NADPH synthase

NADPH-diaphorase activity is the ability of an enzyme to reduce soluble tetrazolium salts to an insoluble, visible formazan. This activity is being used by many laboratories to localize NO synthase histochemically. 

NO synthases (NOS, L-arginine, NADPH oxygen oxi-doreductases, nitric oxide forming EC 1.14.13.39) represent a family of enzymes that catalyze the formation of nitric oxide (NO) from the amino acid L-arginine. In mammals, three isoforms of NOS have been identified. They are termed neuronal NOS (nNOS, NOS I, NOS1), inducible NOS (iNOS, NOS H, NOS2), and endothelial... 

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... 

A relationship between polyol pathway activity and reduction in endothelium-dependent relaxation in aorta from chronic STZ-diabetic rats has recently been reported (Cameron and Cotter, 1992). In agreement with several previous studies (Oyama et al., 1986 Kamata et al., 1989), endothelial-dependent relaxation was defective in the diabetic rats but the deficit was prevented by prior treatment with an AR inhibitor. The mechanism underlying the defect has been speculated to be due to decreased production of endothelium-derived relaxing factor (EDRF) or nitric oxide, NO (Hattori et al., 1991). It has been speculated that these vascular abnormalities may lead to diminished blood flow in susceptible tissues and contribute to the development of some diabetic complications. NO is synthesized from the amino-acid L-arginine by a calcium-dependent NO synthase, which requires NADPH as a cofactor. Competition for NADPH from the polyol pathway would take place during times of sustained hyperglycaemia and... 

NO is a gaseous neurotransmitter implicated in signaling in the central and peripheral nervous system as well as in the immune system and the vasculature. NO is formed from L-arginine by nitric oxide synthase (NOS). There are three isoforms of NOS. All isoforms require NADPH as a cofactor, use L-arginine as a substrate, and are inhibited by Nw-nitro-L-arginine methyl ester (L-NAME). The three isoforms are separate gene products. One isoform of NOS is a cytosolic, calcium/calmodulin-independent, inducible enzyme (iNOS). It is found in macrophages, neutrophils, vascular smooth muscle, and endothelia. The iNOS... 

One of the sex pheromone components of the housefly, Musca domestica, is Z9-21 H that is found on the cuticular surface of the fly. This compound is formed by the elongation of Z9-18 CoA using malonyl-CoA and NADPH to Z15-24 CoA which is decarboxylated to form Z9-21 Hc (Fig. 3) [78-80]. Other pheromone components include an epoxide and ketone that are produced from Z9-21 Hc by a cytochrome P450 [81,82] and methyl-branched alkanes that are produced by the substitution of methylmalonyl-CoA in place of malonyl-CoA at specific points during chain elongation [83,84]. A novel microsomal fatty acid synthase is involved in production of methyl-branched alkanes in most insects [85-87]. This fatty acid synthase is different from the ubiquitous soluble fatty acid synthase that produces saturated straight chain fatty acids in that it is found in the microsomes and prefers methylmalonyl-CoA. The amino acids valine and isoleucine can provide the carbon skeletons for methylmalonyl-CoA as well as propionate [83]. 

Fig. 1. The metabolic cycle for the synthesis and degradation of poly(3HB). (1) 3-ketothiolase (2) NADPH-dependent acetoacetyl-CoA reductase (3) poly(3HB) synthase (4) NADH-dependent acetoacetyl-CoA reductase (5), (6) enolases (7) depolymerase (8) d-(-)-3-hydroxybutyrate dehydrogenase (9) acetoacetyl-CoA synthetase (10) succinyl-CoA transferase (11) citrate synthase (12) see Sect. 3...

At least two enzymes compete for acetyl-CoA - the citrate synthase and 3-ke-tothiolase. The affinities of these enzymes differ for acetyl-CoA (Table l),and at low concentrations of it the citrate synthase reaction tends to dominate, provided that the concentration of 2/H/ is not inhibiting. The fine regulation of the citrate synthases of various poly(3HB) accumulating bacteria has been studied [ 14, 47, 48]. They appear to be controlled by cellular energy status indicators (ATP, NADH, NADPH) and/or intermediates of the TCA cycle. The 3-ketothio-lase has also been investigated [10-14,49, 50]. This enzyme is, above all, inhibited by CoASH [10,14,49]. This important feature will be further considered below. 

Peroxynitrite reacts with heme proteins such as prostacycline synthase (PGI2), microperoxidase, and the heme thiolate protein P450 to form a ferryl nitrogen dioxide complex as an intermediate [120]. Peroxynitrite also reacts with acetaldehyde with the rate constant of 680 1 mol 1 s" 1 forming a hypothetical adduct, which is decomposed into acetate, formate, and methyl radicals [121]. The oxidation of NADH and NADPH by peroxynitrite most certainly occurs by free radical mechanism [122,123], Kirsch and de Groot [122] concluded that peroxynitrite oxidized NADH by a one-electron transfer mechanism to form NAD and superoxide ... 

The existence of nitric oxide synthase (NOS) in phagocytes (see below) provides a different kind of stimulation and the inhibition of NADPH oxidase. It has been found [72] that the low physiological concentrations of peroxynitrite formed from NO and superoxide stimulated superoxide production by PMA-activated human PMNs through the ERK MAPK pathway, while higher peroxynitrite concentrations inhibited it. Moreover, NADPH oxidase was inhibited by lidocaine, a sodium-blocker, in OZ-activated neutrophils through the suppression of p47phox translocation [73]. 

Holland et al. [125] have shown that the potent vascular smooth muscle cell mitogen and phospholipase A2 activator thrombin stimulated superoxide production in human endothelial cells, which was inhibited by the NADPH oxidase inhibitors. Similarly, thrombin enhanced the production of oxygen species and the expression of )Alphos and Rac2 subunits of NADPH oxidase in VSMCs [126,127]. Greene et al. [128] demonstrated that the activator of NO synthase neuropeptide bradykinin is also able to stimulate NADPH oxidase in VSMCs. Similar to XO, NADPH oxidase enhanced superoxide production in pulmonary artery smooth muscle cells upon exposure to hypoxia [129]. 

LOX-dependent superoxide production was also registered under ex vivo conditions [55]. It has been shown that the intravenous administration of lipopolysaccharide to rats stimulated superoxide production by alveolar and peritoneal macrophages. O Donnell and Azzi [56] proposed that a relatively high rate of superoxide production by cultured human fibroblasts in the presence of NADH was relevant to 15-LOX-catalyzed oxidation of unsaturated acids and was independent of NADPH oxidase, prostaglandin H synthase, xanthine oxidase, and cytochrome P-450 activation or mitochondrial respiration. LOX might also be involved in the superoxide production by epidermal growth factor-stimulated pheochromo-cytoma cells [57]. 

O Donnell et al. [70] found that LOX and not cyclooxygenase, cytochrome P-450, NO synthase, NADPH oxidase, xanthine oxidase, ribonucleotide reductase, or mitochondrial respiratory chain is responsible for TNF-a-mediated apoptosis of murine fibrosarcoma cells. 15-LOX activity was found to increase sharply in heart, lung, and vascular tissues of rabbits by hypercholesterolemia [71], Schnurr et al. [72] demonstrated that there is an inverse regulation of 12/15-LOXs and phospholipid hydroperoxide glutathione peroxidases in cells, which balanced the intracellular concentration of oxidized lipids. 

Similar to LOXs, cyclooxygenases may catalyze superoxide production in the presence of NADH and NADPH [49]. It has been shown [88] that prostaglandin H synthase produced oxygen radicals and hydrogen peroxide during the transformation of 2(3)-tcrt-butyl-4-... 

In order to produce PHAs in plants it is necessary to introduce the biosynthetic enzymes from bacteria. PHB represents the best characterized and simplest form of PHA, and the synthetic pathway (Figure 4.2) has been extensively studied in Ralstonia eutropha. 30,31 Starting from acetyl-CoA, a P-ketothiolase is required in order to form acetoacetyl-CoA. This is then reduced by a NADPH-dependent acetoacetyl-CoA reductase, which gives rise to 3-hydroxybutyryl-CoA. The latter intermediate is the substrate for the polymerization reaction catalyzed by polyhydroxybutyrate synthase.30 In Ralstonia eutropha, the thiolase, reductase, and synthase genes make up an operon.31... 

---