Oscillation NADH concentrations

Fig. 3.4 The glycolytic pathway produces NADH which under regular conditions is oxidized to NAD+ while reducing acetaldehyde (ACA) to ethanol (EtOH), thereby in turn reducing NAD+ in order to keep hexose catabolism running. The actual cytosolic NADH concentration is determined by the respective conversion rates of the enzymes involved in the oxidation and regeneration of the compound. If these enzymes convert additional non-natural substrates (xenobiotics, i.e. drugs), the conversion rate changes. As a consequence, the cytosolic NADH concentration differs from the natural condition. Furthermore, if a xenobiotic acts as an enzyme inhibitor, e.g. for ADH, then NAD+ regeneration is substantially affected, which eventually results in altered cytosolic NADH concentration. Therefore the presence of a xenobiotic in the cell is conceivably a perturbation factor. Under the conditions where glycolytic oscillations...

The upshot on the oscillation is a direct measure for the extent of perturbation on the metabolic network upon the uptake of a PAC. Glycolytic oscillations that are systematically perturbed by altered environmental conditions, i.e. exposure to the xenobiotic, constitute a direct and easily accessible measure of the intracellular behavior since the frequency and amplitude of oscillating metabolite concentrations and fluxes depend on both the perturbation and on most intracellular processes due to the coupled energy (ATP) and redox (NADH) balances (Fig. 3.4). 

Fig. 3.6 Vanishing oscillations and flux re-routing for increasing ketone concentration, (a) NADH concentration oscillates between two solid curves, the unstable steady state is denoted by the thin dashed curve, (b) L-Carbinol (solid) and D-carbinol (dashed) fluxes, (c) C3 carbon fluxes where time averages are shown in the oscillatory region.

Oscillations in NADH concentrations as well as in other glycolytic intermediates are observed experimentally by Pye and Chance (1966). In addition double-frequency oscillations in NADH are observed, Fig. III.24. 

Yamazaki and collaborators studied the horseradish peroxidase catalyzed oxidation of NADH by oxygen, 02, in an open system, and observed oscillations in concentrations of oxygen (1965) and of NADH (1967). Degn (1968, 1969) also observed oscillations in a similar peroxidase catalyzed reaction. Degn and Mayer (1969) investigated the theoretical considerations of these reactions and more recently Olsen and Degn (1977) observed chaotic oscillations in the peroxidase catalyzed oxidation of NADH by 02 in an open system, Fig. III.31. 

Notice that the partial oxygen pressure remains constant and that in both situations large amplitude oscillations in NADH and some amino acid concentrations are observed. 

In this section we discuss the model predictions for the ketone ethyl acetoacetate (1). With the ketone absent ([Ket]x = 0 mM), the extended model reproduces all previous results with oscillations of all system variables above [Glc]xo > 18.5 mM [53]. Figure 3.6 shows the system s response to a fixed glucose concentration [Glc]xo at 30 mM and an increase of [Ket]x to 1 mM. The oscillations vanish at [Ket]x = 0.23 mM in a supercritical Hopf bifurcation and the steady state is stable for [Ket]x > 0.23 mM. Figure 3.6a shows the minimum and maximum concentrations of NADH as two thick curves, while in all other panels the time averages of the plotted variables are shown, not the minimum and maximum values. Since the addition of ketone provides an alternative mode of oxidation of NADH, the concentration of NADH is decreasing in Fig. 3.6a whereas the fluxes of carbinol production are increasing in Fig. 3.6b. 

Oscillations in glycolytic system have been observed experimentally by numerous investigators. An early observation is by Duysens and Amesz (1957). By adding glucose (GLU), they observed oscillations in the concentration of reduced phos-phopyridine nucleotide (NADH). Later, Chance et al. (1964) also reported NADH oscillations in a cell-free extract, and Ghosh and Chance observed oscillations in fructose-1,6-diphosphate (FDP) and glucose-6-phosphate (G6P), see Fig. III. 18. 

Fig. III.3I. Chaotic oscillations in horesradish peroxidase catalyzed oxidation of NADH by 02. Cases a), b) and c) are for decreasing concentrations of peroxidase. (From Olsen and Degn (1977))...

In analyzing oscillations in peroxidase catalyzed aerobic oxidation of NADH, Fedkina et al. (1981) experimentally obtained oscillatory results in concentrations of peroxidase compound Co(III) and 02 and NADH. They studied the influence of temperature change on the oscillations. 

Hung and Ross [6] carried out experiments in which some of the proposed tests are applied to assign roles to four of the detectable species in the HRP system oxygen, NADH, the native enzyme (Per +), and an oxidized form of the native enzyme called compound III (coIII). These tests include comparing the relative amplitude and phase shifts of the oscillations of these compounds, concentration shift regulation and destabilization experiments, qualitative pulsed species response, and quench experiments. 

The phase shifts are also derived from the data in fig. 11.9. Oxygen and NADH are approximately in phase during oscillations, that is, both the maxima and minima of their oscillations occur at the same time coIII, on the other hand, has a different waveform and while its maximum is delayed relative to oxygen and NADH, its minimum occurs at about the same time. On the whole, oscillations of coIII are lagging the native enzyme is nearly antiphase with respect to coIII, which suggests that any other enzymatic intermediates are present in small concentrations. Table 11.8 summarizes the symbolic phase shift relations. 

Fig. 11.12 Measurement of the quench in oscillations of the oxygen concentration due to an addition of 1.5 xL of air to the reacting solution at the arrow. External constraints and initial conditions 20.0 xL/s oxygen flow rate, 21.0 [xL/h NADH flow, 0.54 xM of HRP, 10 xM of DCP, 0.1 xM of MB and 7.5 mL of solution in the reactor. (From [6].)...

This is another method of distinguishing between essential and nonessential species of type B and C. The quench perturbation of essential species and nonessential species of type A is much smaller than that of type B and C nonessential species. Oxygen quenches the oscillations temporarily if a small amount of it is added at a phase when its concentration is decreasing (see fig. 11.12). Adding varying amounts of NADH to the system at different oscillatory phases does not quench the oscillations and confirms their assignments. 

Studies with yeast, heart, and brain have shown that concentrations of intermediates within the glycolytic pathway often follow an oscillating function. Continuous spectrophotometric recording techniques for determining the NAD" /NADH ratio in cell-free extracts first revealed oscillations of the NADH level in these systems. These studies then led to the discovery of glycolytic oscillations in yeast cell and cell-free extracts, beef heart extracts, rat skeletal muscle extracts, and in ascites tumor cells, with concentrations of intermediates varying in the range between 10 and 10 M (Chance et al., 1973). 

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