NADPH oxidation

It was shown in Section 2.3.4.3 that poly(aniline)/poly(vinylsulfonate) films are also electrocatalytic surfaces for the oxidation NADPH. Assuming that the kinetics of this system are similar to those for the NADH system, the data have been analysed using the uninhibited kinetic model. From comparison to the NADH data, this film is thick (e 1) and the data span both Cases II and IV. Therefore, all the data were fitted using the expression for the Case II/IV boundary (equation (2.21) Table 2.4) using equation (2.4) to calculate the concentration of NAD(P)H at the film/solution interface. The fit is shown in Fig. 2.30 and the parameters generated by this fit are given in Table 2.9. 

Analytical models of modified electrodes for NADH oxidation 

Comparison with the corresponding values for NADH oxidation shows that the parameters are quite similar as expected. Since the experiment was carried out using the same polymer film, the fact that the parameters obtained are not identical indicates slight differences in the kinetics for the two substrates. 

Starting from a knowledge of the homogeneous mechanism and kinetics, we have shown in this chapter how we can identify possible approaches to 

Best-fit parameters from the analysis of the currents for NAD(P)H oxidation using the uninhibited 2 parameter fit at poty(aniline)/poly(vinylsulfonate)-modified electrodes. The values were obtained by non-linear least squares fits of the experimental data in Fig. 2.30 to equations (2.4) and (2.21). Also shown are the best-fit parameters from the uninhibited 2 parameter fit analysis of the currents for NADH oxidation at the same poly(aniline)/poly(v-inylsulfonate)-modified electrode (n is the number of data points used) 

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Forstermann U (2006) Janus-faced role of endothelial NO synthase in vascular disease - Uncoupling of NADPH oxidation from NO synthesis and its pharmacological reversal. Biol Chem 387 1521-1533... 

In addition to moving acetyl-CoA from the mitochondria to the cytoplasm, this cycle also converts an NADH to an NADPH. If we assume that the amount of ATP that we could get from NADH and NADPH oxidation is the same, making NADPH from NADH and NADP+ doesn t cost any energy. So we can conclude that the cost of just moving the acetyl-CoA out of the mitochondria is 2 ATPs per acetyl-CoA. 

Shen T, Hollenberg PF. The mechanism of stimulation of NADPH oxidation during the mechanism-based inactivation of cytochrome P450 2B1 by N-methylcarbazole redox cycling and DNA scission. Chem Res Toxicol 1994 7(2) 231-238. 

Poyer JL, Floyd RA, McCay PB, etal. 1978. Spin-trapping of the trichloromethyl radical produced during enzymic NADPH oxidation in the presence of carbon tetrachloride or bromotrichloromethane. Biochim Biophys Acta 539 402- 409. 

Terpene Induction of mldgut microsomal cytochrome P-450 and pyrethrum-dependent NADPH oxidation In southern armyworm larvae (42)... 

Diet Cytochrome P-450 (nmole/mg protein) X-max (nm) NADPH oxidation (nmole/mln,mg protein)... 

Table VII shows the Increase In cytochrome P-450 content In mlcrosomes from southern armyworm larval midguts resulting from dietary exposure to several cyclic monoterpenes ( ). It also shows a closely corresponding Increase In the rate of NADPH oxidation when pyrethrum Is the substrate (R) being oxidised. The microsomal cytochrome P-450 system Is arranged as outlined In Figure 6, consisting of a terminal heme-lron protein that In the oxidised (Fe3+) state binds the substrate (R). The complex undergoes two reductions during which bound molecular oxygen Is converted to free radical species, one of which Is Inserted In the substrate molecule, and the other one forms water. The reductions...

NADPH oxidation and NO synthesis by the enzyme, it supports a role for reduction of the heme iron in catalysis, and may explain why NOS functions only as an NADPH-dependent reductase in the absence of bound calmodulin (Klatt et ai, 1993). The mechanism of calmodulin gating is envisioned to involve a conformational change between the reductase and oxygenase domains of NOS, such that an electron transfer between the terminal flavin and heme iron becomes possible. Calmodulin may also have a distinct role within the NOS reductase domain, in that its binding dramatically increases reductase activity of the enzyme toward cytochrome c (Klatt et al., 1993 Heinzel et al., 1992). However, it is clear that several other NOS functions occur independent of calmodulin, including the binding of L-arginine and NADPH, and transfer of NADPH-derived electrons into the flavins (Abu-Soud and Stuehr, 1993). 

Babior, who has studied this enzyme at several stages of its purification, found in lysates of PMNs which were activated with zymosan that of eight potential biological reductants only reduced pyridine nucleotides supported the formation of O ". The K , for NADPH was less than the K , for NADH and the activity was decreased in preparations from three patients with chronic granulomatous disease. In accord with predictions based on reaction 7, 0.55 molecule of O7 was measured per molecule of NADPH oxidized under conditions of saturating concentrations of cytochrome c The enzyme which was extracted with Triton X-100 from a granule-rich fraction from activated PMNs, required an external source of FAD for the formation of O from NADPH . Riboflavin and FMN would not substitute. Flavin adenine dinucleotide was proposed as a necessary cofactor, which was probably lost when the enzyme was treated with the detergent. 

However, the reactive metabolite will cause other changes as well as binding to protein. Thus, NAPQI will react both chemically and enzymatically with GSH to form a conjugate and will also oxidize it to GSSG and in turn be reduced back to paracetamol. This cyclical process may explain the occurrence of extensive depletion of GSH. NADPH will also reduce NAPQI and in turn be oxidized to NADP, although reduction via GSH is probably preferential. NADPH oxidation may also result from GSSG reduction via GSH peroxidase (Fig. 7.18). 

It was a surprise to discover that a mutant of E. coli lacking thioredoxin can still reduce ribonucleotides. In the mutant cells thioredoxin is replaced by glutaredoxin, whose active site disulfide linkage is reduced by glutathione rather than directly by NADPH. Oxidized glutathione is, in turn, reduced by NADPH and glutathione reductase. Thus, the end result is the same with respect to ribonucleotide reduction. 

Thyroid Hormone. Treatment of rats with thyroxin increases hepatic microsomal NADPH oxidation in both male and female rats, with the increase being greater in females. Cytochrome P450 content decreases in the male but not in the female. Hyperthyroidism causes a decrease in gender-dependent monooxygenase reactions and appears to interfere with the ability of androgens to increase the activity of the enzymes responsible. Gender differences are not seen in the response of mice and rabbits to... 

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