Microsomal NADH-cytochrome B5 Reductase Research Articles

Monooxygenase system in Guerin’s carcinoma of rats under conditions of ω-3 polyunsaturated fatty acids administration.

The aim of the study was to determine the variations of function in components of monooxygenase system (MOS) of rat Guerin’s carcinoma under ω-3 polyunsaturated fatty acids (PUFAs) administration. The activity of Guerin’s carcinoma microsomal NADH-cytochrome b5 reductase, the content and the rate of cytochrome b5 oxidation-reduction, the content and the rate of cytochrome Р450 oxidation-reduction have been investigated in rats with tumor under conditions of ω-3 PUFAs administration. ω-3 PUFAs supplementation before and after transplantation of Guerin’s carcinoma resulted in the increase of NADH-cytochrome b5 reductase activity and decrease of cytochrome b5 level in the Guerin’s carcinoma microsomal fraction in the logarithmic phases of carcinogenesis as compared to the tumor-bearing rats. Increased activity of NADH-cytochrome b5 reductase facilitates higher electron flow in redox-chain of MOS. Under decreased cytochrome b5 levels the electrons are transferred to oxygen, which leads to heightened generation of superoxide (O2•-) in comparison to control. It was shown, that the decrease of cytochrome P450 level in the Guerin’s carcinoma microsomal fraction in the logarithmic phases of oncogenesis under ω-3 PUFAs administration may be associated with its transition into an inactive form – cytochrome P420. This decrease in cytochrome P450 coincides with increased generation of superoxide by MOS oxygenase chain.

Functional activity of NADH-dependent reductase system in liver and guerin’s carcinoma microsomal fraction in rats exposed to preliminary irradiation

The activity of liver and Guerin's carcinoma microsomal NADH-dependent reductase system has been investigated in tumor-bearing rats exposed to preliminary irradiation. Preliminary irradiation of rats (before transplantation of Guerin's carcinoma) resulted in the decrease of NADH-cytochrome b5 reductase activity and contents of cytochrome b5 in the Guerin's carcinoma microsomal fraction in the logarithmic phases of oncogenesis compared with the non-irradiated tumor-bearing rats. The effect of irradiation preceding transplantation of the tumor to rats results in the increase of enzymatic activities of liver microsomal NADH-cytochrome b5 reductase in the latent and logarithmic phases of tumor growth as compared with non-irradiated tumor-bearing rats. At the same time the contents of cytochrome b5 decreases. During longer periods after irradiation the investigated parameters approached to those in the group of non-irradiated tumor-bearing animals.

Evaluation of bioreductive activation of anticancer drugs idarubicin and mitomycin C by NADH-cytochrome b5 reductase and cytochrome P450 2B4

This study attempted to investigate the ability of microsomal NADH-cytochrome b5 reductase and cytochrome P450 2B4 to reductively activate idarubicin and mitomycin C.In vitro plasmid DNA damage experiments and assays using purified hepatic enzymes were employed to examine their respective roles in the metabolic activation of anticancer drugs.Mitomycin C was found to be not a good substrate for microsomal b5 reductase unlike P450 reductase. It produced low amounts of strand breaks in DNA when incubated with b5 reductase and its one-electron reduction by purified enzyme was found as negligible. Our findings revealed that P450 reductase-mediated metabolism of idarubicin resulted in a large increase in single-strand DNA breaks, whereas, b5 reductase neither catalyzed the reduction of idarubicin nor mediated the formation of DNA damage in the presence of idarubicin. The reconstitution studies, on the other hand, have identified rabbit liver CYP2B4 isozyme as being a potential candidate enzyme for reductive bioactivation of idarubicin and mitomycin C.Thus, the present novel findings strongly suggest that while b5 reductase could not play a key role in the cytotoxic and/or antitumor effects of idarubicin and mitomycin C, CYP2B4 could potentiate their activity in combination with P450 reductase.

Evaluation of data in terms of two-dimensional random walk model: Interaction between NADH-cytochrome b5 reductase and cytochrome b5

Normally, bimolecular reactions are analyzed in terms of the Smoluchowski theory. However, when one attempts to generalize this analysis to cases where diffusion proceeds in two other than in three dimensions, one soon encounters severe conceptual difficulties. Although kinetic studies of membrane enzymes are generally difficult because the usual kinetic formalism refers to nonaggregated homogenous solutions, a major goal of our research is to define the molecular mechanism(s) by which alterations in membrane-bound substrate contents affect the enzyme activity in the same membrane. For that purpose, a simplified random-walk model was adopted in the present work. The enzyme reaction in the two-dimensional membrane could be calculated theoretically by applying the classical analysis of heat equation. As a result, the theoretical rate equation well accounting experimental findings was derived on the model of the liver microsomal NADH-cytochrome b5 reductase reaction. Furthermore, it was found that the modification of the simple rigid-sphere collision theory by including a term called the steric factor was not necessary in this derived equation.

Purification and Partial Characterization of Microsomal NADH-Cytochrome b5 Reductase from Higher Plant Catharanthus roseus

A simple three step procedure was used to purify microsomal NADH-cytochrome b5 (ferricyanide) reductase to homogeneity from the higher plant C. roseus. The microsomal bound reductase was solubilized using zwitterionic detergent-CHAPS. The solubilized reductase was subjected to affinity chromatography on octylamino Sepharose 4B, blue 2-Sepharose CL-6B and NAD+-Agarose. The homogeneous enzyme has an apparent molecular weight of 33,000 as estimated by SDS-PAGE. The purified enzyme catalyzes the reduction of purified cytochrome b5 from C. roseus in the presence of NADH. The reductase also readily transfers electrons from NADH to ferricyanide (Km 56 μM), 2,6-dichlorophenolindophenol (Km 65 μM) and cytochrome c via cytochrome b5 but not to menadione.

The Role of Reactive Oxygen Species in the Antitumor Activity of Bleomycin

Calf thymus DNA was incubated with bleomycin and FeCl3 in the presence of isolated rat liver microsomal NADH-cytochrome b5 reductase, cytochrome b5 and NADH which catalyze redox cycling of the bleomycin-Fe-complex. Furthermore, isolated rat liver nuclei were incubated with bleomycin, FeCl3 and NADH, a system in which redox cycling of bleomycin-Fe leads to DNA damage. In both systems free bases from DNA were released. Furthermore, 8-hydroxy-guanine was also found in the supernatant. On the other hand, 8-hydroxy-deoxyguanosine was detected in DNA of cell nuclei indicating that hydroxylation of the guanine molecule occurred in intact DNA. The release of bases correlated with the release of malondialydehyde as well as with NADH and oxygen consumption. These results indicate that NADH-cytochrome b5 reductase catalyzes redox cycling of the bleomycin-Fe-complex which results in the formation of reactive oxygen species which oxidize deoxyribose as well as bases of DNA. Both mechanisms may contribute to the cytotoxic and cytostatic effects of bleomycin observed in intact cells.

Palmitoyl-CoA elongation in brain microsomes: dependence on cytochrome b5 and NADH-cytochrome b5 reductase.

Experiments were performed to demonstrate the involvement of electron transport system in fatty acid elongation in rat brain microsomes. Mercuric chloride and p-chloromercuriphenylsulfonate, inhibitors on NADH-cytochrome b5 reductase, at 32 microM inhibited NADH-supported palmitoyl-CoA elongation to 30 and 60% of control activity, respectively, whereas NADPH-supported palmitoyl-CoA elongation was unaffected by these mercurials. An antibody to rat liver NADH-cytochrome b5 reductase inhibited brain microsomal NADH-cytochrome b5 reductase activity and NADH-dependent palmitoyl-CoA elongation. Treatment of brain microsomes with trypsin diminished the cytochrome b5 content; NADH- and NADPH-cytochrome c reductase activities were significantly decreased, but the decrease in NADH-cytochrome b5 reductase activity was relatively small. Whereas essentially no incorporation of malonyl-CoA into palmitoyl-CoA was observed with trypsin-treated microsomes, addition of detergent-solubilized cytochrome b5 resulted in a recovery of fatty acid elongation. These results indicate the presence of an electron transport system, NADH-NADH-cytochrome b5 reductase-cytochrome b5-fatty acid elongation, in brain microsomes.

Complete amino acid sequence of steer liver microsomal NADH-cytochrome b5 reductase.

The complete covalent structure of liver microsomal NADH-cytochrome b5 reductase from steer liver microsomes was determined. Cleavage at methionyl bonds gave 10 peptides accounting for all the residues of the protein. Acid cleavage of the reductase at the Asp-Pro bonds gave three peptides accounting for all the CNBr peptides in the molecule. Subfragmentation of these peptides by chemical and enzymatic cleavage provided overlaps which established all the fragments in an unambiguous sequence of 300 residues, corresponding to Mr 34,110. Limited tryptic digestion cleaved reductase at residues 28 and 119, yielding a preparation having two noncovalently linked peptides having a conformation which binds flavin and retains the structural features essential for NADH-cytochrome b5 activity. A model for the secondary structure of cytochrome b5 reductase is proposed that is based on computer-assisted analysis of the amino acid sequence. In this model the beta-turns are predominant and there is some 25% alpha and 30% beta structure.

Cytochrome reductase activities in rat brain microsomes during development.

Postnatal developmental alterations of microsomal NADH-cytochrome b5 reductase and NADPH-cytochrome c reductase activities were determined in the brain of rats. The reductase activities increased from a low level in the immature brain to a maximum level at 23 to 30 days of age, and then decreased slightly to a plateau. The periods of the activity increments were in accord with those of the enhancement of microsomal fatty acid elongation. The specific activities of these reductases were high in cerebral hemispheres and medulla oblongata, intermediate in midbrain, and lowest in cerebellum of the four regions of 20-day-old rat brain.

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.

Tryptic cleavage of rat liver sulfite oxidase. Isolation and characterization of molybdenum and heme domains.

Treatment of rat liver sulfite oxidase with trypsin leads to loss of ability to oxidize sulfite in the presence of cytochrome c as electron acceptor. Ability to oxidize sulfite with ferricyanide as acceptor is undiminished, while sulfite leads to O2 activity is partially retained. Gel filtration of the proteolytic products has led to the isolation of two major fragments of dissimilar size derived from sulfite oxidase. The smaller fragment has a molecular weight of 9500 and appears to be monomeric when detached from sulfite oxidase. It contains the heme in its cytochrome b5 structure, has no sulfite oxidase activity, and is reducible with dithionite but not with sulfite. The heme fragment can mediate electron transfer between pig liver microsomal NADH cytochrome b5 reductase and cytochrome c. The larger fragment has a molecular weight of 47,400 under denaturing conditions but elutes from Sephadex G-200 as a dimer. It contains no heme but retains all of the molybdenum and the modified sulfite-oxidizing capacity present in the proteolytic mixture. All of the EPR properties of the molybdenum center of native sulfite oxidase are retained in the molybdenum fragment. The molybdenum center is a weak chromophore with an absorption sectrum suggestive of coordination with sulfur ligands. Reduction by sulfite generates a spectrum attributable to molybdenum (V). Spectra of oxidized and sulfite-reduced preparations are sensitive to anions and pH. NH2-terminal analysis of native sulfite oxidase and the two tryptic fragments has permitted the conclusion that the sequence represented by the heme fragment is the NH2 terminus of native enzyme. These studies have demonstrated that the two cofactor moieties of sulfite oxidase are contained in distinct domains which are covalently held in contiguity by means of an exposed hinge region. Isolation of functional heme and molybdenum domains of sulfite oxidase after tryptic cleavage has demonstrated conclusively that the cytochrome b5 region of the molecule is required for electron transfer to the physiological acceptor, cytochrome c.

NADH-cytochrome c reductase activity in cultured human lymphocytes : Similarity to the liver microsomal NADH-cytochrome b5 reductase and cytochrome b5 enzyme system

A NADH-cytochrome c reductase activity was increased upon mitogen stimulation of human lymphocytes. The activity was not inhibited by antimycin A or rotenone but was specifically inhibited by antibodies elicited against rat liver NADH-cytochrome b 5 reductase or cytochrome b 5. The activity was linear with cellular homogenates up to 5.2 × 10 6 cells/ml and had abroad pH optimum of 7.7. The presence of 3-methylcholanthrene in mitogen stimulation media had no effect on the NADH-cytochrome c reductase activity but differentially induced the benzo(a)pyrene hydroxylase (AHH) activity. The reductase activity was present in nonstimulated cells and appears not to be significantly increased in activity per cell upon mitogen-stimulation of the peripheral lymphocyte.

Evidence for Random Distribution and Translational Movement of Cytochrome b5 in Endoplasmic Reticulum

Abstract Evidence is presented that electron transfer between different molecules of cytochrome b5 does not occur at an appreciable rate in microsomes. In view of this fact, the earlier observation that a large excess of functional amphipathic cytochrome b5 can be bound to microsomes suggests that cytochrome b5 and NADH-cytochrome b5 reductase are randomly distributed on the microsomal membrane. This model requires translational diffusion of these proteins within the membrane prior to interaction. The dependence of the rate of reduction of microsomal cytochrome b5 by NADH upon the concentration of cytochrome b5 and the effect of partial inactivation of microsomal NADH-cytochrome b5 reductase on the extent of cytochrome b5 reduction are both consistent with this model for the microsomal membrane. The evidence indicates that the interaction of the active sites of cytochrome b5 and reductase is not significantly altered by binding to the membrane and suggests that the hydrophilic portions of these proteins, although securely bound to the membrane by means of a hydrophobic peptide, may be capable of independent and significant motion in the aqueous phase.

Studies on the Function of Cell Membrane: (1) Leakage of Nadh-Cytochrome c Reductase into Plasma from Liver Cells in CCL4-Poisoned Rats

NADH-cytochrome c reductase was not observed in normal rat plasma. NADH-cytochrome c reductase appeared in the plasma of rats after CCl4 administration. The time-course pattern of the enzyme activity was quite different between the sexes the elevation of the activity was markedly enhanced by fasting. NADH-cytochrome c reductase activity detected in the circulating plasma of CCl4-poisoned rats originated from the microsomal NADH-cytochrome b5 reductase system in liver cells. Mechanisms of the leakage of this enzyme into the plasma are discussed.

STUDIES ON THE FUNCTION OF CELL MEMBRANE

NADH-cytochrome c reductase was not observed in normal rat plasma. NADH-cytochrome c reductase appeared in the plasma of rats after CCl4 administration. The time-course pattern of the enzyme activity was quite different between the sexes the elevation of the activity was markedly enhanced by fasting. NADH-cytochrome c reductase activity detected in the circulating plasma of CCl4-poisoned rats originated from the microsomal NADH-cytochrome b5 reductase system in liver cells. Mechanisms of the leakage of this enzyme into the plasma are discussed.

The Binding of Cytochrome b5 to Liver Microsomes

Abstract The 40–44 hydrophobic peptide segment, present in cytochrome b5 obtained by nonhydrolytic means, but absent from lipase or trypsin-extracted cytochrome b5, has been found to be required for the binding of a 10- to 20-fold molar excess of cytochrome b5 to liver microsomes. At saturation, the bound cytochrome b5 constitutes nearly 20% of the total protein of the vesicle preparations. This extra bound hemoprotein is indistinguishable from endogenous cytochrome b5 as an electron acceptor from microsomal NADH cytochrome b5 reductase and as an electron donor for external cytochrome c or the rat liver microsomal stearyl coenzyme A desaturase. On the basis of earlier work of others and present data, it appears that these enzymes are localized on the outside of microsomal vesicles. A model, consistent with these data, proposes that the extra, as well as endogenous cytochrome b5, is bound to microsomes by interactions between the nonpolar peptide segment and the phospholipid of the membrane. This permits interactions of the polar catalytic heme binding segment at the surface of the outer membrane with either reductase, cytochrome c, or the desaturase system. The latter catalytic events require only the translational movement of the nonpolar portion of the protein in the lipid membrane of several molecular diameters.

Studies on cytochrome b-555 from larvae of the housefly, Musca domestica L. I. Purification and properties of cytochrome b-555.

1. Cytochrome b-555 was highly purified from larvae of the housefly, Musca domestica L. The absorption spectra of the purified cytochrome b-555 showed peaks at 358–360, 414, and 530mμ in the oxidized form, and at 424, 528, and 555mμ in the reduced. The α-band of the reduced form was asymmetric at room temperature and at neutral pH, but split into two distinct peaks at 552 and 556 mμ at liquid nitrogen temperature. The purified cytochrome contained protoheme as the prosthetic group and combined neither with carbon monoxide nor with cyanide. 2. In the ultracentrifugation, the cytochrome preparation showed a symmetrical pattern and its sedimentation coefficient, S20, w, was 1.43S. The molecular weight was determined to be 13, 700 by the gel filtration method. 3. Cytochrome b-555 was reduced readily by sodium dithionite, slowly by cysteine, and anaerobically by ascorbate, but was not reduced non-enzymatically by NADH. The reduced form was oxidized by potassium ferricyanide, beef heart ferricytochrome c, and very slowly by air (the apparent first-order velocity constant was 0.0046 sec−1). The midpoint redox potential (E'o) of cytochrome b-555 was +0.006volt at pH7.0 and at 12°C. 4. Purified cytochrome b-555 was reduced by NADH in the presence of the larval microsomal fraction or NADH-cytochrome b5 reductase [EC 1.6.2.2] purified from rat liver microsomea. The reaction was inhibited neither by antimycin A nor by rotenone as in the case of microsomal NADH-cytochrome b5 reductase systems of housefly larvae and mammalian liver. 5. Purified cytochrome b-555 seemed to be a solubilized form of cytochrome b5 which existed both in the mitochondrial and microsomal fractions prepared from the larvae. Solubilization of larval cytochrome b5 from the cytoplasmic membranes was discussed.

An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study.

Preparations of rat-liver mitochondria catalyze the oxidation of exogenous NADH by added cytochrome c or ferricyanide by a reaction that is insensitive to the respiratory chain inhibitors, antimycin A, amytal, and rotenone, and is not coupled to phosphorylation. Experiments with tritiated NADH are described which demonstrate that this "external" pathway of NADH oxidation resembles stereochemically the NADH-cytochrome c reductase system of liver microsomes, and differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distributation data are presented which substantiate the conclusion that microsomal contamination cannot account for the rotenone-insensitive NADH-cytochrome c reductase activity observed with the mitochondria. A procedure is developed, based on swelling and shrinking of the mitochondria followed by sonication and density gradient centrifugation, which permits the separation of two particulate subfractions, one containing the bulk of the respiratory chain components, and the other the bulk of the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, and that the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to, or identical with, the microsomal NADH-cytochrome b(5) reductase and cytochrome b(5).