Kinetic properties of guinea pig liver microsomal NADPH-cytochrome P-450 reductase

NADPH-cytochrome P-450 reductase was purified to apparent homogeneity from detergent-solubilized guinea pig liver microsomes. The reductase had a mol. wt of 78,000 and contained one mole each of FAD and FMN. Electron transfer activity to cytochrome c was optimal at a pH of 8.0 and an ionic strength of 0.43. The results of kinetic experiments were consistent with a ternary-complex mechanism for the interaction of the reductase with cytochrome c and NADPH. Km values for NADPH and cytochrome c were 3.1 and 26.7 microM, respectively. Inhibition by NADP+ and 2'-AMP was competitive with respect to NADPH; Ki values were 12.1 microM for NADP+ and 46.7 microM for 2'-AMP.     

Amino acid sequence of COOH-terminal 20K Da fragment from pig liver microsomal NADPH-cytochrome P-450 reductase

We have determined the complete amino acid sequence of a 20K Da COOH-terminal fragment of porcine NADPH-cytochrome P-450 reductase. The 20K Da fragment is probably produced by a proteolytic cleavage of the intact protein in porcine liver microsomes, and since the cleavage does not affect enzymatic activity, the fragment has been studied as a distinct domain. The sequence comprises 175 amino acids including three cysteine residues, one of which has been previously identified as protected by NADPH from S-carboxymethylation. The NADPH-protected cysteine lies in a stretch of 12 residues with partial homology to glutathione reductase, and is adjacent to a hydrophobic region containing a glycine-rich stretch homologous to other FAD-containing proteins. The predicted secondary structure over this entire region is beta-sheet/beta-turn/beta-sheet/alpha-helix/beta-sheet/beta-turn/alpha-h elix corresponding to hydrophobic residues 21-28/glycine-rich residues 29-33/residues 34-38/residues 39-54/residues 56-61/NADPH-protected cysteine residues 62-78/residues 71-82. It is possible that the 20K Da domain provided a significant portion of the sequence responsible for binding FAD and NADPH in the intact enzyme. This data provides a basis for further active site studies.     

Localization of cytochrome c-binding domain on NADPH-cytochrome P-450 reductase

A covalent complex between purified rat liver microsomal NADPH-cytochrome P-450 reductase and horse cytochrome c was formed through cross-linking studies with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at low ionic strength. The purified cross-linked derivative shows that this product is a 1:1 complex containing one molecule each of the flavoprotein and cytochrome. The covalent complex had almost completely blocked the electron transfer from NADPH to exogenous cytochrome c or the rabbit liver microsomal cytochrome P-450 induced by phenobarbital, indicating that the cross-linked cytochrome c covers the electron-accepting site of the reductase. These results suggest that the covalently cross-linked derivative is a valid model of the noncovalent electron transfer complex. Although the exact number and site of the cross-linked location were not determinable, in cytochrome c the amide bond originates from Lys-13 and in reductase it might be at any one of six different side chain carboxyl groups in the two neighboring cluster acidic residues, Asp-207, -208, and -209, and Glu-213, Glu-214, and Asp-215. It is therefore proposed that the six clustered carboxyl groups on reductase are in an exposed location near the area where one heme edge comes close to the molecular surface.     

Preparation of homogeneous NADPH-cytochrome P-450 reductase from rat liver.

NADPH-cytochrome P-450 reductase with capacity to support cytochrome P-450-dependent drug metabolism and to reduce artificial electron acceptors has been purified to apparent homogeneity by solubilization with Renex 690 and chromatography on DEAE-Sephadex, Agarose and QAE-Sephadex. The purified protein migrates as a single band on native and SDS-polyacrylamide gel electrophoresis, exhibits a minimum molecular weight of 80,000 daltons and contains 1 molecule each of FAD and FMN per 80,000 molecular weight. The specific activity for cytochrome $\text{c}$ as electron acceptor is 48.8 μmoles per min and for substrate hydroxylation of benzphetamine measured as NADPH oxidation in the presence of cytochrome P-450 and phosphatidylcholine is 2.5 μmoles per min.     

Isolation, characterization and partial sequence of cyanogen bromide fragments and thiol peptides from pig kidney D-amino-acid oxidase.

A partial characterization of the primary structure of D-amino-acid oxidase (D-Amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3.) from hog kidney has been achieved by a CNBr cleavage of the 14C-carboxymethylated protein. Four fragments have been isolated and purified and their alignment made possible by overlapping with methionine-containing peptides derived from tryptic digestion of the 14C-carboxymethylated protein. A partial sequencing of the CNBr fragments has been carried out by the automated Edman procedure and by manual sequence analysis. Chymotryptic peptides containing the 5 alkylated thiols of the monomer enzyme (Curti, B., Ronchi, S., branzoli, U., Ferri, G. and Williams, Jr., C. H. (1973) Biochim. Biophys. Acta 327, 266-273) have been isolated and their sequence determined. The present results do not show any significant homologies with the known sequences of other flavoproteins.     

Properties of NADPH-cytochrome P-450 reductase purified from rabbit liver microsomes.

NADPH-cytochrome P-450 reductase has been purified to electrophoretic homogeneity from rabbit liver microsomes by a procedure that may be used in conjunction with the isolation of the major forms of cytochrome P-450. The purified reductase is active in a reconstituted hydroxylation system containing P-450LM₂ or P-450LM₄. The enzyme contains one molecule each of FMN and FAD per polypeptide chain having an apparent minimal molecular weight of 74,000. Immunological techniques provided evidence for only a single form of the reductase; lower molecular weight forms occasionally seen are believed to be due to degradation by contaminating microsomal or bacterial proteases. Upon anaerobic photochemical reduction, the rabbit liver reductase undergoes spectral changes highly similar to those previously described by Vermilion and Coon for the rat liver enzyme; the fully reduced rabbit liver enzyme is converted to the three-electron-reduced form by the addition of NADP and then to the stable one-electron-reduced form by exposure to oxygen. The CD spectra of the fully oxidized enzyme, one-electron-reduced form (air-stable semiquinone), three-electron-reduced form, and fully reduced form are presented. The results obtained provide evidence that the FMN and FAD are in highly different environments in the enzyme, as also indicated by the different redox potentials and oxygen reactivities of the flavins.     

Highly purified detergent-solubilized NADPH-cytochrome P-450 reductase from phenobarbital-induced rat liver microsomes

NADPH-cytochrome P-450 reductase was highly purified from liver microsomes of phenobarbital-induced rats by column chromatography on DEAE-cellulose, DEAE-Sephadex A-50, and hydroxylapatite in the presence of deoxycholate or Renex 690, a nonionic detergent. The purified enzyme gave a single major band with a molecular weight of 79,000 daltons on SDS-polyacrylamide gel electrophoresis. FMN and FAD were present in about equal amounts. The most active reductase preparation catalyzed the reduction of 40.9 μmoles of cytochrome $\text{c}$ per min per mg of protein and, as an indirect measure of cytochrome P-450 reduction, the oxidation of 2.0 μmoles of NADPH per min per mg of protein in a reconstituted hydroxylation system containing benzphetamine as the substrate.     

Complete amino acid sequence of NADPH-cytochrome P-450 reductase from porcine hepatic microsomes

The complete amino acid sequence of porcine hepatic microsomal NADPH-cytochrome P-450 reductase has been determined by microsequence analysis on several sets of proteolytic fragments. Sequence studies were performed initially on a 20-kilodalton (kDa) fragment and then on 80-kDa fragment. The amino-terminal end of the mature protein was blocked with an acetyl group, followed by 676 amino acid residues. It has been revealed that the COOH-terminal 20-kDa fragment has been derived from original enzyme by cleavage at the Asn-Gly (residues 502-503) linkage by an unknown mechanism. An NADPH-protected cysteine residue is located at residue 565, near a region exhibiting high sequence homology with ferredoxin-NADP+ reductase. The FMN and FAD binding regions are possibly located in the amino-terminal region and the middle part of the protein molecule, respectively, as suggested by Porter and Kasper [Porter, T. D., & Kasper, C. B. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 973-977]. When this sequence is compared with that of rat enzyme, 60 amino acid residues are substituted, probably due to species differences. However, total sequence homology between these enzymes is 90%. Hydropathy plot analysis reveals that two regions from residues 27-43 and from residues 523-544 exhibit a high degree of hydrophobicity, suggesting membrane binding or interaction with cytochrome P-450.     

Molecular cloning and sequence analysis of full-length cDNA for rabbit liver NADPH-cytochrome P-450 reductase mRNA

The nucleotide sequence of the mRNA for NADPH-cytochrome P-450 reductase from rabbit liver was determined from a full-length cDNA clone (pFP105). The clone contains 2,269 nucleotides complementary to rabbit liver reductase mRNA. The single open reading frame of 2,037 nucleotides codes for a 679-amino acid polypeptide with a calculated molecular weight of 76,583 daltons. The cloned cDNA contains the complete 3'-noncoding region of 193 nucleotides, including 68 nucleotides of poly(A), and 39 nucleotides of the 5'-noncoding region. The nucleotide sequence in the coding region of cDNA of rabbit reductase (pFP105) showed 85% homology to that of rat reductase (Porter, T.D. & Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U.S. 82, 973-977, and Murakami, H. et al. (1986) DNA 5, 1-10). Rabbit reductase has one more amino acid residue than the rat enzyme, and the amino acid compositions of the two enzymes are similar. The amino acid sequence of the rabbit enzyme showed 91% identity with that of the rat enzyme. The segment related to binding of FMN and FAD was well conserved among rabbit, rat, and pig reductases. The sequence related to AMP moiety-binding was also conserved among these species, and was found in the amino acid sequence of NADH-cytochrome b5 reductase, another flavoenzyme in the microsomal electron transport system.     

Purification and some properties of NADPH-cytochrome P-450 reductase from crab-eating monkey liver microsomes

NADPH-cytochrome P-450 reductase was purified to 30.8 units/mg from monkey liver microsomes. The purified reductase showed one major protein band (78,000) and two minor ones (58,000 and 20,000) on analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Monkey, rat, and guinea pig reductases were not immunochemically identical to each other judged from Ouchterlony double diffusion analysis and immunotitration with regard to NADPH-cytochrome c reductase activity.     

Interaction of liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase in the presence and absence of lipid

Phospholipid has been reported to be necessary for optimal catalytic activity of a number of mammalian cytochrome P-450 (P-450) systems. We also confirm that a number of individual phospholipids and mixtures, used as soluble monomers or phospholipid vesicles, show activation of 7-ethoxycoumarin O-deethylase activity by an enzyme system composed of rat liver microsomal P-450PB-B and NADPH-P-450 reductase. However, by preincubating a mixture of P-450 and NADPH-P-450 reductase at high concentrations, optimal activity can be obtained in the absence of phospholipid. The catalytic activity of the complex formed is concentration dependent in the absence of lipid or in the presence of soluble lipid. The activity in phospholipid vesicles is optimal and concentration independent. The apparent Km for NADPH-P-450 reductase in P-450-dependent oxidation systems is lowered severalfold in the presence of phospholipid. The apparent Km for the P-450 substrate, 7-ethoxycoumarin, and the temperature dependence of 7-ethoxycoumarin O-deethylase activity were unaffected by the addition of phospholipid to a preformed complex of P-450PB-B and NADPH-P-450 reductase. The effect of lipid on a number of other P-450 isozymes was also examined and in no case did lipid enhance the catalytic activity of the preformed complex. These results lead to the conclusion that the major effect of phospholipids in P-450-based enzyme systems is the facilitation of an active P-450:NADPH-P-450 reductase complex. This is the first report that maximum P-450 supported monooxygenase activity can be obtained in the absence of phospholipid.     

Relationship between state of aggregation and catalytic activity for cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase

The detergent 1-O-n-octyl-beta-D-glucopyranoside (octylglucoside) was found to replace the phospholipid requirement in the demethylation of benzphetamine by cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase purified from phenobarbital-treated rabbit liver. At low enzyme concentration (0.1 microM) in the absence of glycerol and phosphate, the maximum rate of benzphetamine-specific NADPH oxidation was approximately 35% of that observed in the presence of dilauroylglyceryl-3-phosphoryl choline. At higher enzyme concentration (2.5 microM) and in the presence of 0.15 M phosphate, 20% glycerol, octylglucoside was as effective as phospholipid in stimulating the production of formaldehyde from benzphetamine. The detergent concentration required for maximal enzymatic activity was 2.5-4.0 g/liter, depending on the cytochrome preparation used. At higher octylglucoside concentrations (5-7 g/liter), activity decreased to zero, although neither enzyme appeared to be irreversibly denatured at these detergent concentrations. Sedimentation equilibrium experiments with P-450LM2 alone or in the presence of equimolar reductase showed that increasing octylglucoside levels promoted disaggregation of the cytochrome. Pentamers and hexamers predominated at detergent concentrations where maximal activity was observed, while higher levels of detergent where activity was absent produced cytochrome dimers and, ultimately, monomers. The reductase was monomeric at detergent levels between at least 3 and 7 g/liter. Moreover, both gel filtration and sedimentation equilibrium experiments demonstrated that a stable complex between P-450LM2 and its reductase was not formed at octylglucoside concentrations where high activity was evident. These results are consistent with a model of P-450/reductase interaction in which functional aggregates of three to six cytochrome polypeptides move laterally in the microsomal membrane and interact with the reductase by random collision.     

A rapid one-step purification of NADPH-cytochrome c (P-450) reductase from rat liver microsomes

NADPH-cytochrome c (P-450) reductase from liver microsomes of phenobarbital-treated rats has been purified in a single step by affinity chromatography on agarose-hexane-adenosine 2',5'-diphosphate. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme assay, and radioimmunoassay the protein obtained by this single step procedure is as pure as that isolated by multicolumn procedures.     

Modification of carboxyl groups on NADPH-cytochrome P-450 reductase involved in binding of cytochromes c and P-450 LM2

Carboxyl groups of NADPH-cytochrome P-450 reductase have been modified with the water-soluble carbodiimide EDC. Although there is no significant loss in DCPIP reduction the activity with cytochrome c and cytochrome P-450 LM2 as electron acceptors was inhibited by about 60 and 85%, respectively (1 h incubation time, 20 mM EDC). The inactivation by EDC was nearly completely prevented in the presence of cytochrome P-450 LM2, but not by bovine serum albumin. These results and crosslinking studies suggest that carboxyl groups of NADPH-cytochrome P-450 reductase are involved in charge-pair interactions to cytochrome c and to at least two amino groups of cytochrome P-450 LM2.     

Purification and characterization of NADPH-cytochrome P-450 reductase from rat epidermis

NADPH-cytochrome P-450 oxidoreductase (P-450 red) transfers reducing equivalents from NADPH to cytochrome P-450 (P-450) in the monooxygenase system. Detergent solubilized proteins from the membrane fraction of neonatal rat epidermis were purified by 2′,5′-ADP-agarose affinity column chromatography. The purified protein showed an apparent homogeneity on sodium dodecylsulfate-polyacrylamide gel electrophoresis and molecular weight was estimated to be 78 kDa. NADPH-cytochrome c reductase activity increased by 95-fold in the purified enzyme. Epidermal P-450 red in vitro reconstituted benzo(a)pyrene hydroxylase activity in a dose dependent manner with P-450 purified from either rat liver or epidermis. Western blot analysis demonstrated that epidermal P-450 red immunologically cross reacts to liver P-450 red. Immunohistochemical staining showed that the enzyme was predominantly localized in the epidermis. The intensity of immunohistochemical staining of rat skin sections and tissue distribution did not change in the skin treated with β-naphtoflavone, which results in a substantial increase in P-450 1A1 activity. Quantitative assessment of P-450 red in treated and untreated epidermis also showed no change. These findings indicate that constitutive P-450 red, fully capable of supporting P-450, exists in rat epidermis, and can function in metabolism of endogenous and exogenous compounds.     

Preparation and characterization of FAD-dependent NADPH-cytochrome P-450 reductase

NADPH-cytochrome P-450 reductase releases FAD upon dilution into slightly acidic potassium bromide. Chromatography on high performance hydroxylapatite resolved the FAD-dependent reductase from holoreductase. The FAD dependence was matched by a low FAD content, with the ratio of FAD to FMN as low as 0.015. The aporeductase had negligible activity toward cytochrome c, ferricyanide, menadione, dichlorophenolindophenol, nitro blue tetrazolium, and an analogue of NADP, acetylpyridine adenine dinucleotide phosphate. A 4-min incubation in FAD reconstituted from one-half to all of the enzyme activity, as compared to the untreated reductase, depending upon the substrate. After a 2-h reconstitution, the reductase eluted from hydroxylapatite at the same location in the elution profile as did the untreated holoreductase. The reconstituted reductase had little flavin dependence, was nearly equimolar in FMN and FAD, and had close to the specific activity, per mol of flavin, of untreated reductase. The dependence upon FAD implies that FMN is not a competent electron acceptor from NADPH. Thus, the FAD site must be the only point of electron uptake from NADPH.     

NADPH-cytochrome P-450 reductase from rat liver: purification by affinity chromatography and characterization.

(NADPH)-cytochrome P-450 reductase was purified to apparent homogeneity by a procedure utilizing nicotinamide adenine dinucleotide phosphate (NADP)-Sepharose affinity column chromatography. The purified flavoprotein has a molecular weight of 79 700 and catalyzes cytochrome P-450 dependent drug metabolism, as well as reduction of exogenous electron acceptors. Aerobic titration of cytochrome P-450 reductase with NADPH indicates that an air-stable reduced form of the enzyme is generated by the addition of 0.5 mol of NADPH per mole of flavin, as judged by spectral characteristics. Further addition of NADPH causes no other changes in the absorbance spectrum. A Km value for NADPH of 5 micron was observed when either cytochrome P-450 or cytochrome c was employed as electron acceptor. A Km value of 8 +/- 2 micron was determined for cytochrome c and a Km of 0.09 +/- 0.01 micron was estimated for cytochrome P-450.     

Interaction between cytochrome P-450 (P-450C21) and NADPH-cytochrome P-450 reductase from adrenocortical microsomes in a reconstituted system

The interaction between P-450C21 and NADPH-cytochrome P-450 reductase, both purified from bovine adrenocortical microsomes, has been investigated in a reconstituted system with a nonionic detergent, Emulgen 913, by kinetic analysis and gel filtrations. Steady state kinetic data in progesterone 21-hydroxylation showed formation of an equimolar complex between the two enzyme proteins at low Emulgen concentration. Steady state kinetic studies on the electron transfer from NADPH to P-450C21 via the reductase showed that a stable complex formation between the two enzyme proteins was not involved in the steady state electron transfer at high Emulgen concentration. In stopped flow experiments, a time course of the P-450C21 reduction showed biphasic kinetics composed of fast and slow phases. The dependence of kinetic parameters on Emulgen concentration indicates that the fast phase corresponds to the electron transfer within the complex and the slow phase to the electron transfer through a random collision between P-450C21 and the reductase. The stable complex formation between P-450C21 and the reductase has been clearly demonstrated by gel filtration. The stable complex was composed of several molecules of the two enzyme proteins at an equimolar ratio, which was active for progesterone 21-hydroxylation and had a tendency to dissociate at high Emulgen concentration.