Differences in carbohydrate moieties of high molecular weight glycoproteins of human lymphocytes of T and B origins revealed by monoclonal autoantibodies with anti-I and anti-i specificities

NADPH reduced rabbit liver microsomal enzymes catalyzed anaerobic dehalogenation of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) to produce CF₂CHCl and CF₃CH₂Cl. Anaerobic dehalogenation was optimal at pH7.4 and was blocked by either oxygen or carbon monoxide. The degree of inhibition of anaerobic dehalogenation by carbon monoxide was closely correlated to the proportion of carbon monoxide complex of cytochrome P450. Anaerobic dehalogenation was enhanced by pretreatment of the animals with phenobarbital but not with methylcholanthrene.     

The interaction of microsomal cytochrome P450 2B4 with its redox partners, cytochrome P450 reductase and cytochrome b(5)

Cytochrome P450 2B4 is a microsomal protein with a multi-step reaction cycle similar to that observed in the majority of other cytochromes P450. The cytochrome P450 2B4-substrate complex is reduced from the ferric to the ferrous form by cytochrome P450 reductase. After binding oxygen, the oxyferrous protein accepts a second electron which is provided by either cytochrome P450 reductase or cytochrome b₅. In both instances, product formation occurs. When the second electron is donated by cytochrome b₅, catalysis (product formation) is ∼10- to 100-fold faster than in the presence of cytochrome P450 reductase. This allows less time for side product formation (hydrogen peroxide and superoxide) and improves by ∼15% the coupling of NADPH consumption to product formation. Cytochrome b₅ has also been shown to compete with cytochrome P450 reductase for a binding site on the proximal surface of cytochrome P450 2B4. These two different effects of cytochrome b₅ on cytochrome P450 2B4 reactivity can explain how cytochrome b₅ is able to stimulate, inhibit, or have no effect on cytochrome P450 2B4 activity. At low molar ratios (<1) of cytochrome b₅ to cytochrome P450 reductase, the more rapid catalysis results in enhanced substrate metabolism. In contrast, at high molar ratios (>1) of cytochrome b₅ to cytochrome P450 reductase, cytochrome b₅ inhibits activity by binding to the proximal surface of cytochrome P450 and preventing the reductase from reducing ferric cytochrome P450 to the ferrous protein, thereby aborting the catalytic reaction cycle. When the stimulatory and inhibitory effects of cytochrome b₅ are equal, it will appear to have no effect on the enzymatic activity. It is hypothesized that cytochrome b₅ stimulates catalysis by causing a conformational change in the active site, which allows the active oxidizing oxyferryl species of cytochrome P450 to be formed more rapidly than in the presence of reductase.     

Mechanism of the microsomal reduction of carbon tetrachloride and halothane

The source of the hydrogen atoms in reduced metabolites of carbon tetrachloride and halothane has been studied. This was approached by measuring deuterium incorporation into chloroform and 2-chloro-1,1,1-trifluoroethane formed as microsomal metabolites of carbon tetrachloride and halothane, respectively, in a medium enriched in deuterium oxide. GC/MS analysis showed no deuterium enrichment of chloroform when hepatic microsomal fractions from control rats were used; however, small increases in enrichment were seen when microsomes from phenobarbitalor benzpyrene-treated rats were employed. No detectable deuterium incorporation into 2-chloro-1,1,1-trifluoroethane was observed. These results suggest that carbanions are not formed as major intermediates and suggest that one-electron transfer reactions predominate in the reductive metabolism of carbon tetrachloride and halothane.     

Quantitative analysis of volatile halothane metabolites in biological tissues by gas chromatography

A simple and sensitive gas chromatographic method for the determination of 2-chloro-1, 1-difluoroethylene (CDE) and 2-chloro-1,1,1-trifluoroethane (CTE), two highly volatile metabolites of halothane, in blood, liver and isolated hepatic microscomes is described. The entire head-space in equilibrium with a known volume or weight of the sample is injected into the gas chromatograph equipped with a flame ionization detector. Quantification is accomplished with standards prepared by fortifying blank samples with known concentrations of CDE and CTE which are treated under the same conditions as the samples. Detection limits for CDE and CTE were 2 pmole/ml in blood and 10 pmole/g in liver and the mean relative standard deviations are no greater than ± 6% except for CTE in hepatic microsomes (± 9%). A preliminary study of blood CDE and CTE levels in humans anesthetized with halothane is reported.     

Anaerobic dehalogenation of halothane by reconstituted liver microsomal cytochrome P-450 enzyme system

Cytochrome P-450 from liver microsomes of phenobarbital-treated rabbits catalyzed anaerobic dehalogenation of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) when combined with NADPH and NADPH-cytochrome P-450 reductase. Cytochromes P-450B₁ and P-448 from liver microsomes of untreated rabbits were less active. Triton X-100 accelerated the reaction. Unlike anaerobic dehalogenation of halothane in microsomes, the major product was 2-chloro-1,1,1-trifluoroethane and 2-chloro-1,1-difluoroethylene was negligible. These products were not detected under aerobic conditions, and dehalogenation activity was inhibited by carbon monoxide, phenyl isocyanide and metyrapone.     

Photoreactions of cytochrome b6 in systems using resolved chloroplast electron-transfer complexes

Photoreactions of cytochrome b₆ have been studied using resolved chloroplast electron-transfer complexes. In the presence of Photosystem (PS) II and the cytochrome b₆-f complex, photoreduction of the cytochrome can be observed. No soluble components are required for this reaction. Cytochrome b₆ photoreduction was found to be inhibited by quinone analogs, which inhibit at the Rieske iron-sulfur center of the cytochrome complex, by the addition of ascorbate and by depletion of the Rieske center and bound plastoquinone from the cytochrome complex. Photoreduction of cytochrome b₆ can also be demonstrated in the presence of the cytochrome complex and PS I. This photoreduction requires plastocyanin and a low-potential electron donor, such as durohydroquinone. Cytochrome b₆ photoreduction in the presence of PS I is inhibited by quinone analogs which interact with the Rieske iron-sulfur center. These results are discussed in terms of a Q-cycle mechanism in which plastosemiquinone serves as the reductant for cytochrome b₆ via an oxidant-induced reductive pathway.     

Cytochrome b5 reductase–cytochrome b5 as an active P450 redox enzyme system in Phanerochaete chrysosporium: Atypical properties and in vivo evidence of electron transfer capability to CYP63A2

Two central redox enzyme systems exist to reduce eukaryotic P450 enzymes, the P450 oxidoreductase (POR) and the cyt b₅ reductase–cyt b₅. In fungi, limited information is available for the cyt b₅ reductase–cyt b₅ system. Here we characterized the kinetic mechanism of (cyt b₅r)–cyt b₅ redox system from the model white-rot fungus Phanerochaete chrysosporium (Pc) and made a quantitative comparison to the POR system. We determined that Pc-cyt b₅r followed a “ping-pong” mechanism and could directly reduce cytochrome c. However, unlike other cyt b₅ reductases, Pc-cyt b₅r lacked the typical ferricyanide reduction activity, a standard for cyt b₅ reductases. Through co-expression in yeast, we demonstrated that the Pc-cyt b₅r–cyt b₅ complex is capable of transferring electrons to Pc-P450 CYP63A2 for its benzo(a)pyrene monooxygenation activity and that the efficiency was comparable to POR. In fact, both redox systems supported oxidation of an estimated one-third of the added benzo(a)pyrene amount. To our knowledge, this is the first report to indicate that the cyt b₅r–cyt b₅ complex of fungi is capable of transferring electrons to a P450 monooxygenase. Furthermore, this is the first eukaryotic quantitative comparison of the two P450 redox enzyme systems (POR and cyt b₅r–cyt b₅) in terms of supporting a P450 monooxygenase activity.     

Plasma membranes from intestinal microvilli and erythrocytes contain cytochromes b5 and P-420

The presence of cytochromes b₅, P-450 and P-420 and activities of NADH- and NADPH-cytochrome c reductases were determined in plasma membranes isolated from microvilli of the chick and rat intestinal epithelium and erythrocyte membranes from chick, rat and man. The results are compared with the amounts of these components found in microsomal fractions from intestinal epithelium and in nuclear membranes from chick erythrocytes. Plasma membranes from intestinal microvilli and from erythrocytes contained significant amounts of NADH-cytochrome c reductase activity and of a pigment spectrophotometrically indistinguishable from rat liver microsomal cytochrome b₅. In addition, cytochrome b₅ fragments were prepared from the membranes by limited trypsin digestion and consisted of two to four components with M_r values in the range 10 000–13 500. In low-temperature difference spectra, the presence of a second cytochrome was noted which was similar to cytochrome P-420. Cytochrome P-450 and NADPH-cytochrome c reductase activities were not detected in plasma membrane fractions in significant concentrations but were present in the corresponding endomembrane fractions. These findings in highly purified, well defined plasma membrane fractions, in which contamination by endomembranes is minimal, strengthen the evidence for the existence of cytochrome-containing redox systems in plasma membranes of various cells and suggest that such redox components are general components of the cell surface. Possible functions and origins of these redox components in plasma membranes are discussed.     

Involvement of plastoquinone bound within the isolated cytochrome b6-f complex from chloroplasts in oxidant-induced reduction of cytochrome b6

(1) Oxidant-induced reduction of cytochrome b₆ is completely dependent on a reduced component within the isolated cytochrome b₆-f complex. This component can be reduced by dithionite or by NADH/N-methylphenazonium methosulfate. It is a 2H⁺/2e⁻ carrier with a midpoint potential of 100 mV at pH 7.0, which is very similar to the midpoint potential of the plastoquinone pool in chloroplasts. (2) Oxidant-induced reduction of cytochrome b₆ is stimulated by plastoquinol-1 as well as by plastoquinol-9. The midpoint potential of the transient reduction of cytochrome b₆, however, was not shifted by added plastoquinol. (3) Quinone analysis of the purified cytochrome b₆-f complex revealed about one plastoquinone per cytochrome f. The endogenous quinone is heterogeneous, a form more polar than plastoquinone-A, probably plastoquinone-C, dominating, This is different from the thylakoid membrane where plastoquinone-A is the main quinone. (4) The endogenous quinone can be extracted from the lyophilized cytochrome b₆-f complex by acetone, but not by hydrocarbon solvents. Oxidant-induced reduction of cytochrome b₆ was observed in the lyophilized and hexane-extracted complex, but was lost in the acetone-extracted complex. Reconstitution was achieved either with plastoquinol-1 or plastoquinol-9, suggesting that a plastoquinol molecule is involved in oxidant-induced reduction of cytochrome b₆.     

Site-Directed Mutagenesis of Cytochrome b5 for Studies of Its Interaction with Cytochrome P450

We have shown earlier that microsomal cytochrome b ₅ can form a specific complex with mitochondrial cytochrome P450 (cytochrome P450scc). The formation of the complex between these two heme proteins was proved spectrophotometrically, by affinity chromatography on immobilized cytochrome b ₅, and by measuring the cholesterol side-chain cleavage activity of cytochrome P450scc in a reconstituted system in the presence of cytochrome b ₅. To further study the mechanism of interaction of these heme proteins and evaluate the role of negatively charged amino acid residues Glu42, Glu48, and Asp65 of cytochrome b ₅, which are located at the site responsible for interaction with electron transfer partners, we used sitedirected mutagenesis to replace residues Glu42 and Glu48 with lysine and residue Asp65 with alanine. The resulting mutant forms of cytochrome b ₅ were expressed in E. coli, and full-length and truncated forms (shortened from the C-terminal sequence due to cleavage of 40 amino acid residues) of these cytochrome b ₅ mutants were purified. Addition of the truncated forms of cytochrome b ₅ (which do not contain the hydrophobic C-terminal sequence responsible for interaction with the membrane) to the reconstituted system containing cytochrome P450scc caused practically no stimulation of catalytic activity, indicating an important role of the hydrophobic fragment of cytochrome b ₅ in its interaction with cytochrome P450scc. However, full-length cytochrome b ₅ and the full-length Glu48Lys and Asp65Ala mutant forms of cytochrome b ₅ stimulated the cholesterol side-chain cleavage reaction catalyzed by cytochrome P450scc by 100%, suggesting that residues Glu48 and Asp65 of cytochrome b ₅ are not directly involved in its interaction with cytochrome P450scc. The replacement of Glu42 for lysine, however, made the Glu42Lys mutant form of cytochrome b ₅ about 40% less effective in stimulation of the cholesterol side-chain cleavage activity of cytochrome P450scc, indicating that residue Glu42 of cytochrome b ₅ is involved in electrostatic interactions with cytochrome P450scc. Residues Glu42 and Glu48 of cytochrome b ₅ appear to participate in electrostatic interaction with microsomal type cytochrome P450.     

Preparation of a biologically active apo-cytochrome b5 via heterologous expression in Escherichia coli

Cytochrome b₅ (b₅) has been shown to modulate many cytochrome P450 (CYP)-dependent reactions. In order to elucidate the mechanism of such modulations, it is necessary to evaluate not only the effect of native b₅ on CYP-catalyzed reactions, but also that of the apo-cytochrome b₅ (apo-b₅). Therefore, the apo-b₅ protein was prepared using a heterologous expression in Escherichia coli. The gene for rabbit b₅ was constructed from synthetic oligonucleotides using polymerase chain reaction (PCR), cloned into pUC19 plasmid and amplified in DH5α cells. The gene sequence was verified by DNA sequencing. The sequence coding b₅ was cleaved from pUC19 by NdeI and XhoI restriction endonucleases and subcloned to the expression vector pET22b. This vector was used to transform E. coli BL-21 (DE3) Gold cells by heat shock. Expression of b₅ was induced with isopropyl β-d-1-thiogalactopyranoside (IPTG). The b₅ protein, produced predominantly in its apo-form, was purified from isolated membranes of E. coli cells by chromatography on a column of DEAE–Sepharose. Using such procedures, the homogenous preparation of apo-b₅ protein was obtained. Oxidized and reduced forms of the apo-b₅ reconstituted with heme exhibit the same absorbance spectra as native b₅. The prepared recombinant apo-b₅ reconstituted with heme can be reduced by NADPH:CYP reductase. The reconstituted apo-b₅ is also fully biologically active, exhibiting the comparable stimulation effect on the CYP3A4 enzymatic activity towards oxidation of 1-phenylazo-2-hydroxynaphthalene (Sudan I) as native rabbit and human b₅.     

Microcalorimetric studies of the interactions between cytochromes c and c1 and of their interactions with phospholipids

Thermotropic properties of purified cytochrome c₁ and cytochrome c have been studied by differential scanning calorimetry under various conditions. Both cytochromes exhibit a single endothermodenaturation peak in the differential scanning calorimetric thermogram. Thermodenaturation temperatures are ionic strength, pH, and redox state dependent. The ferrocytochromes are more stable toward thermodenaturation than the ferricytochromes. The enthalpy changes of thermodenaturation of ferro- and ferricytochrome c₁ are markedly dependent on the ionic strength of the solution. The effect of the ionic strength of solution on the enthalpy change of thermodenaturation of cytochrome c is rather insignificant. The formation of a complex between cytochromes c and c₁ at lower ionic strength causes a significant destabilization of the former and a slight stabilization of the latter. The destabilization of cytochrome c upon mixing with cytochrome c₁ was also observed at high ionic strength, under which conditions no stable complex was detected by physical separation. This suggests formation of a transient complex between these two cytochromes. When cytochrome c was complexed with phospholipids, no change in the thermodenaturation temperature was observed, but a great increase in the enthalpy change of thermodenaturation resulted.     

The cytochrome P450-containing monooxygenase of Trichosporon cutaneum: Occurrence and properties

Trichosporon cutaneum metabolizes glucose purely oxidatively and cytochrome P450 was not detected in the reduced CO-difference spectrum of whole cells. However, in the isolated microsomal fraction the corresponding monooxygenase was present as shown by the appearence of cytochrome P450, NADPH-cytochrome c (P450) reductase and cytochrome b₅. The absorption maximum of the terminal oxidase in the reduced CO-difference spectrum shifted between 447 and 448 nm. Derepression of biosynthesis of all components was achieved by transition of the cells from carbon- to oxygen-limited growth in continuous culture. The monooxygenase exhibited aminopyrine demethylation activity but not -hydroxylation activity of lauric acid. With respect to the growth limiting nutrient (carbon and oxygen respectively), mitochondrial cytochrome content showed an analogous behavior as cytochrome P450 and cytochrome b₅.     

Purification and characterization of liver microsomal cytochrome P-450 from untreated male rats

Different forms of cytochrome P-450 from untreated male rats were simultaneously purified to homogeneity using the HPLC technique. The absorption maximum, molecular weight, NH₂-terminal sequence and catalytic activity of them were determined. The NH₂-terminal sequences of six forms of cytochrome P-450 (designated P450 UT-1, UT-2, UT-4, UT-5, UT-7 and UT-8) indicate that these cytochrome P-450 isozymes are of different molecular species. The hydrophobicity values of the NH₂-terminal sequences of P450 UT-1 and P450 UT-8 were lower than that of other forms. P450 UT-8 has the highest molecular weight, 54 000, of the six forms of P-450. P450 UT-2 was active in demethylation of benzphetmaine, 450 UT-4 was active in the metabolism of 7-ethoxycoumarin and p-nitroanisole. P450 UT-1 ad P450 UT-2 were active in the 2α- and 16α-hydroxylation of testosterone, whereas P450 UT-4 was active in the 6β-, 7α- and 15α-hydroxylation of the same steroid. We believe that P450 UT-1, P450 UT-7 and P450 UT-8 are as yet unrecognized forms of cytochrome P-450.     

Cosubstrate effects in reductive dehalogenation byPseudomonas putida G786 expressing cytochrome P-450CAM

Cytochrome P-450_CAM was shown to be the primary catalyst mediating reductive dehalogenation of polychlorinated ethanes byPseudomonas putida G786. Under anaerobic conditions, the enzyme catalyzed reductive elimination reactionsin vivo with the substrates hexachloroethane, pentachloroethane, and 1,1,1,2-tetrachloroethane; the products were tetrachloroethylene, trichloroethylene, and 1,1-dichloroethylene, respectively.In vivo reaction rates were determined. No reaction was observed with 1,1,2,2-tetrachloroethane or 1,1,1-trichloroethane. Purified cytochrome P-450_CAM was used to measure dissociation constants of polychlorinated ethanes for the enzyme active site. Observed rates and dissociation constants were used to predict the course of a reaction with the three substrates simultaneously. Data obtained from experiments withP. putida G786 generally followed the simulated reaction curves. Oxygen suppressed the reductive dechlorination reactions and, in the case of 1,1,1,2-tetrachloroethane, 2,2,2-trichloroacetaldehyde was formed. Significant rates of reductive dechlorination were observed at 5% oxygen suggesting that these reactions could occur under partially aerobic conditions. These studies highlight the potential to use an aerobic bacterium,P. putida G786, under a range of oxygen tensions to reductively dehalogenate mixed wastes which are only degraded at very low rates by obligately anaerobic bacteria.Abbreviations GC/MS Gas chromatography/mass spectrometry - P-450_CAM Cytochrome m of the camphor oxidizing system ofP. putida - pca Polychlorinated ethane     

Ascaris suum NADH-methemo(myo)globin reductase systems recovering differential functions of hemoglobin and myoglobin, adapting to environmental hypoxia

We reported previously that Ascaris suum cytochrome b₅, specifically expressed in this nematode at the adult stage and dually localized in extracellular perienteric fluid and hypodermis, is involved in both perienteric NADH-methemoglobin and cytosolic NADH-metmyoglobin reduction, where cytochrome b₅ functions as an electron carrier between NADH-mediated cytochrome b₅ reductase and substrates, methemo(myo)globins to reduce the nonfunctional globins back to functional ferrous hemo(myo)globins. To further characterize NADH-methemo(myo)globin reductase systems, the midpoint potentials of A. suum perienteric hemoglobin and body wall myoglobin, as well as the affinities of Ascaris methemoglobin and metmyoglobin toward cytochrome b₅, were evaluated using potentiometric titration and surface plasmon resonance techniques, respectively. Midpoint potentials of + 7.2 mV and + 19.5 mV were obtained for Ascaris perienteric hemoglobin and body wall myoglobin, respectively. The affinities of Ascaris perienteric methemoglobin and body wall metmyoglobin toward the nematode cytochrome b₅ were comparable to that for mammalian hemoglobin and cytochrome b₅; association constants were 0.585 × 10³ M^− 1 and 2.32 × 10³ M^− 1, respectively, with rapid equilibration kinetics. These observations highlight the physiological importance of A. suum perienteric NADH-methemoglobin and cytosolic metmyoglobin reductase systems. Differential roles of A. suum perienteric hemoglobin and body wall myoglobin are also discussed from the viewpoint of oxygen homeostasis under hypoxic conditions.     

Purification of a new cytochrome P-450 from human liver microsomes

Using a classical methodlogy of purification consisting of three chromatographic steps (Octyl-Sepharose, DEAE-cellulose, CM-cellulos) we have purified a new cytochrome P-450 from human liver microsomes. It was called cytochrome P-450₉. It has been proven to be different from all preceedingly purified human liver microsomal cytochrome P-450 isozymes by its immunological and electrophoretical properties. It does not cross-react with any rat liver cytochrome P-450 and anti-cytochrome P-450₉, does not recognize rat liver microsomes; thus this cytochrome P-450₉ is specific to humans. This cytochrome P-450 isozyme exists in low amounts in human liver microsomes and exhibits an important quatitative polymorphism. In reconstituted system, cytochrome P-450₉ is able to hydroxylate all substrates tested but is not specific on any; its exatc role in xenobiotic metabolism in man remains to be elucidated.     

A 2.6 kb intron separates the signal peptide coding sequence of an anther-specific protein from the rest of the gene in sunflower

Summary Metabolism of sulfonylurea herbicides by Streptomyces griseolus ATCC 11796 is carried out via two cytochromes P-450, P-450_SU1 and P-450_SU2. Mutants of S. griseolus, selected by their reduced ability to metabolize a fluorescent sulfonylurea, do not synthesize cytochrome P-450_SU1 when grown in the presence of sulfonylureas. Genetic evidence indicated that this phenotype was the result of a deletion of > 15 kb of DNA, including the structural genes for cytochrome P-450_SU1 and an associated ferredoxin Fd-1 (suaC and suaB, respectively). In the absence of this monooxygenase system, the mutants described here respond to the presence of sulfonylureas or phenobarbital in the growth medium with the expression of only the suhC,B gene products (cytochrome P-450_SU2 and Fd-2), previously observed only as minor components in wild-type cells treated with sulfonylurea. These strains have enabled an analysis of sulfonylurea metabolism mediated by cytochrome P-450_SU2 in the absence of P-450_SU1, yielding an in vivo delineation of the roles of the two different cytochrome P-450 systems in herbicide metabolism by S. griseolus.     

Direct expression in Escherichia coli and characterization of bovine adrenodoxins with modified amino-terminal regions

Four forms of bovine adrenodoxin with modified amino-termini obtained by direct expression of cDNAs in Escherichia coli are Ad(Met¹), Ad(Met⁻¹), Ad(Met⁻¹²), and Ad(Met⁶). The shoulder numbers represent this site of translation initiator Met at the amino-termini. The adrenodoxins, except for Ad(Met⁻¹), were purified from the cell lysate and the ratios of A₄₁₄-to-A₂₇₆ of the purified proteins were over 0.92. NADPH-cytochrome c reductase activities of the three forms of adrenodoxin in the presence of adrenodoxin reductase were the same as that of purified bovine adrenocortical adrenodoxin. However, as cytochrome P-450_SCC reduction catalyzed by Ad(Met⁰) was about 60% or that by Ad(Met¹), the contribution of the amino-terminal region for the electron transfer or binding to cytochrome P-450_SCC would need to be considered.