NADPH-dependent microsomal reductases

Thus, superoxide itself is obviously too inert to be a direct initiator of lipid peroxidation. However, it may be converted into some reactive species in superoxide-dependent oxidative processes. It has been suggested that superoxide can initiate lipid peroxidation by reducing ferric into ferrous iron, which is able to catalyze the formation of free hydroxyl radicals via the Fenton reaction. The possibility of hydroxyl-initiated lipid peroxidation was considered in earlier studies. For example, Lai and Piette [8] identified hydroxyl radicals in NADPH-dependent microsomal lipid peroxidation by EPR spectroscopy using the spin-trapping agents DMPO and phenyl-tcrt-butylnitrone. They proposed that hydroxyl radicals are generated by the Fenton reaction between ferrous ions and hydrogen peroxide formed by the dismutation of superoxide. Later on, the formation of hydroxyl radicals was shown in the oxidation of NADPH catalyzed by microsomal NADPH-cytochrome P-450 reductase [9,10]. 

Other types of reduction catalyzed by non-microsomal enzymes have also been described for xenobiotics. Thus, reduction of aldehydes and ketones may be carried out either by alcohol dehydrogenase or NADPH-dependent cytosolic reductases present in the liver. Sulfoxides and sulfides may be reduced by cytosolic enzymes, in the latter case involving glutathione and glutathione reductase. Double bonds in unsaturated compounds and epoxides may also be reduced. Metals, such as pentavalent arsenic, can also be reduced. 

Since the distinctive metabolism of testosterone provides the most plausible explanation of the diversity of androgenic responses, brief reference should be made to the enzymes involved. Such a summary is presented in Table 2. Crucial enzymes include microsomal and nuclear NADPH-dependent 5a-reductase, the microsomal... 

Biorcduction of nitro compounds is carried out by NADPH-dependent microsomal and soluble nitro reductases present in the liver. A multicomponent hepatic microsomal reductase system requiring NADPH appears to be responsible for azo reduction. " " In addition, bacterial reductases present in the intestine can reduce nitro and azo compounds, especially those that are absorbed poorly or excreted mainly in the bile. - ... 

Purified pig liver NADPH-cytochrome P-450 reductase had a low level of chromate-reductase acitivity. This enzyme (Eo = - 320 mV, - 190 mV), like the NADPH cofactor (-320 mV), although thermodynamically capable of reducing chromate must have a kinetic barrier to the reaction. Upon adding purified PB-induced rat liver cytochrome P-450 to the purified reductase the NADPH-dependent chromate reductase activity was greatly increased.. Thus, reconstitution studies with the purified enzymes confirm our previous studies with microsomes which implicated cytochrome P-450 as the chromate reductase. Cytochrome P-450 appears to be able to transfer electrons to chromate in a facile manner. 

Nitroreductase activity (substrate / -nitrobenzoic acid) was present in the hepatopancreas of H. americanus (Table 20), mainly in the cytosol (79% of total activity), but also in the microsomes (11%) and mitochondria (10%) (Elmamlouk and Gessner 1976). The mitochondrial activity was more active with NADH than with NADPH, whereas the reverse was seen for the cytosolic enzyme. Differential inhibitory and stimulatory effects of FAD and FMN indicated the existence of distinct NADH- and NADPH-dependent cytosolic reductases. The activity of the cytosolic NADPH-dependent reductase was increased under anaerobic conditions. 

A NADPH-dependent A reductase which converts ergosta-5,7,22,-24(28)-tetraenol into ergosterol (12-H) is present in yeast microsomes (Jarman eta/., 1975). 

Squalene epoxidase, like most enzymes responsible for the later steps of sterol biosynthesis [43, 51], is membrane-bound which makes its purification in native form challenging. The purification is additionally complicated by the presence of a large number of cytochrome P450 and other enzymes that have similar hydro-phobicity and size as squalene epoxidase and are hence difficult to remove [52]. Most studies have been carried out with rat liver microsome squalene epoxidase either partially purified or as a homogenate of the cell membrane fraction. In vitro reconstitution of squalene epoxidase activity is absolutely dependent on molecular oxygen, NADPH, FAD, and NADPH-cytochrome c reductase [52, 53]. In this respect, squalene epoxidase resembles the cytochrome P450 enzymes described... 

Superoxide generation was detected via the NADPH-dependent SOD-inhibitable epinephrine oxidation and spin trapping [15,16], Grover and Piette [17] proposed that superoxide is produced equally by both FAD and FMN of cytochrome P-450 reductase. However, from comparison of the reduction potentials of FAD (-328 mV) and FMN (190 mV) one might expect FAD to be the most efficient superoxide producer. Recently, the importance of the microsomal cytochrome h558 reductase-catalyzed superoxide production has been shown in bovine cardiac myocytes [18]. 

This mechanism is now considered to be of importance for the protection of LDL against oxidation stress, Chapter 25.) The antioxidant effect of ubiquinones on lipid peroxidation was first shown in 1980 [237]. In 1987 Solaini et al. [238] showed that the depletion of beef heart mitochondria from ubiquinone enhanced the iron adriamycin-initiated lipid peroxidation whereas the reincorporation of ubiquinone in mitochondria depressed lipid peroxidation. It was concluded that ubiquinone is able to protect mitochondria against the prooxidant effect of adriamycin. Inhibition of in vitro and in vivo liposomal, microsomal, and mitochondrial lipid peroxidation has also been shown in studies by Beyer [239] and Frei et al. [240]. Later on, it was suggested that ubihydroquinones inhibit lipid peroxidation only in cooperation with vitamin E [241]. However, simultaneous presence of ubihydroquinone and vitamin E apparently is not always necessary [242], although the synergistic interaction of these antioxidants may take place (see below). It has been shown that the enzymatic reduction of ubiquinones to ubihydroquinones is catalyzed by NADH-dependent plasma membrane reductase and NADPH-dependent cytosolic ubiquinone reductase [243,244]. 

Table I). The levels of both, cytochrome P-L50 (Table i) and its NADPH (reduced nicotinamide adenine dinucleotide phosphate) requiring reducing component (Figure l)(which can be measured as NADPH dependent cytochrome c reductase) are substantial in fish liver microsomes, although lower than in mammals. NADPH cytochrome c reductase level in trout Salmo trutta lacustris) is 20 nmol cytochrome c reduced/mg microsomal protein/min the corresponding activity in male Sprague Dawley rat liver microsomes is 96 nmol cytochrome c reduced/mg microsomal protein/min (lU). 

All are probably bound to the microsomal membranes.503 507a An NADPH-dependent reductase reduces vitamin K quinone to its hydroquinone form. Conversion of Glu residues to Gla residues requires this reduced vitamin K as well as 02 and C02. During the carboxy-lation reaction the reduced vitamin K is converted into vitamin K 2,3-epoxide (Eq. 15-55).508 The mechanism is uncertain but a peroxide intermediate such as that shown in Eq. 15-56 is probably involved. This could be used to generate a hydroxide ion adjacent to the pro-S -H of the glutamate side chain of the substrate. This hydrogen could be abstracted by the OH to form... 

Nitrobenzene reductase activity has been detected in the fat body, gut, and Malpighian tubules of the Madagascar cockroach, G. portentosa (Rose and Young, 1973). Anaerobic conditions are essential for activity. The enzymes in the microsomes are strongly NADH dependent, whereas those in the soluble fraction are strongly NADPH dependent. Activity is enhanced by the addition of flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) or riboflavin. It appears that the true substrate for the nitroreductase is FMN and that the reduction of the nitro compounds occurs nonenzymatically (Figure 8.15). Similar results are obtained using azofuchsin as substrate. 

Monooxvgenases. These enzymes are important in the detoxication of pyrethroid, carbamate, organophosphorus and other classes of insecticides (2). They are microsomal, membrane associated enzymes which catalyze reactions in which one atom from molecular oxygen is inserted into the insecticide and the second oxygen atom is reduced to form water. Catalysis depends on the close association of the heme-containing cytochrome P450 terminal oxidase with NADPH cytochrome C reductase for electron transport, and it also depends on availability of NADPH and oxygen. 

Oxidative cleavage of the O-alkyl linkage in glycerolipids is catalyzed by a microsomal tetrahydropteridine (Pte-H4)-dependent alkyl monooxygenase (Fig. 12) (T.-C. Lee, 1981). The required cofactor, Pte H4, is regenerated from Pte-Hj by an NADPH-linked pteridine reductase, a cytosolic enzyme. Oxidative attack on the ether bond in lipids is similar to the enzymatic mechanism described for the hydroxylation of phenylalanine. Fatty aldehydes produced via the cleavage reaction can be either oxidized to the corresponding acid or reduced to the alcohol by appropriate enzymes. 

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