Reconstitution of the reduced nicotinamide adenine dinucleotide phosphate- and reduced nicotinamide adenine dinucleotide-peroxidase activities from solubilized components of rat liver microsomes

Hrycay, Eugene G.; Jonen, H. G.; Lu, Anthony Y. H.; Levin, Wayne

doi:10.1016/0003-9861(75)90374-4

Cited by 39 publications

(7 citation statements)

References 28 publications

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“…We have shown that cross-resistance to MMC in a multidrug-resistant variant of P388 mouse leukaemia (P388/R-84) is independent of either deficient drug activation or reduced drug accumulation, but seems to be influenced by GSH/GST levels (Singh et al, 1992;Xu & Singh, 1992a,b). Subsequently, using a panel of three unrelated human bladder cancer cell lines (J82, HT-1 197 and SCaBER) differing in their sensitivities to MMC, we noticed a good correlation between MMC cytotoxicity and GST level, activity being highest in the relatively insensitive cell line (SCaBER) and lowest in the comparatively sensitive cell line, J82 (Xu et al, 1993 (Xu & Singh, 1992a, b Enzyme assays NADPH cytochrome P450 reductase activity was determined by the procedure described by Hrycay et al (1975). DT diaphorase was measured according to the method described by Ernster (1967) using 2,6-dichlorophenol indophenol (DCPIP) as a substrate.…”

mentioning

confidence: 81%

Mechanism of differential sensitivity of human bladder cancer cells to mitomycin C and its analogue

Gupta²,

Singh³

1994

Br J Cancer

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confidence: 81%

Mechanism of differential sensitivity of human bladder cancer cells to mitomycin C and its analogue

Gupta²,

Singh³

1994

Br J Cancer

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“…Rahimtula et al (20) have proposed the occurrence of higher valence states in such reactions, ad Rahimtula ad O'Brien (22) have suggested that a peroxidase-type mechanism may be in operation. The reconstitution of NADPH-and NADH-peroxidase activities from solubilized components of rat liver microsomes has recently been reported (42).…”

Section: Hydroxylationmentioning

confidence: 99%

Studies on hydroperoxide-dependent substrate hydroxylation by purified liver microsomal cytochrome P-450

Nordblom

White

Coon

1976

Archives of Biochemistry and Biophysics

349

110

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Highly purified liver microsomal cytochrome P-450 catalyzes the hydroperoxide-dependent hydroxylation of a variety of substrates in the absence of NADPH, NADPHcytochrome P-450 reductase, and molecular oxygen. The addition of phosphatidylcholine is necessary for maximal activity. The absence of flavoproteins and cytochrome b, from the cytochromeP-450 preparations rules out the involvement of other known microsomal electron carriers. The ferrous form of cytochrome P-450 is not involved in peroxidedependent hydroxylation reactions, as indicated by the lack of inhibition by carbon monoxide. With cumene hydroperoxide present, a variety of substrates is attacked, including N-methylaniline, N,N-dimethylaniline, cyclohexane, benzphetamine, and aminopyrine.With benzphetamine as the substrate, cumene hydroperoxide may be replaced by other peroxides, including hydrogen peroxide, or by peracids or sodium chlorite. A study of the stoichiometry indicated that equimolar amounts ofN-methylaniline, formaldehyde, and cumyl alcohol (o,o-dimethylbenzyl alcohol) are formed in the reaction of N,N-dimethylaniline with cumene hydroperoxide. Since H,180 is incorporated only slightly into cyclohexanol in the reaction of cyclohexane with cumene hydroperoxide, it appears that the oxygen atom in cyclohexanol is derived primarily from the peroxide. The data obtained are in accord with a peroxidase-like mechanism for the action of cytochrome P-450.The mixed function oxidase of liver microsomal membranes is capable of hydroxylating or otherwise metabolizing fatty acids and steroids as well as a variety of foreign compounds, including drugs, insecticides, alkanes, anesthetics, and carcinogens. This enzyme system was resolved into three components, P-450LM ,3 NADPHcytochrome P-450 reductase, and phosphatidylcholine (3-61, and more recently our

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“…It is possible that the active species could be a hydroperoxide radical and that hydrogen removal via a radical mechanism may be involved in the mechanism. Heme centers, such as the cytochrome P-450 system, can act as peroxidases (25), and tbutyl-OOH may be acting to oxygenate a heme center that can then act to oxidatively remove the glutamyl -y proton. In any event, the formation of an epoxide from a hydroperoxide is chemically favorable, and it is reasonable to consider using this energy to at least partially drive the carboxylation reaction.…”

Section: Discussionmentioning

confidence: 99%

Vitamin K-dependent carboxylase: evidence for a hydroperoxide intermediate in the reaction.

Larson¹,

Suttie²

1978

Proc. Natl. Acad. Sci. U.S.A.

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Vitamin K is an essential cofactor for a microsomal carboxylase that converts glutamyl residues in endogenous precursor proteins to y-carboxyglutamyl residues in completed proteins. The same microsomal preparations convert vitamin K to its 2,3-epoxide, and it has been suggested that these two reactions (carboxylation and epoxidation) are coupled. Glutathione peroxidase, which reduces hydrogen peroxide and organic hydroperoxides, inhibits both of these reactions in a preparation of microsomes solubilized by Triton X-100. Catalase has no effect. In the absence of vitamin K, and in the presence of NADPH, tert-butyl hydroperoxide acts as a weak vitamin K analog. At lower concentrations, tert-butyl hydroperoxide is an apparent competitive inhibitor of vitamin K for both the carboxylase and epoxidase reactions. These data are consistent with the hypothesis that both of these vitamin K-requiring reactions involve a common oxygenated intermediate, and that a hydroperoxide of the vitamin is the species involved. Vitamin K functions in the postribosomal modification of liver microsomal precursor proteins to form biologically active prothrombin, and the other vitamin K-dependent plasma clotting factors: VII, IX, and X (1, 2). The vitamin K-dependent modification of these precursors involves the carboxylation of specific glutamyl residues to form y-carboxyglutamyl residues in the completed proteins, and in vitro microsomal systems that carry out this vitamin K-dependent carboxylation have been developed (3)(4)(5)(6)(7). These systems, which have now been solubilized (8-13), have utilized the endogenous microsomal precursor protein(s) as a substrate for the carboxylase; more recently, synthetic peptides that are analogous to Glu-containing regions of the prothrombin precursor have been used as substrates (14,15). This carboxylation reaction requires the reduced form of vitamin K, 02, and CO2 but appears to have no ATP, coenzyme A, or biotin requirement. The same microsomal preparations that catalyze the carboxylation also convert vitamin K to its 2,3-epoxide (16-18). It has been suggested (16) that the epoxidation reaction may be coupled to the carboxylation reaction, and the evidence that relates to this hypothesis has recently been reviewed (19). One possible mode of coupling of these two reactions would be through a common intermediate. This report presents evidence that is consistent with the hypothesis that a hydroperoxide of the vitamin is involved in both of these reactions, and suggests possible molecular roles for the vitamin in this unique carboxylase.MATERIALS AND METHODS Incubation Conditions. Microsomal pellets were prepared from 7-to 10-day vitamin K-deficient rat livers as previously described (14). The microsome pellets were solubilized in SIK (0.25 M sucrose/0.025 M imidazole/0.5 M KC1, pH 7.2) buffer containing 1.5% (vol/vol) Triton X-100 and centrifuged at 105,000 X g for 60 min to remove insoluble material. Incubations consisted of 0.4 ml of this supernatant and 0.1 ml of SIK buffer containing NAD...

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Reconstitution of the reduced nicotinamide adenine dinucleotide phosphate- and reduced nicotinamide adenine dinucleotide-peroxidase activities from solubilized components of rat liver microsomes

Cited by 39 publications

References 28 publications

Mechanism of differential sensitivity of human bladder cancer cells to mitomycin C and its analogue

Mechanism of differential sensitivity of human bladder cancer cells to mitomycin C and its analogue

Studies on hydroperoxide-dependent substrate hydroxylation by purified liver microsomal cytochrome P-450

Vitamin K-dependent carboxylase: evidence for a hydroperoxide intermediate in the reaction.

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