NADH-concentration

The answer is that many of the enzymes are sensitive to elevated NADH concentrations. Glycolysis and the bridging reaction will produce NADH in a relatively high quantity. 

The NAD /NADH concentration ratio in the cytosol of the liver is maintained at a value of about 1000 but oxidation of ethanol can lower this ratio by at least tenfold. Many dehydrogenase reactions are close to equilibrium so that, for those that react with NAD /NADH, the concentrations of all the other substrates and products will be affected by a change in the NAD /NADH concentration ratio. Hence, a decrease in the NAD /NADH concentration ratio will lower the concentration of the oxidised reactant and raise that of the reduced reactant of aU the dehydrogenation reactions in the cytosol ... 

Fig. 7. Enzyme-coupled assay in which the hydrolase-catalyzed reaction releases acetic acid. The latter is converted by acetyl-CoA synthetase (ACS) into acetyl-CoA in the presence of (ATP) and coenzyme A (CoA). Citrate synthase (CS) catalyzes the reaction between acetyl-CoA and oxaloacetate to give citrate. The oxaloacetate required for this reaction is formed from L-malate and NAD in the presence of L-malate dehydrogenase (l-MDH). Initial rates of acetic acid formation can thus be determined by the increase in adsorption at 340 nm due to the increase in NADH concentration. Use of optically pure (Ry- or (5)-acetates allows the determination of the apparent enantioselectivity i app i81)-...

Kurkijarvi et al. were the first to demonstrate the feasibility of seg-mented-flow bioluminescence assays by use of a bioreactor packed with bacterial bioluminescent enzymes immobilized on Sepharose 4B [60]. The packed glass colunrn used was placed in front of the photomultiplier tube of a luminometer. The luminescence signal obtained was linearly related to the NADH concentration from 1 pmol to 10 nmol for sample volumes of 2-20 pL. In the region of 400 NADH assays could be performed with a single enzyme column, with no appreciable change in sensitivity or accuracy. However, problems arising from packing or disruption of the matrix were encountered after 4 days of intensive use. 

Some early kinetic studies on the enzymic reaction indicated that LADH exhibits pre-steady state half-of-the-sites reactivity. Bernard et al. reported that two distinct kinetic processes, well separated in rate, were observed for the conversion of reactants into products under conditions of excess enzyme.1367 They also reported that each of the two phases corresponded to conversion of exactly one half of the limiting concentration of substrate being converted to products. On the basis of this they proposed two possible models, the favoured one based on catalytically non-equivalent but interconvertible states of the two binding sites, with the possibility that the asymmetry of the sites may be induced by coenzyme binding. Further evidence for this non-equivalence of the subunits was obtained in similar subsequent studies using a chromophoric nitroso substrate, p-nitroso-A,JV-dimethylaniline with limiting NADH concentrations.1368... 

However, in their study of intermediates in the enzymic reduction of acetaldehyde, Shore and Gutfreund could find no inequivalence in the binding sites of the subunits at all NADH concentrations studied.1369 This conclusion that the two active sites are kinetically equivalent is supported by kinetic studies by Hadom et al.1370 and by Kvassman and Pettersson. 1 Work by Kordal and Parsons also supports this conclusion.13" They devised a method of persuading 3H-labelled NADH to bind to one site per enzyme molecule and then, using a stopped-flow technique, to react this with excess unlabelled product. Full site reactivity was observed in either direction. They concluded that no half site reactivity was observed, and that there was no indication of subunit asymmetry induced by either the coenzyme binding or by chemical reaction. 

The test kit for the determination of acetic acid released is used according to the manufacturer s protocol (see also below). Spectropho to metric determination of NADH concentration is performed at 340 nm in the milliliter scale, e. g., in an Ultrospec 3000 photometer, and on the microliter scale in a fluorimeter, e. g. FLUOstar. 

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...

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.

A complicating factor in studying the oxidation of deuterated NADH derivatives is the possibility that nonstereospecific exchange of C(4) hydrogen between the product NAD+ and the reactant NADH would take place. To minimize exchange under our reaction conditions, we used NADH concentrations less than 0.5mAf. The results of our experiments are listed in Table III. It is quite clear from Table III that flavopapain IV showed considerable, but not complete, prefer-... 

To determine the effect of acid catalysed decomposition of NADH on the electrochemical response in our experiments, the decrease in oxidation current for NADH was recorded as a function of time. The results of this experiment were compared with the decrease in NADH concentration as spectrophotometrically determined. The rates of decrease of the current and the concentration of NADH are both first-order and occur on similar timescales (Fig. 2.14). Analysis of the data for the two experiments provide first-order rate constants of 1.68 and 1.16 x 10-4 s-1 for the electrochemical and spectrophotometric measurements, respectively. The small difference between these two constants can be explained by the additional consumption of NADH by reaction at the electrode during the electrochemical measurement. This electrochemical process is also a first-order rate process, and the extent of the effect can be determined by using the treatment of Hitchman and Albery [50] for electrolysis using a rotating disc electrode. The results are consistent with the observed difference in the two rate constants. 

Fig. 2.16. Plot of the current recorded at +0.1 V at a poly(aniline)/poly(vinylsulfonate)-coated glassy carbon electrode (deposition charge ISO mC, geometric area 0.38 cm2) rotated at 9 Hz in 0.1 mol dm-3 citrate/phosphate buffer at pH 7 as a function of the NADH concentration. The currents are corrected for the background current (<0.2 p.A) in each case. Data for three different films prepared under identical conditions are shown. The inset shows the current for oxidation of NADH at +0.1 V at an uncoated glassy carbon electrode treated identically to the coated electrode except that no aniline monomer was added to...

First, the effect of film thickness on the amperometric response was studied. The effect of film thickness has been assessed for eight different film thicknesses at four different NADH concentrations. For thin films, Fig. 2.18 shows that the response increases with film thickness reaching a... 

The effect of variation of the rotation speed on the current response is shown in Fig. 2.26. The results are for a film of the same thickness as those in Fig. 2.24. We therefore fit the data to the equation for the Case 11/IV boundary (equation (2.21) in Table 2.3). Again, the effect of rotation rate on the NADH concentration at the film/solution interface was determined and accounted for by using equation (2.4). The best fits of the theory to experiment are shown by the lines in Fig. 2.26, and the corresponding values of ca,[site]DsKs and KM/Ks are given in Table 2.6 along with results from a separate replicate experiment. 

Finally, the data for all the experiments carried out at fixed rotation rate for nine film thicknesses, with and without added NAD+ at a range of NADH concentrations up to 5 mmol dm-3 (in all a total of 343 separate data points), were fitted to equations (2.4)-(2.8) for the uninhibited fit, with the addition of equations (2.15), (2.17), (2.18) and (2.19) for the inhibited fit using three adjustable parameters. Figure 2.28 shows a three-dimensional representation of the data, shown by the points, and the best fit to the theory, shown by the surface. The resulting best-fit parameters are given in Table 2.8. The position of the case boundary and the different cases were calculated from the best-fit parameters and are shown in Fig. 2.28. 

Fig. 2.29. Experimental data from poly(aniline)/poly(vinylsulfonate) film deposited on a glassy carbon electrode (0.38 cm2, Qi — 150 mC). The response to increasing NADH concentration as shown by the points. The lines represent the uninhibited... 

For this purpose an analytical thin-layer cell and a laboratory-scale batch reactor were successfully used. The coupled system was controlled by the NADH concentration level. The major disadvantage of the process is the poor stability of the hydrogenase. 

In the model of Termonia and Ross, glucose, inorganic phosphate, NAD, and NADH concentrations are not modeled explicitly and assumed fixed. The kinetic equations for the remaining reactants are... 

We can see from Figure 6.3 that the relative NADH concentration is the more important controller of steady state TCA cycle flux, in agreement with experimental observations [124], When ADP concentration is low, a variation in [NAD //V from 0 to 1 produces a change in JDH from 0 to nearly 1.59 mmol sec-1 (1 mito)-1. When NAD concentration is near zero, the rate of NADH production is not sensitive to ADP. Yet the flux is by no means insensitive to ADP. Neither NAD nor ADP represents a sole independent controller of the system. 

The concentrations of NAD and NADH are not modeled in the above equations. Since NADH-generating fluxes are not included, this model holds NAD and NADH concentrations at fixed concentrations. The behavior of the model as a function of NADH redox state is explored below. 

The relationship between the work rate (rate of delivery of ATP out of the mitochondrion) and [ADPC] is illustrated in Figure 7.12. Flux through the ANT transporter increases with [ADPC], with higher flux possible at higher NADH concentration, due to the effect of NADH on AT, which drives ANT. 

---