Nicotinamide Adenine Dinucleotide-cytochrome B5 Reductase Research Articles

Characterization of a novel nicotinamide adenine dinucleotide-cytochrome b5 reductase mutation associated with canine hereditary methemoglobinemia.

Hereditary methemoglobinemia associated with nicotinamide adenine dinucleotide-cytochrome b5 reductase (b5R) deficiency is a rare autosomal recessive disorder in animals. Recently, nonsynonymous b5R gene (CYB5R3) variants have been reported to be associated with canine and feline hereditary methemoglobinemia. However, the underlying molecular mechanisms of canine and feline methemoglobinemia caused by these nonsynonymous variants have not yet been reported. Previously, we reported a Pomeranian dog family with hereditary methemoglobinemia, carrying CYB5R3 mutation of an A>C transition at codon 194 in exon 7, replacing an isoleucine residue with leucine (p.Ile194Leu). In this study, we investigated the enzymatic and structural properties of the soluble form of wild-type and Ile194Leu canine b5Rs to characterize the effects of this missense mutation. Our results showed that the kinetic properties of the mutant enzyme were not affected by this amino acid substitution. The secondary structure of the wild-type and Ile194Leu b5Rs detected by circular dichroism showed a similar pattern. However, the mutant enzyme exhibited decreased heat stability and increased susceptibility to trypsin hydrolysis. Moreover, the thermostability and unfolding measurements indicated that the mutant enzyme was more sensitive to temperature-dependent denaturation than the wild-type b5R. We concluded from these results that unstable mutant enzyme properties with normal enzymatic activity would be associated with hereditary methemoglobinemia in the Pomeranian dog family.

Influences of maternal ethanol intake on maternal and perinatal hepatic heme and drug metabolizing enzymes in rats.

The effect of ethanol on maternal and neonatal hepatic heme and drug metabolizing systems was determined. Ethanol (16%, w/v) was administered orally as drinking solution to pregnant or lactating rats at different pre- and post-natal stages. The dams and pups were sacrificed on days 7, 14 and 21 after parturition, respectively. Ethanol administration to lactating rats from just after birth caused an appreciable decrease in the maternal and neonatal body and liver weights. In addition, the activities of nicotinamide adenine dinucleotide phosphate-cytochrome c reductase, nicotinamide adenine dinucleotide-cytochrome b5 reductase and heme oxygenase were significantly enhanced in the livers of neonates whose mothers were exposed to the ethanol during only first week of lactation, but those activities were not altered in the maternal livers. However, no remarkable alterations were observed in the contents of cytochrome P-450 and b5, and the activities of aminopyrine demethylase, aniline hydroxylase and delta-aminolevulinic acid synthetase in the livers of neonates from mothers who had received ethanol during lactation period or last week of gestation, although the activities of aminopyrine demethylase and aniline hydroxylase were enhanced significantly in lactating dams by ethanol consumption for 14 d after parturition.

Reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase: location of the hydrophobic, membrane-binding region at the carboxyl-terminal end and the masked amino terminus

Microsomal NADH-cytochrome b5 reductase is an amphiphilic protein consisting of a hydrophilic (catalytic) region and a hydrophobic (membrane-binding) segment. Digestion of the reductase purified from rabbit liver microsomes with carboxypeptidase Y (CPY), but not with aminopeptidases, resulted in the abolishment of the capacities of the reductase to bind to phosphatidylcholine liposomes and to reconstitute an active NADH-cytochrome c reductase system upon mixing with cytochrome b5. The NADH-ferricyanide reductase activity of the flavoprotein was, however, inactivated only slightly by the CPY digestion. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid analyses indicated that the CPY treatment removed about 30 amino acid residues from the tcooh terminus of the reductase and that about 70% of the amino acids released were hydrophobic. It is concluded that the hydrophobic region of the reductase, responsible for both membrane binding and effective reconstitution of NADH-cytochrome c reductase activity, is located at the COOH-terminal portion of the molecule. No NH2-terminal residue could be detected in the intact and CPY-modified reductase preparations. The location of the hydrophobic, membrane-binding segment at the COOH-terminal end and the masked NH2 terminus have also been reported for cytochrome b5, another microsomal membrane protein.

Reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase and cytochrome b5 as electron carriers in NADH-supported cytochrome P-450-dependent enzyme activities in liver microsomes

The role of NADH-cytochrome b 5 reductase and cytochrome b 5 as electron carriers in NADH-supported electron transport reactions in rat liver microsomes has been examined by measuring three enzyme activities: NADH-cytochrome P-450 reductase, NADH-peroxidase, and NADH-cytochrome c reductase. The first two reactions are known to involve the participation of an NADH-specific reductase and cytochrome P-450 whereas the third requires the reductase and cytochrome b 5. Antibody prepared against NADH-cytochrome b 5 reductase markedly inhibited the NADH-peroxidase and NADH-cytochrome c reductase activities suggesting the involvement of this NADH-specific reductase in these reactions. Liver microsomes prepared from phenobarbital-pretreated rats were digested with subtilisin to remove cytochrome b 5 and the submicrosomal particles were collected by centrifugation. The specific content of cytochrome b 5 in the digested particles was about 5% of that originally present in liver microsomes and all three enzyme activities showed similar decreases whereas NADH-ferricyanide reductase activity (an activity associated with the flavoenzyme NADH-cytochrome b 5 reductase) remained virtually unchanged. Binding of an excess of detergent-purified cytochrome b 5 to the submicrosomal particles at 37 °C for 20 min followed by centrifugation and enzymic measurements revealed a striking increase in the three enzyme activities. Further evidence for cytochrome b 5 involvement in the NADH-peroxidase reaction was the marked inhibition by antibody prepared against the hemoprotein. These results suggest that in microsomal NADH-supported cytochrome P-450-dependent electron transport reactions, cytochrome b 5 functions as an intermediate electron carrier between NADH-cytochrome b 5 reductase and cytochrome P-450.

The Binding of Reduced Nicotinamide Adenine Dinucleotide-Cytochrome b5 Reductase to Hepatic Microsomes

Abstract Incubation of microsomes with the amphipathic form of NADH-cytochrome b5 reductase results in the binding of a maximum of 7.2 ± 0.5 nmoles of reductase per mg of microsomal protein representing a 100-fold increase over endogenous enzyme levels. Interactions with the phospholipid portion of the membrane are involved in this binding and extra bound and endogenous reductase are apparently functionally identical. The binding of reductase is similar to the binding of cytochrome b5 to microsomes and the model for the microsomal membrane formulated on the basis of cytochrome b5 binding is therefore extended to include reductase. This model, involving random distribution and translational diffusion of these proteins on the surface of the membrane, is supported by experiments showing that extra bound reductase and cytochrome b5 interact catalytically and exclude each other from the microsomal membrane.

Microsomal electron transport: II. Reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase and cytochrome P-450 as electron carriers in microsomal NADH-peroxidase activity

An electron transport system that catalyzes the oxidation of NADH by organic hydroperoxides has been discovered in rat liver microsomes. The rate of NADH oxidation by hydroperoxides was first-order with respect to microsomal protein concentration and a Km value for NADH of less than 3 μ m was obtained. Examination of the hydroperoxide specificity revealed that cumene hydroperoxide and various steroid hydroperoxides were effective substrates for the enzyme system. A Km value for cumene hydroperoxide of about 0.6 m m was obtained. The activity was inhibited 50% by 0.45 m m potassium cyanide but was insensitive to inhibition by carbon monoxide. NADH was a more efficient electron donor for peroxidase activity than NADPH. Evidence suggesting the involvement of NADH-cytochrome b5 reductase (EC 1.6.2.2) in the NADH-peroxidase reaction included: (1) a marked inhibition by 0.05 m m p-hydroxymercuribenzoate with partial protection by NADH; (2) inhibition by antibody to the flavoenzyme; (3) a similar Km for NADH in both activities; (4) a similar distribution of both activities in the supernatant and pellet fractions after digestion of liver microsomes with trypsin; and (5) a requirement of the flavoenzyme in the reconstituted enzyme system. Cytochrome b5 apparently was not involved in the NADH-peroxidase enzyme system since removal of the hemoprotein from liver microsomes by trypsin produced no inhibitory effect; addition of purified cytochrome b5 to either microsomes or a microsomal preparation devoid of the hemoprotein had no stimulatory effect; and antibody preparated against the hemoprotein did not inhibit the reaction rate. Evidence against the involvement of NADPH-cytochrome c reductase in the reaction was the lack of inhibition by NADP+ and by antibody prepared against the flavoprotein. Evidence for the participation of a cytochrome P-450 species in the NADH-peroxidase enzyme system included inhibition by type I, type II, and modified type II compounds; inhibition by reagents converting cytochrome P-450 to cytochrome P-420; and marked stimulation by in vivo phenobarbital treatment. The NADH-reduced form of cytochrome P-450 was oxidized very rapidly by cumene hydroperoxide under a CO atmosphere. The participation of two distinct species of cytochrome P-450 in the NADH- and NADPH-dependent peroxidase enzyme systems of liver microsomes is suggested.

Lipid-Protein Interactions in the Reconstitution of the Microsomal Reduced Nicotinamide Adenine Dinucleotide-Cytochrome b5 Reductase System

Abstract Interactions between the lipid bilayer and the hydrophobic portions of cytochrome b5 and NADH-cytochrome b5 reductase have been found to be essential for, and, in fact, to control the over-all rate of reduction of cytochrome b5 by NADH in hepatic microsomes. The present data and earlier work from this laboratory show that the reoxidation of reduced cytochrome b5 reductase by cytochrome b5 is the rate-limiting step in the reduction of microsomal cytochrome b5. Removal of phospholipid markedly decreases the rate of cytochrome b5 reduction and reconstitution with liposomes completely restores the structural and functional characteristics of the NADH-cytochrome b5 reductase electron transport pathway. These findings, along with the observation of a similar effect of liposomes on the NADH-dependent reduction of purified cytochrome b5 by the purified reductase, indicate that the amphipathic nature of these proteins is responsible for the lipid dependence of this reaction.

A Form of Reduced Nicotinamide Adenine Dinucleotide-Cytochrome b5 Reductase Containing Both the Catalytic Site and an Additional Hydrophobic Membrane-binding Segment

Cytochrome b5 reductase from calf liver has been purified by a nonhydrolytic procedure which involves Triton X-100 extraction of microsomes and DEAE-chromatography in the presence of Triton X-100 and deoxycholate. The reductase obtained in this manner has a monomeric molecular weight of 43,000 as compared to a molecular weight of 33,000 for reductase obtained by lysosomal extraction of microsomes. The additional mass present in the new preparation is due to an additional 99 amino acid residues, about 65% of which are hydrophobic and most of which occur in a single amino acid sequence. This hydrophobic amino acid sequence confers upon the reductase a marked tendency to polymerize in aqueous media and permits it to bind to microsomal membranes. The detergent-extracted reductase is catalytically similar to lysosomal-extracted reductase with ferricyanide as an electron acceptor, but its reaction with cytochrome b5 is slowed by polymerization. Chymotryptic cleavage of detergent-extracted reductase releases a core enzyme, which is chromatographically indistinguishable from lysosomal-extracted reductase, and polymers of hydrophobic peptides, the largest of which has a monomeric molecular weight of about 10,000. This is similar to our earlier studies with detergent-extracted cytochrome b5 and constitutes the second example of a membrane protein which has a distinctly amphipathic structure.

The Reaction Sequence in Electron Transfer in the Reduced Nicotinamide Adenine Dinucleotide-Cytochrome b5 Reductase System

The Reaction Sequence in Electron Transfer in the Reduced Nicotinamide Adenine Dinucleotide-Cytochrome b5 Reductase System