fig. 5A ). The rate of NADPH production seems to be regulated within a range of the anabolic reduction charge of 0.2 to 0.3 unit; production is high (steady state growth value) at an anabolic reduction charge of 0.4 to 0.5 and low at an anabolic reduction charge of 0.6 to 0.7, as indicated by the experiments with chloramphenicol and rifampicin (Fig.  7, A and B The cells adapt from one set of balanced nucleotide utilizing and producing rates regulated by the steady state charges, toward a new set of rates controlled by the same steady state charges, as seen in Fig. 4, Fig. 5, A and B, Fig.  6B. Since the maximal rates of metabolic reactions are determined by the total amount of enzymes present, these results suggest that the amounts of enzymes charging and discharging the transfer nucleotides may be regulated by the relevant charge, by induction, or repression of synthesis of the enzymes.