Simultaneous purification and characterization of cytochrome b5 reductase and cytochrome b5 from sheep liver

Cytochrome b5 was purified from detergent solubilized sheep liver microsomes by using three successive DEAE-cellulose, and Sephadex G-100 column chromatographies. It was purified 54-fold and the yield was 23.5% with respect to microsomes. The apparent Mr of cytochrome b5 was estimated to be 16,200 +/- 500 by SDS-PAGE. Absolute absorption spectrum of the purified cytochrome b5 showed maximal absorption at 412 nm and dithionite-reduced cytochrome b5 gave peaks at 557, 526.5 and 423 nm. The ability of the purified sheep liver cytochrome b5 to transfer electrons from NADH-cytochrome b5 reductase to cytochrome c was investigated. The K(m) and Vmax values were calculated to be 0.088 microM cytochrome b5 and 315.8 microM cytochrome c reduced/min/mg enzyme, respectively. Also the reduction of cytochrome b5 by reductase was studied and K(m) and Vmax values were determined to be 5 microM cytochrome b5 and 5200 nmol cytochrome b5 reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating concentration of cytochrome b5 were found to be 0.0017 mM NADH and 6944 nmol cytochrome b5 reduced/min/mg enzyme, respectively. NADH-cytochrome b5 reductase was also partially purified from the same source, detergent solubilized sheep liver microsomes, by using two successive DEAE-cellulose, and 5'-ADP-agarose affinity column chromatographies. It was purified 144-fold and the yield was 7% with respect to microsomes. The apparent monomer Mr of reductase was estimated to be 34,000 by SDS-PAGE. When ferricyanide was used as an electron acceptor, reductase showed maximum activity between 6.8 and 7.5. The K(m) and Vmax values of the enzyme for ferricyanide were calculated as 0.024 mM ferricyanide and 673 mumol ferricyanide reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating amounts of ferricyanide were found to be 0.020 mM NADH and 699 mumol ferricyanide reduced/min/mg enzyme, respectively.     

Cytochrome b5 reductase and cytochrome b5 support the CYP2E1-mediated activation of nitrosamines in a recombinant Ames test

With CYP2E1 in vitro both the first and the second electron of the catalytic cycle can come from cytochrome b(5) via either NADPH-cytochrome P450 reductase or NADH-cytochrome b(5) reductase, and the presence of cytochrome b(5) stimulates CYP2E1 turnover both in vitro and in vivo. To determine whether electron input via the NADH-dependent pathway was similarly functional in whole cells and necessary for the stimulation by cytochrome b(5), we constructed five plasmids designed to express human CYP2E1 in various combinations with cytochrome b(5) reductase, cytochrome b(5), and cytochrome P450 reductase. CYP2E1 activity in Salmonella typhimurium cells transformed with each plasmid was assessed by mutagenic reversion frequency in the presence of dimethylnitrosamine. A fivefold increase in reversion frequency when cytochrome b(5) was coexpressed with P450 reductase was abolished by disruption of heme-binding in cytochrome b(5) by site-directed mutagenesis (His68Ala), suggesting that electron transfer to cytochrome b(5) was necessary for the stimulation. Addition of cytochrome b(5) reductase to the cytochrome b(5)/P450 reductase coexpression plasmid did not further increase the stimulation by cytochrome b(5), but b(5) reductase could support CYP2E1 activity in the absence of P450 reductase at a level equivalent to that obtained with just CYP2E1 and P450 reductase. Neither cytochrome b(5) reductase nor cytochrome b(5) alone could support CYP2E1 activity. These results demonstrate that the cytochrome b(5) reductase/cytochrome b(5) pathway can support CYP2E1 activity in bacterial cells.     

The oxidation-reduction characteristics of cytochrome b5 in hen liver microsomes.

Hen liver microsomes contained 0.20 nmol of cytochromeb₅ per mg of protein. Upon addition of NADH about 95% cytochrome b₅ was reduced very fast with a rate constant of 206 s^?1When ferricyanide was added to the reaction system the cytochrome stayed in the oxidized form until the ferricyanide reduction was almost completed. The reduced cytochrome b₅ in microsomes was oxidized very rapidly by ferricyanide. The rate constant of 4.5 × 10⁸m^?1 s^?1, calculated on the basis of assumption that ferricyanide reacts directly with the cytochrome, was found to be more than 100 times higher than that of the reaction between ferricyanide and soluble cytochrome b₅. To explain the results, therefore, the reverse electron flow from cytochrome b₅ to the flavin coenzyme in microsomes was assumed.By three independent methods the specific activity of the microsomes was measured at about 20 nmol of NADH oxidized per s per mg of protein and it was concluded that the reduction of the flavin coenzyme of cytochrome b₅ reductase by NADH is rate-limiting in the NADH-cytochrome b₅ and NADH-ferricyanide reductase reactions of hen liver microsomes. In the NADH-ferricyanide reductase reaction the apparent Michaelis constant for NADH was 2.8 μm and that for ferricyanide was too low to be measured. In the NADH-cytochrome c reductase reaction the maximum velocity was 2.86 nmol of cytochrome c reduced per s per mg of protein and the apparent Michaelis constant for cytochrome c was 3.8 μm.     

Purification and characterization of cholesterol 7 alpha-hydroxylase cytochrome P-450 of untreated rabbit liver microsomes

Cytochrome P-450 which catalyzes the 7 alpha-hydroxylation of cholesterol was purified from liver microsomes of untreated rabbits. The minimum molecular weight of the cytochrome P-450 was estimated to be 48,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The preparation contained 7 nmol of cytochrome per mg of protein. The oxidized form of the P-450 showed absorption maxima at 568, 535, and 417 nm, which are characteristic of a low spin hemoprotein, while the reduced form showed maxima at 545 and 413 nm. The carbon monoxide complex of the reduced form showed maxima at 550 and 447 nm. The cholesterol 7 alpha-hydroxylase system of untreated rabbit liver microsomes was reconstituted with the purified P-450, NADPH-cytochrome P-450 reductase, and cytochrome b5. The P-450 catalyzed the 7 alpha-hydroxylation of cholesterol 500 times more efficiently than the starting microsomes. The reconstituted hydroxylase system showed a substantial salt dependency. In the presence of cytochrome b5 the activity was maximum at 0.4 M KCl (4.55 nmol product formed/mg of protein per min), whereas in the absence of cytochrome b5 the activity was marginal (0.65 nmol product formed/mg of protein per min) and inhibited by KCl. Thus, cytochrome b5 stimulated the hydroxylase activity by one order of magnitude. These results indicate that cytochrome b5 is an essential component of the cholesterol 7 alpha-hydroxylase system of untreated rabbit liver microsomes.     

Immunological similarity between NADH-cytochrome b5 reductase of erythrocytes and liver microsomes.

In a number of animal species soluble NADH-cytochrome b5 reductase of erythrocytes was compared with membrane-bound NADH-cytochrome b5 reductase of liver microsomes by using an antibody to purified NADH-cytochrome b5 reductase from rat liver microsomes. The results obtained indicated clearly that they are immunologically very similar to each other. The data with erythrocyte ghosts suggested that cytochrome b5 and NADH-cytochrome b5 reductase are also present in the ghost.     

Evidence for a second microsomal trans-2-enoyl coenzyme A reductase in rat liver. NADPH-specific short chain reductase

Evidence for the existence of a previously unknown rat hepatic microsomal reductase, short chain trans-2-enoyl-CoA reductase (SC reductase) is presented. This reductase has a specific requirement for NADPH, is unable to utilize NADH, and catalyzes the conversion of crotonyl-CoA and trans-2-hexenoyl-CoA to butyric acid and hexenoic acid at a rate of 5 and 65 nmol per min per mg of microsomal protein, respectively. Highly purified NADPH cytochrome P-450 reductase incorporated into liposomes prepared from dilauroyl phosphatidylcholine in the presence or absence of cytochrome P-450 possesses no SC reductase activity. These liposomal preparations did, however, catalyze mixed function oxidations of benzphetamine and testosterone. Rabbit antibody to rat liver NADPH cytochrome P-450 reductase had little to no effect on the conversion of crotonyl-CoA and trans-2-hexenoyl-CoA, suggesting that the SC reductase accepts reducing equivalents directly from NADPH. When acetoacetyl-CoA was incubated with hepatic microsomes and either NADH or NADPH, no formation of butyrate was detected; however, when both cofactors were present, a rate of formation of 3 nmol of butyrate was determined per min per mg of microsomal protein. These results suggest the presence of a previously unknown short chain beta-ketoreductase which catalyzes the reduction of short chain beta-keto acids, only in the presence of NADH. Our results also indicate that the electrons from NADH to the beta-ketoreductase bypass cytochrome b5. The physiological significance is discussed in terms of lipogenesis and ketone body utilization by the liver.     

Effect of divalent cations on NADH-dependent and NADPH-dependent cytochrome b5 reduction by hepatic microsomes

The effect of Ca2+ or Mg2+ on cytochrome b5 reduction by porcine liver microsomes was examined using trypsin-solubilized cytochrome b5 as a substrate. The reduction of exogenous cytochrome b5 by microsomes was low at 1.2 microM cytochrome b5 (3.9 or 2.7 nmol/min/mg protein, respectively, with NADH or NADPH). The addition of CaCl2 greatly enhanced either NADH-dependent or NADPH-dependent cytochrome b5 reduction. At 2 mM CaCl2, the reduction rate was increased to 23- or 18-fold of control, respectively with NADH or NADPH. The concentration for half-maximal effect (EC50) was 0.5 or 0.6 mM in the NADH or NADPH systems, respectively. MgCl2 also stimulated cytochrome b5 reduction with a EC50 value of 1.0 mM in the NADH system or 0.6 mM in the NADPH system. The comparison with the result with KCl indicated that the activation by CaCl2 or MgCl2 is caused mainly by their divalent cation moiety. The Km value for cytochrome b5 was decreased and the Vmax was increased by calcium with either the NADH- or the NADPH-dependent system. NADH-ferricyanide reductase activity was not affected by calcium, but NADPH-ferricyanide reductase activity was stimulated as well as NADPH-cytochrome c reductase activity. In the presence of Triton X-100, divalent cations were inhibitory in NADH-dependent cytochrome b5 reduction, and in contrast, stimulative in NADPH-dependent reaction. These findings suggest that the activation of cytochrome b5 reduction by divalent cations in the NADH system is mainly due to an increasing accessibility of the substrate, and in the NADPH system, in addition to this, a direct effect of divalent cations on NADPH-cytochrome P450 reductase is also involved.     

Purification and properties of the intact form of NADH-cytochrome b5 reductase from rabbit liver microsomes.

NADH-cytochrome b5 reductase [EC 1.6.2.2] has been solubilized with Triton X-100 and purified to homogeneity from rabbit liver microsomes. The purified enzyme is essentially free of the detergent and phospholipids and exists in aqueous media as an oligomeric aggregate of about 13 S. Its monomeric molecular weight is about 33,000 and 1 mole of FAD is associated with 1 mole of the monomeric unit. The enzyme catalyzes the reductions by NADH of ferricyanide and 2,6-dichlorophenol indophenol at an activity ratio of 1 : 0.09. Although the intact form of cytochrome b5 is a poorer electron acceptor than its hydrophilic fragment for the purified flavoprotein, electron transfer from the reductase to the intact cytochrome can be markedly stimulated by detergents or phospholipids, which also cause profound enhancement of the NADH-cytochrome c reductase activity reconstituted from the reducatse and cytochrome b5. Upon digestion with trypsin [EC 3.4.21.4], the ability of the reductase to form an active NADH-cytochrome c reductase system with the intact form of cytochrome b5 and Triton X-100 is rapidly lost. This loss of the reconstitution capability can be prevented by preincubation of the reductase with phosphatidylcholine liposomes. Trypsin digestion also results in the cleavage of the reductase molecule to a protein having a molecular weight of about 25,000 and a smaller fragment. The purified flavoprotein can bind to liver microsomes, liver mitochondria, sonicated human erythrocyte ghosts, and phosphatidylcholine liposomes. The reductase solubilized directly from liver microsomes by lysosomal digestion however, is devoid of membrane-binding capacity. It is concluded that the intact form of NADH-cytochrome b5 reductase is an amphipathic protein and its hydrophobic moiety, which is removable by lysosomal digestion, is responsible for the tight binding of the reductase to microsomes and for its normal functioning in the membrane.     

Propylthiouracil, a selective inhibitor of NADH-cytochrome b5 reductase

Propylthiouracil inhibited the activity of NADH-cytochrome b5 reductase of rat liver microsomes using potassium ferricyanide as electron acceptor. On the other hand, NADPH-cytochrome P-450 reductase activity was not affected by the compound. NADH-supported reduction of cytochrome b5 was also inhibited by propylthiouracil in the reconstituted system consisting of cytochrome b5 and partially purified NADH-cytochrome b5 reductase.     

Searching the Cytochrome P450 Enzymes for the Metabolism of Meranzin Hydrate: A Prospective Antidepressant Originating from Chaihu-Shugan-San

Meranzin hydrate (MH), an absorbed bioactive compound from the Traditional Chinese Medicine (TCM) Chaihu-Shugan-San (CSS), was first isolated in our laboratory and was found to possess anti-depression activity. However, the role of cytochrome P450s (CYPs) in the metabolism of MH was unclear. In this study, we screened the CYPs for the metabolism of MH in vitro by human liver microsomes (HLMs) or human recombinant CYPs. MH inhibited the enzyme activities of CYP1A2 and CYP2C19 in a concentration-dependent manner in the HLMs. The K_m and V_max values of MH were 10.3±1.3 µM and 99.1±3.3 nmol/mg protein/min, respectively, for the HLMs; 8.0±1.6 µM and 112.4±5.7 nmol/nmol P450/min, respectively, for CYP1A2; and 25.9±6.6 µM and 134.3±12.4 nmol/nmol P450/min, respectively, for CYP2C19. Other human CYP isoforms including CYP2A6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 showed minimal or no effect on MH metabolism. The results suggested that MH was simultaneously a substrate and an inhibitor of CYP1A2 and CYP2C9, and MH had the potential to perpetrate drug-drug interactions with other CYP1A2 and CYP2C19 substrates.     

Studies on the microsomal electron-transport system of anaerobically grown yeast. IV. Purification and characterization of NADH-cytochrome b5 reductase.

The presence of NADH-cytochrome b5 reductase [EC 1.6.2.2] in microsomes from anaerobically grown yeast was confirmed by its isolation and purification. The purified preparation of the reductase showed an apparent molecular weight of 27,000 daltons. The reductase appeared to contain loosely-bound FAD as a prosthetic group. The reductase required NADH as a specific electron donor, and could reduce some redox dyes as well as cytochrom b5. The reductase, however, could not reduce cytochrome c. Michaelis constants of the reductase for NADH and calf liver cytochrome b5 were 6.3 and 1.5 micron M, respectively, and optimal pH for cytochrome b5 reduction was 5.6. Although some differences exist between the properties of NADH-cytochrome b5 reductase from yeast and from mammalia, the results indicate a functional similarity of the present enzyme to mammalian NADH-cytochrome b5 reductase in the microsomal electron-transport system.     

Induction of rat hepatic drug metabolizing enzymes by dimethylcyclosiloxanes

Low molecular weight dimethylcyclosiloxanes (DMCS) are important precursors in the synthesis of polydimethysiloxane polymers widely used in industry, and in medical and personal care products. The objective of this study was to characterize the ability of two DMCS, octamethylcyclosiloxane (D4) and decamethylcyclopentasiloxane (D5) to induce drug metabolizing enzymes in rats. Male and female Sprague-Dawley rats were administered 1, 5, 20, or 100 mg/kg D4 or D5 in corn oil daily by gavage for 4 days. Changes in the levels of activity and/or immunoreactivity of CYP1A1/2, CYP2B1/2, CYP3A1/2 and NADPH cytochrome P450 reductase in liver microsomes were examined. Significant increases were observed in the liver to body weight ratio in female rats administered either D4 or D5 at doses > or = 20 mg/kg. Increases in the liver to body weight ratio were observed in male rats treated with > or = 100 mg/kg D5 but not with D4. Relatively large increases in CYP2B1/2 enzymatic activity and immunoreactive protein were observed with increasing concentrations of both D4 and D5. Significant increases in 7-pentoxyresorufin O-depentylase (PROD) activity were also detected in male and female rats given D4 at doses > or = 5 mg/kg. D5 increased PROD activity in male rats at doses > or = 20 mg/kg and in female rats at doses > or = 5 mg/kg. 7-Ethoxyresorufin O-deethylase (EROD) activity was increased in both male and female rats receiving > or = 20 mg/kg D4 or > or = 5 mg/kg D5; however, no changes were detected in CYP1A1/2 immunoreactive protein in rats of either sex. D4 and D5 caused significant increases in CYP3A1/2 immunoreactive protein in only male rats treated with 100 mg/kg of either compound. However, significant increases were detected in CYP3A1/2 immunoreactive protein in female rats at D4 doses > or = 20 mg/kg and D5 doses > or = 5 mg/kg. Induction of NADPH cytochrome P-450 reductase immunoreactive protein was observed with D4 in female rats and in both male and female rats with D5. Induction of CYP2B/1/2, CYP3A1/2 and NADPH cytochrome P450 reductase was observed in rats treated with 50 mg/kg phenobarbital by intraperitoneal injection. Maximal CYP2B induction detected with D4 was approximately 50% of the increase observed with phenobarbital. In summary, D4 and D5 induced CYP2B1/2 in adult rat liver in a manner similar to that observed with phenobarbital; however, differences were observed between D4 and D5 in their ability to induce CYP3A1/2 and NADPH cytochrome P450 reductase. Female rats were more sensitive to the inductive properties of low doses of both DMCS than male rats whereas male rats were more responsive to phenobarbital induction.     

Participation of a cytochrome b5-like hemoprotein of outer mitochondrial membrane (OM cytochrome b) in NADH-semidehydroascorbic acid reductase activity of rat liver

The participation of a cytochrome b₅-like hemoprotein of outer mitochondrial membrane (OM cytochrome b) in the NADH-semidehydroascorbate (SDA) reductase activity of rat liver was studied. NADH-SDA reductase activity was strongly inhibited by antibodies against OM cytochrome b and NADH-cytochrome b₅ reductase, whereas no inhibition was caused by anti-cytochrome b₅ antibody. NADH-SDA reductase exhibited the same distribution pattern as OM cytochrome b-mediated rotenone-insensitive NADH-cytochrome c reductase activity among various subcellular fractions and submitochondrial fractions. Both activities were localized in outer mitochondrial membrane. These observations suggest that OM cytochrome b-mediated rotenone-insensitive NADH-cytochrome c reductase system participates in the NADH-SDA reductase activity of rat liver.     

Sulfide:quinone oxidoreductase in membranes of the hyperthermophilic bacterium Aquifex aeolicus (VF5)

The sulfide-dependent reduction of exogenous ubiquinone by membranes of the hyperthermophilic chemotrophic bacterium Aquifex aeolicus (VF5), the sulfide-dependent consumption of oxygen and the reduction of cytochromes by sulfide in membranes were studied. Sulfide reduced decyl-ubiquinone with a maximal rate of up to 3.5 micromol (mg protein)(-1) min(-1) at 20 degrees C. Rates of 220 nmol (mg protein)(-1) min(-1)] for the sulfide-dependent consumption of oxygen and 480 nmol (mg protein)(-1) min(-1) for the oxidation of sulfide at 20 C were estimated. The reactions were sensitive towards 2-n-nonyl-4-hydroxyquinoline-N-oxide, but insensitive towards cyanide. Both reduction of decyl-ubiquinone and consumption of oxygen by sulfide rapidly increased with increasing temperature. For the sulfide-dependent respiratory activity, a sulfide-to-oxygen ratio of 2.3+/-0.2 was measured. This indicates that sulfide was oxidized to the level of zero-valent sulfur. Reduction of cytochromes by sulfide was monitored with an LED-array spectrophotometer. Reduction of cytochrome b was stimulated by 2-n-nonyl-4-hydroxyquinoline-N-oxide in the presence of excess sulfide under oxic conditions. This "oxidant-induced reduction" of cytochrome b suggests that electron transport from sulfide to oxygen in A. aeolicus employs the cytochrome bc complex via the quinone pool. Comparison of the amino acid sequence with the sequence of the sulfide:quinone oxidoreductase from Rhodobacter capsulatus and of the flavocytochrome c from Allochromatium vinosum revealed that the sulfide:quinone oxidoreductase from A. aeolicus belongs to the glutathione reductase family of flavoproteins.     

Purification of NADH-cytochrome b5 reductase from sheep lung and its electrophoretic, spectral and some other properties

1. NADH-cytochrome b5 reductase was purified from sheep lung microsomes in the presence of non-ionic and ionic detergents, Emulgen 913 and cholate, respectively. 2. The purification procedure involved the ion-exchange chromatography of the detergent solubilized microsomes on DEAE-cellulose. 3. Further purification and concentration of lung reductase was carried out with a second DEAE-cellulose column followed by the affinity column chromatography of partially purified reductase on 5'-ADP-agarose column. 4. The specific activity of sheep lung reductase was 638 mumol ferricyanide reduced/min/mg protein and the yield was 6% of the initial activity in microsomes. 5. The SDS-polyacrylamide gel electrophoresis of the purified lung reductase showed one protein band having the monomer mol. wt of 34,500 +/- 1500. In the presence of 0.4% deoxycholate, it existed as an active dimer having a mol. wt of 68,500. 6. Trypsin treated lung reductase showed two extra protein bands of mol. wts of 28,000 and 25,000 on 10% SDS-polyacrylamide gels. 7. The purified enzyme was found to contain FAD as prosthetic group and the absorption spectrum of lung reductase showed two peaks at 390 and 461 nm which were typical for flavoproteins and a shoulder at 490 nm. 8. The maximal activity of lung reductase was observed between pH 6.5-8.0 and at pH 6.8, when ferricyanide and partially purified sheep lung cytochrome b5 was used as electron acceptors, respectively.     

Purification and characterization of recombinant rat hepatic CYP4F1.

CYP4F1 was discovered by Chen and Hardwick (Arch. Biochem. Biophys. 300, 18-23, 1993) as a new CYP4 cytochrome P450 (P450) preferentially expressed in rat hepatomas. However, the catalytic function of this P450 remained poorly defined. We have purified recombinant CYP4F1 protein to a specific content of 12 nmol of P450/mg of protein from transfected yeast cells by chromatography of solubilized microsomes on an amino-n-hexyl Sepharose 4B column, followed by sequential HPLC on a DEAE column and two hydroxylapatite columns. The purified P450 was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 53 kDa. The enzyme catalyzed the omega-hydroxylation of leukotriene B(4) with a K(m) of 134 microM and a V(max) of 6.5 nmol/min/nmol of P450 in the presence of rabbit hepatic NADPH-P450 reductase and cytochrome b(5). In addition, 6-trans-LTB(4), lipoxin A(4), prostaglandin A(1), and several hydroxyeicosatetraenoic acids (HETEs) were also omega-hydroxylated. Of several eicosanoids examined, 8-HETE was the most efficient substrate, with a K(m) of 18.6 microM and a V(max) of 15.8 nmol/min/nmol of P450. In contrast, no activity was detected toward lipoxin B(4), laurate, palmitate, arachidonate, and benzphetamine. The results suggest that CYP4F1 participates in the hepatic inactivation of several bioactive eicosanoids.     

Expression of cytochrome P450 2A6 in Escherichia coli: purification, spectral and catalytic characterization, and preparation of polyclonal antibodies.

Cytochrome P450 (CYP) 2A6 is the principal human enzyme catalyzing coumarin 7-hydroxylation and is known to be involved in the metabolism of halothane, nicotine, and metabolic activation of butadiene and nitrosamines. In this paper expression of CYP2A6 in Escherichia coli is reported. In order to achieve expression, the N-terminus of protein was modified by PCR mutagenesis. The N-terminal variant with only a single amino acid change showed expression of 210 nmol of CYP2A6/liter of culture. Recombinant CYP2A6 protein was purified to electrophoretic homogeneity and further characterized. Absolute spectra were typical for CYP proteins and indicated low spin characteristics of isolated protein. Due to a hydrophobic segment the N-terminal amino acid sequence of recombinant CYP2A6 was blocked. The N-terminal formylmethionine block was removed by mild acid treatment. Purified CYP2A6 had good catalytic activity toward marker substrate coumarin in a reconstituted system (K(m) = 1.48 +/- 0.37 microM, V(max) = 3.36 +/- 0.18 nmol product/min/nmol CYP). Its activity in the reconstituted system was stimulated by the presence of cytochrome b(5) and glutathione. CYP2A6 was shown to metabolize chlorzoxazone in the reconstituted system with activity of 0.32 nmol of product/min/nmol of CYP, and thus caution should be taken when interpretation of CYP2E1 in vivo phenotyping data is performed. Rabbit polyclonal antibodies were produced against recombinant CYP2A6 and proved to be very useful for immunoblotting and immunoinhibition studies. Availability of this expression system and specific antibodies should facilitate characterization of the role of CYP2A6 in the metabolism of chemicals and in the study biological relevance of genetic polymorphisms of this enzyme.     

Components of the cytochrome P450-dependent monooxygenase system and 'NADPH-independent benzo[a]pyrene hydroxylase' activity in a wide range of marine invertebrate species

Levels of components of the cytochrome P450 (CYP)-dependent monooxygenase system were characterised in microsomes of major biotransformation tissues, or whole bodies, of 33 species from six phyla of aquatic invertebrates. The phylogenetic distribution of benzo[a]pyrene hydroxylase (BPH) activity in the absence of added NADPH (so-called 'NADPH-independent BPH activity') and presence of NADPH was also examined. Microsomal protein yield was higher in individual tissues than whole tissues. The main components (total CYP and cytochrome b5; NADPH-dependent cytochrome c (CYP) reductase, NADH-dependent cytochrome c reductase and NADH-dependent ferricyanide (b5) reductase activities) were found in most species of the Porifera, Cnidaria, Mollusca, Polychaeta, Crustacea and Echinodermata examined. The so-called '418-peak' of the carbon-monoxide difference spectrum of reduced microsomes was found in all species, indicating the wide distribution of this protein. Total CYP levels (pmol mg(-1) protein; mean+/-SEM) were similar in molluscs (50+/-7), crustaceans (61+/-11) and echinoderms (56+/-9), with the exception of high levels (223-266) in two crustacean species. NADPH-dependent BPH activity (pmol min(-1) mg(-1) protein) was found in 32 species, being lowest in Porifera and Cnidaria (3-4), intermediate in Mollusca (7.8+/-1.3), and highest in Crustacea (25+/-4) and Echinodermata (15+/-4). NADPH-independent BPH activity was evident in 13 out of 15 molluscan species examined, with the addition of NADPH either stimulating (8 species) or inhibiting (5 species) the activity. NADPH-independent BPH activity was also seen in two poriferan species and indicated in three crustacean species, suggesting that the phenomenon is not solely restricted to the Mollusca.     

The interaction of NADH-cytochrome b5 reductase and cytochrome b5 bound to egg lecithin liposomes.

Incubation of liposomes prepared by sonication of egg lecithin with the amphipathic form of cytochrome b5 results in the binding of a maximum of 244 molecules of cytochrome b5 per liposomal vesicle. Interactions of the phospholipid with the hydrophobic segment of cytochrome b5 are involved in this binding which does not disrupt the liposome. When a small amount of NADH-cytochrome b5 reductase is bound liposomes simultaneously with cytochrome b5, the two proteins catalyze the reduction of cytochrome c by NADH. A qualitative kinetic analysis reveals that all of the cytochrome b5 interacts with reductase, a result consistent with these protein undergoing translational diffusion in the plane of the membrane. This system and the purified stearyl coenzyme A desaturase provide a model to study the dynamics of protein andlipid interactions in this membrane-bound oxidative sequence.     

Kinetic mechanism of guinea pig neutrophil 5-lipoxygenase

The kinetic mechanism of guinea pig neutrophil 5-lipoxygenase was investigated using a continuous spectrophotometric assay that monitors product diene formation at 236 nm due to substrate oxygenation. Progress curves for reactions with both arachidonic acid and eicosapentaenoic acid are characterized by 1-3-min lag phases in the attainment of steady-state velocities and product inhibition, as indicated by the total cessation of the reaction prior to complete depletion of substrate. The dependence of the steady-state velocity on arachidonic acid concentration appears to follow Michaelis-Menten kinetics, with Vmax = 4.2 +/- 0.4 nmol of 5-hydroxy-6,8,11,14-eicosatetraenoic acid/min/mg of protein and Ks = 25 +/- 4 microM. The addition of Ca2+ results in an overall activation: lag phases are shortened to 10-20 s, Vmax increases to 24 +/- 2 nmol/min/mg of protein, and Ks decreases to 7.7 +/- 1.7 microM; and a change in a mechanism to one involving substrate inhibition (Kss = 13 +/- 1 microM). The observed activation by Ca2+ has a half-maximal response at around 30 microM. In the presence of Ca2+, ATP causes an increase in Vmax to 30 +/- 4 nmol/min/mg of protein without changing Ks or Kss and a reduction of the lag to less than 5 s. The half-maximal response for ATP is 31 +/- 7 microM. Oxygenation of eicosapentaenoic acid in the presence of Ca2+ and ATP occurs with similar kinetics, except for significantly less substrate inhibition: Vmax = 31 +/- 6 nmol/min/mg of protein, Ks = 7 +/- 1 microM, and Kss = 33 +/- 2 microM. This is the first report suggesting a kinetic mechanism for 5-lipoxygenase, which accounts for substrate inhibition, regulation by Ca2+, and ATP and substrate specificity.