Microsome-catalyzed NADH

Figure 10. Microsome-catalyzed NADH reduction of Cl(NHo)r,Ru(lll) cmd subsequent formation of (Isonicotinamide)(NH3)sRu(II) A, reaction run in air B, reaction run under Ar C, reaction run under Ar in presence of metyrapone D, reaction run under N2 (3S)...

Recent studies suggest that many factors may affect hydroxyl radical generation by microsomes. Reinke et al. [34] demonstrated that the hydroxyl radical-mediated oxidation of ethanol in rat liver microsomes depended on phosphate or Tris buffer. Cytochrome bs can also participate in the microsomal production of hydroxyl radicals catalyzed by NADH-cytochrome bs reductase [35,36]. Considering the numerous demonstrations of hydroxyl radical formation in microsomes, it becomes obvious that this is not a genuine enzymatic process because it depends on the presence or absence of free iron. Consequently, in vitro experiments in buffers containing iron ions can significantly differ from real biological systems. 

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]. 

The NADH- and oxygen-dependent microsomal metabolism of the di-, tri- and tetraethyl substituted derivatives of germanium, tin and lead was shown to give rise to ethylene as a major product and ethane as a minor product27. These reactions were shown to be catalyzed by the liver microsomal fractions. 

Indicine IV-oxide (169) (Scheme 36) is a clinically important pyrrolizidine alkaloid being used in the treatment of neoplasms. The compound is an attractive drug candidate because it does not have the acute toxicity observed in other pyrrolizidine alkaloids. Indicine IV-oxide apparently demonstrates increased biological activity and toxicity after reduction to the tertiary amine. Duffel and Gillespie (90) demonstrated that horseradish peroxidase catalyzes the reduction of indicine IV-oxide to indicine in an anaerobic reaction requiring a reduced pyridine nucleotide (either NADH or NADPH) and a flavin coenzyme (FMN or FAD). Rat liver microsomes and the 100,000 x g supernatant fraction also catalyze the reduction of the IV-oxide, and cofactor requirements and inhibition characteristics with these enzyme systems are similar to those exhibited by horseradish peroxidase. Sodium azide inhibited the TV-oxide reduction reaction, while aminotriazole did not. With rat liver microsomes, IV-octylamine decreased... 

Polyunsaturated fatty acid synthesis is catalyzed by acyl-lipid-desaturases, also named front-end desaturases due to their action mechanism, which proceeds via introduction of double bonds into preformed acyl chains by oxygen and electron-donor dependent desaturation, between the carboxyl group and the pre-existing unsaturation which acts as substrate. For many microsomal desaturases, the electron donors are cytochrome b5, and a small hemoprotein that operates in numerous redox reactions in plants, involving NADH-dependent acyl-group desaturation [200]. 

The microsomal fraction of liver contains a monooxygenase system consisting of cytochrome P-450 (EC 1.14.14.1), NADPH-cytochrome P-450 reductase (EC 1.6.2.4), and phospholipid. This system catalyzes the hydroxylation of a large number of both foreign and endogenous compounds in a mixed-function oxidation reaction using molecular oxygen and NADPH or NADH as electron donors ... 

Heme oxygenase, which catalyzes the conversion of free heme groups to biliverdin and CO, functions as part of a microsomal electron transport system similar to that of cytochrome 1 450-(FP = NADPH-cytochrome P450 reductase.) Heme oxygenase requires 3 02 and 5 NADPH. Biliverdin reductase can use NADPH or NADH as a reductant. 

The enzymes necessary for the conversion of famesyl pyrophosphate to squalene are called squalene synthetase . The enzymes necessary for the two reactions have not been resolved nor has either been purified to a significant extent, and it is not yet certain if the two reactions are catalyzed by two discrete entities. Squalene synthetase has an absolute requirement for a divalent cation, Mg " and Mn " being the best. A reduced pyridine nucleotide (NADH or NADPH) is required for the reduction of presqualene pyrophosphate to squalene. Yeast microsomes with Mn " and no reduced pyridine nucleotide will form dehydrosqualene instead [70]. The conversion of famesyl pyrophosphate to presqualene pyrophosphate is enhanced several-fold by the reduced pyridine nucleotide [71]. Also, some organic solvents as well as detergents increase this activity. 

Studies on metabolic stability using hepatocyte suspensions are not feasible for automation/HTS, but these studies do provide rather complete profiles of hepatic biotransformation without the supplements of cofactors and cosubstrates. The use of S9 in metabolic stability studies can be evaluated in a manner similar to that used for the microsomal assays, but with the possible addition of a broader panel of cofactors or cosubstrates. These include NADPH for CYP/FMO-mediated reactions, NADH for xanthine oxidoreductase and quinone oxidoreductase 2, NADPH-dependent reductions by carbonyl reductases, and NADPH/NADH-dependent reductions catalyzed by aldo-keto reductases, uridine 5 -diphosphate... 

Three indoleacetaldehyde reductases were purified from cucumber seedlings [ 1,4]. The enzyme requiring NADH as a cofactor occurred in the cytosol one of the two NADPH-specific reductases was associated with a microsomal fraction. The latter reduced phenylacetaldehyde at about half the rate observed for indoleacetaldehyde and exhibited minor activity on some of the aliphatic aldehydes tested. The NADH-requiring enzyme acted only on indoleacetaldehyde and phenylacetaldehyde. None of the three enzymes would catalyze the reverse oxidation of tryptophol. 

The 3 hydroxysteroid reductase of rat liver which catalyzes the conversion of XXX to cholestanol (XXXI) was localized also in the microsomal fractions, and shown to provide the epimeric alcohols in a ratio of 10 1 (3/3 3a) in the presence of NADPH. The enzyme was not inhibited by cholestanol, but pronounced inhibition was noted with 7-keto- or 7a-hydroxycholestanoI (XXXII) or zl -cholestenone (XXVIII) (120). This enzyme differs from the Cj9 steroid reductase, since the latter utilize NADH equally well and provides predominantly the 3a-ol. 

In mammalian tissues, cytochrome P-450 may catalyze the generation of free radicals via the decomposition of hydroperoxides (Horton and Fairhurst, 1987). The reaction in skeletal muscle microsomes is dependent on the presence of NADH or NADPH, ADP and iron (Rhee, 1988). Also, the NADPH dehydrogenase of the respiratory chain may initiate lipid peroxidation via the generation of superoxide radicals (Horton and Fairhurst, 1987). Furthermore, peroxidases, cyclooxygenases, prostaglandin synthase, methemoglobin and microsomal oxidases in animal tissues have been implicated in the initiation and promotion of lipid peroxidation in muscle tissues (Kanner and Harel, 1985 Hsieh and KinseUa, 1989). 

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