Rabbit liver microsomal lipid peroxidation. The effect of lipid on the rate of peroxidation

Rat and rabbit liver microsomes catalyze an NADPH-cytochrome P-450 reductase-dependent peroxidation of endogenous lipid in the presence of the chelate, ADP-Fe3+. Although liver microsomes from both species contain comparable levels of NADPH-cytochrome P-450 reductase and cytochrome P-450, the rate of lipid peroxidation (assayed by malondialdehyde and lipid hydroperoxide formation) catalyzed by rabbit liver microsomes is only about 40% of that catalyzed by rat liver microsomes. Microsomal lipid peroxidation was reconstituted with liposomes made from extracted microsomal lipid and purified protease-solubilized NADPH-cytochrome P-450 reductase from both rat and rabbit liver microsomes. The results demonstrated that the lower rates of lipid peroxidation catalyzed by rabbit liver microsomes could not be attributed to the specific activity of the reductase. Microsomal lipid from rabbit liver was found to be much less susceptible to lipid peroxidation. This was due to the lower polyunsaturated fatty acid content rather than the presence of antioxidants in rabbit liver microsomal lipid. Gas-liquid chromatographic analysis of fatty acids lost during microsomal lipid peroxidation revealed that the degree of fatty acid unsaturation correlated well with rates of lipid peroxidation.     

Isolation and properties of reduced nicotinamide adenine dinucleotide phosphate-hepatoredoxin reductase of rabbit liver mitochondria.

An NADPH-hepatoredoxin reductase was purified from mitochondria of rabbit hepatocytes. The optical absorption spectrum showed a typical flavoprotein. The NADPH-hepatoredoxin reductase has an FAD as a coenzyme and the molecular weight of the NADPH-hepatoredoxin reductase was estimated to be 51000 by SDS-polyacrylamide gel electrophoresis. The NADPH-hepatoredoxin reductase was immunochemically similar to NADPH-adrenodoxin reductase of bovine and pig adrenocortical mitochondria, but not NADPH-cytochrome P-450 reductase of rabbit liver microsomes. The NADPH-cytochrome c reductase activity of the NADPH-hepatoredoxin reductase and hepatoredoxin complex, unlike NADPH-cytochrome P-450 reductase, was decreased by increasing ionic strength.     

Purification and characterization of NADPH-cytochrome c reductase from porcine polymorphonuclear leukocytes

NADPH-cytochrome c reductase [NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4] was highly purified from the membrane fraction of porcine polymorphonuclear leukocytes by column chromatographies on DEAE cellulose DE-52, 2',5'-ADP-agarose, Sephacryl S-300, and Bio-gel HTP. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, the purified preparation gave a main band with a molecular weight of 80,000. The enzyme contained 0.79 mol of FAD and 0.88 mol of FMN per mol, and was capable of exhibiting a benzphetamine N-demethylation activity in the presence of cytochrome P-450 purified from rabbit liver microsomes and dilauroylphosphatidylcholine, as is the case with liver NADPH-cytochrome P-450 reductase. The cytochrome c reductase activity of the polymorphonuclear leukocytes (PMN) enzyme was precipitated with rabbit anti-guinea pig liver NADPH-cytochrome P-450 reductase IgG followed by addition of guinea pig anti-rabbit IgG antibody. The biochemical and immunological properties of the PMN enzyme so far examined were similar to those of the liver enzyme, although its function in leukocytes has not yet been determined.     

The effects of phosphatase on the components of the cytochrome P-450-dependent microsomal monooxygenase

Incubation of rabbit liver microsomes with alkaline phosphatase resulted in a marked decrease of NADPH-dependent monooxygenase activities. This decrease was found to be correlated with the decrease of NADPH-cytochrome c reductase activity catalyzed by NADPH-cytochrome P-450 reductase. Neither the content of cytochrome P-450, as determined from its CO difference spectrum, nor the peroxide-supported demethylase activity catalyzed by cytochrome P-450 alone was affected by the phosphatase treatment. NADH-cytochrome b5 reductase and cytochrome b5 were not affected by the phosphatase either. NADPH-cytochrome P-450 reductase purified from rabbit liver microsomes lost its NADPH-dependent cytochrome c reductase activity upon incubation with phosphatase in a way similar to that of microsome-bound reductase. Flavin analysis showed that the phosphatase treatment caused a decrease of FMN with concomitant appearance of riboflavin. Alkaline phosphatase, therefore, inactivates the reductase by attacking its FMN, and the inactivation of the reductase, in turn, leads to a decrease of the microsomal monooxygenase activities.     

NADPH-cytochrome P-450 reductase is not a component of the liver microsomal steroid 5-alpha reductase

Liver microsomal steroid 5-alpha-reduction is catalyzed by a NADPH-dependent enzyme system. The requirement of NADPH-cytochrome P-450 reductase to shuttle reduction equivalents from NADPH to steroid 5-alpha-reductase was investigated using an inhibitory antibody against NADPH-cytochrome P-450 reductase. This antibody preparation inhibited cytochrome c reduction in microsomes from female rat liver with an I50 of 0.75 mg antibody/mg of microsomal protein. Benzphetamine N-demethylation and testosterone 6-beta-hydroxylation, two cytochrome P-450-mediated oxidative reactions, were inhibited by the antibody. On the other hand, testosterone 5-alpha-reductase was not affected by the antibody. These results suggest that NADPH-cytochrome P-450 reductase is not an obligatory component of the liver microsomal steroid 5-alpha-reduction.     

The rabbit pulmonary monooxygenase system. Immunochemical and biochemical characterization of enzyme components.

The enzymatic components of the rabbit pulmonary monooxygenase system, cytochromes P-450I and P-450II and NADPH-cytochrome P-450 reductase, are immunochemically distinct proteins. In pulmonary microsomes, the N-demethylation of benzphetamine, amino-pyrine, and ethylmorphine, and the O-deethylation of 7-ethoxycoumarin are dependent only on cytochrome P-450I, and the hydroxylation of coumarin is apparently catalyzed by both cytochromes. Cytochrome P-450II is immunochemically distinct from the major forms of hepatic cytochrome P-450 induced by phenobarbital or 3-methylcholanthrene, whereas cytochrome P-450I is indistinguishable from the former on the basis of physical and catalytic as well as immunochemical characteristics. Pulmonary and hepatic NADPH-cytochrome P-450 reductases also have identical physical, catalytic, and immunochemical properties. The lack of response of the lung monooxygenase system to phenobarbital, therefore, is apparently not due to an inability of the lung to synthesize the enzymes induced by phenobarbital in the liver. The relatively high proportion of cytochrome P-450I in the lung appears to be responsible for the higher rates (per nmol of P-450) of N-demethylation that have been observed in rabbit pulmonary as compared to hepatic microsomal fractions.     

Catalytic properties of cytochrome P-450 from human liver

A partially purified cytochrome P-450 fraction was prepared from the microsomal fraction of human liver. When combined with NADPH, a synthetic phospholipid and NADPH-cytochrome P-450 reductase from rat liver, the cytochrome P-450 fraction from human liver was able to catalyze the following hydroxylations: 11- and 12-hydroxylation of laurate, 12α- and 26-hydroxylation of 5β-cholestane-3α,7α-diol, 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol, and 6β-hydroxylation of androstenedione and progesterone. It was shown that the rate of 11- and 12-hydroxylation of laurate was linear with increasing amounts of cytochrome P-450 and with time in the presence of excess NADPH-cytochrome P-450 reductase and the phospholipid. In the presence of a fixed amount of cytochrome P-450 and the phospholipid, the rate of 11- and 12-hydroxylation increased with increasing concentrations of NADPH-cytochrome P-450 reductase up to a certain level and then remained constant. The requirement of the phospholipid could be increased markedly by centrifugation of the cytochrome P-450 fraction at 100,000g just prior to incubation. It is concluded that cytochrome P-450 from human liver is similar to previously studied cytochrome P-450 from rat liver with respect to catalytic properties and mechanism of reaction.     

Interaction between cytochrome P-448 and NADP-cytochrome P-450 reductase in reconstituted microsomal membranes

The regularities of changes in the functional activity of the microsomal monooxygenase system reconstituted by self-assembly from intact rat liver microsomes solubilized with 4% sodium cholate were studied at variable levels of NADPH-cytochrome P-450 reductase and the 3-methylcholanthrene-induced form of cytochrome P-450. Using antibodies against cytochrome P-448, the role of cytochrome P-448 in the overall reaction of benzopyrene hydroxylation induced in the microsomal membrane by a set of molecular forms of cytochrome P-450 was investigated. The effect of NADPH-cytochrome P-450 reductase and cytochrome P-448 incorporation into reconstituted microsomal membranes on benzpyrene metabolism suggests that in intact microsomal membranes benzopyrene metabolism induced by different forms of cytochrome P-450, with the exception of P-448, is limited by reductase is not the limiting component; however, cytochrome P-448 reveals its maximum activity at the cytochrome to reductase optimal molar ratio of 5:1; above this level, the catalytic activity of cytochrome P-448 is lowered.     

Effect of monooxygenase reactions catalyzed by cytochrome P-450 on the microsomal membrane

Hydroxylation of dimethylaniline in rabbit liver microsomes is accompanied by inactivation of cytochrome P-450 and the formation of products inhibiting the catalytic activity of non-inactivated cytochrome P-450. Other enzymes and electron carriers of microsomal membrane (cytochrome b5, NADH-ferricyanide reductase, NADPH-cytochrome c and NADPH-cytochrome P-450 reductases) as well as glucose-6-phosphatase were not inactivated in the course of the monooxygenase reactions. Phospholipids and microsomal membrane proteins were also unaffected thereby. Consequently, the changes in the microsomal membrane during cytochrome P-450 dependent monooxygenase system functioning are confined to the inactivation of cytochrome P-450.     

Rotation of cytochrome P-450. Complex formation of cytochrome P-450 with NADPH-cytochrome P-450 reductase in liposomes demonstrated by combining protein rotation with antibody-induced cross-linking

Purified rat liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase were co-reconstituted in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles by a cholate dialysis technique. Rotational diffusion of cytochrome P-450 was measured by detecting the decay of absorption anisotropy r(t), after photolysis of the heme X CO complex by a vertically polarized laser flash. All cytochrome P-450 was found to be rotationally mobile when co-reconstituted with equimolar amounts of NADPH-cytochrome P-450 reductase in lipid to cytochrome P-450 ((L/P450)) = 1 (w/w] vesicles. Antibodies against NADPH-cytochrome P-450 reductase were raised. Their specificity was demonstrated by Ouchterlony double diffusion analysis. Antireductase Fab fragments were prepared from antireductase IgG by papain digestion. The N-demethylation of benzphetamine, catalyzed by the proteoliposomes, was significantly inhibited by antireductase IgG and by antireductase Fab fragments. Cross-linking of NADPH-cytochrome P-450 reductase by antireductase IgG resulted in complete immobilization of cytochrome P-450 in L/P450 = 1 vesicles. Antireductase IgG also immobilized cytochrome P-450 in L/P450 = 5 vesicles, although the degree of immobilization was slightly smaller. No immobilization of cytochrome P-450 in L/P450 = 1 vesicles was detected in the presence of antireductase Fab fragments or preimmune IgG. These results further support the proposal of the formation of monomolecular complexes between cytochrome P-450 and NADPH-cytochrome P-450 reductase in liposomal membranes (Gut, J., Richter, C., Cherry, R.J., Winterhalter, K.H., and Kawato, S. (1982) J. Biol. Chem. 257, 7030-7036).     

Physicochemical properties of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase from bovine adrenocortical microsomes.

Adrenocortical NADPH-cytochrome P-450 reductase (EC. 1.6.2.4) was purified from bovine adrenocortical microsomes by detergent solubilization and affinity chromatography. The purified cytochrome P-450 reductase was a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, being electrophoretically homogeneous and pure. The cytochrome P-450 reductase was optically a typical flavoprotein. The absorption peaks were at 274, 380 and 45 nm with shoulders at 290, 360 and 480 nm. The NADPH-cytochrome P-450 reductase was capable of reconstituting the 21-hydroxylase activity of 17 alpha-hydroxyprogesterone in the presence of cytochrome P-45021 of adrenocortical microsomes. The specific activity of the 21-hydroxylase of 17 alpha-hydroxyprogesterone in the reconstituted system using the excess concentration of the cytochrome P-450 reductase, was 15.8 nmol/min per nmol of cytochrome P-45021 at 37 degrees C. The NADPH-cytochrome P-450 reductase, like hepatic microsomal NADPH-cytochrome P-450 reductase, could directly reduce the cytochrome P-45021. The physicochemical properties of the NADPH-cytochrome P-450 reductase were investigated. Its molecular weight was estimated to be 80 000 +/- 1000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical ultracentrifugation. The cytochrome P-450 reductase contained 1 mol each FAD and FMN as coenzymes. Iron, manganese, molybdenum and copper were not detected. The Km values of NADPH and NADH for the NADPH-cytochrome c reductase activity and those of cytochrome c for the activity of NADPH-cytochrome P-450 reductase were determined kinetically. They were 5.3 microM for NADPH, 1.1 mM for NADH, and 9-24 microM for cytochrome c. Chemical modification of the amino acid residues showed that a histidyl and cysteinyl residue are essential for the binding site of NADPH of NADPH-cytochrome P-450 reductase.     

Role of a hydrophobic polypeptide in the N-terminal region of NADPH-cytochrome P-450 reductase in complex formation with P-450LM.

Detergent-solubilized liver microsomal NADPH-cytochrome P-450 reductase is known to retain the ability to catalyze electron transfer to cytochrome P-450, whereas the trypsin-solubilized reductase does not. In the present study, treatment of the highly purified detergent-solubilized rabbit liver enzyme (m.w. 77,700) with trypsin was shown to yield a small peptide (m.w. 6,100) as well as the large peptide (m.w. 71,200) which retains the flavin prosthetic groups. The small peptide, which is hydrophobic in nature as shown by its amino acid composition and solubility properties, is apparently the moiety in the native reductase involved in binding to cytochrome P-450 and to the microsomal membrane. The C-terminal amino acid sequences of the native reductase and large fragment are identical [-Trp-(Leu, Val)-Asp-Ser-COOH], thereby indicating that the hydrophobic peptide is located in the N-terminal region of the enzyme.     

A form of rabbit liver cytochrome P-450 that catalyzes the 7 alpha-hydroxylation of cholesterol

A form of cytochrome P-450 which comigrates with cytochrome P-450LM4 (molecular weight, 55 000) on SDS-polyacrylamide gel was purified from liver microsomes of cholestyramine-treated rabbits. This form of cytochrome P-450 catalyzed the 7 alpha-hydroxylation of cholesterol with an activity of 37.5 pmol/min per nmol cytochrome P-450 in the reconstituted enzyme system containing cytochrome P-450 and NADPH-cytochrome P-450 reductase. The substrate specificity of this form of cytochrome P-450 was compared with cytochrome P-450LM4 isolated from phenobarbital- and beta-naphthoflavone-treated rabbit liver microsomes. The latter two isoenzymes do not catalyze 7 alpha-hydroxylation of cholesterol, but are more active in O-deethylation of 7-ethoxycoumarin and p-nitrophenetole. Ouchterlony double diffusion revealed cross-reactivity between anti-P-450LM4 (phenobarbital) IgG and cytochrome P-450 isolated from cholestyramine- or beta-naphthoflavone-treated rabbit liver microsomes. A two-dimensional iodinated tryptic peptide fingerprint indicated only minor structural differences among these three cytochrome P-450LM4 preparations.     

The effect of some biphenyl solubilizing and suspending agents on biphenyl 4-hydroxylase of rabbit liver microsomes

The effects of ethanol, acetone, dimethylsulfoxide (DMSO), polyoxyethylene sorbitan monooleate (Tween 80), polyoxyethylene sorbitan monolaurate (Tween 20), Triton X-100, and carboxymethyl cellulose (CMC) on the kinetics of biphenyl 4-hydroxylase of rabbit liver microsomes were investigated in an attempt to find a substrate-solubilizing or suspending agent (carrier) which was itself a non-effector of the mixed-function oxidase. The effects of these carriers on the activities of NADPH-cytochrome P-450 reductase, NADPH-cytochrome c reductase, and cytochrome P-450 content were also investigated.Ethanol and DMSO inhibited biphenyl 4-hydroxylase and NADPH-cytochrome P-450 reductase. Acetone inhibited the hydroxylase uncompetitively at concentrations which appeared to stimulate NADPH-cytochrome P-450 reductase. All of the detergents inhibited biphenyl 4-hydroxylase although only Triton X-100 markedly affected the reduction of cytochrome P-450. The interaction of Tween 80 with the hydroxylase gave rise to non-linear Lineweaver-Burk plots although at high concentrations of biphenyl or low concentrations of the detergent the inhibition appeared to be competitive.Biphenyl caused a 2–3-fold stimulation of NADPH-cytochrome P-450 reductase, but in the presence of Tween 80 the stimulation was absent. Since V of biphenyl 4-hydroxylase in the presence of Tween 80 was not significantly different from V in its absence it would appear that the reduction of cytochrome P-450 was not ratelimiting.Of all the carriers studied only CMC was without effect on all aspects of microsomal electron transport investigated. As far as biphenyl 4-hydroxylase is concerned, CMC appears to be the most suitable substrate carrier.     

Studies on the rate-determining factor in testosterone hydroxylation by rat liver microsomal cytochrome P-450: evidence against cytochrome P-450 isozyme:isozyme interactions

The aim of the present study was to examine a recent proposal that inhibitory isozyme:isozyme interactions explain why membrane-bound isozymes of rat liver microsomal cytochrome P-450 exert only a fraction of the catalytic activity they express when purified and reconstituted with saturating amounts of NADPH-cytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine. The different pathways of testosterone hydroxylation catalyzed by cytochromes P-450a (7 alpha-hydroxylation), P-450b (16 beta-hydroxylation), and P-450c (6 beta-hydroxylation) enabled possible inhibitory interactions between these isozymes to be investigated simultaneously with a single substrate. No loss of catalytic activity was observed when purified cytochromes P-450a, P-450b, or P-450c were reconstituted in binary or ternary mixtures under a variety of incubation conditions. When purified cytochromes P-450a, P-450b, and P-450c were reconstituted under conditions that mimicked a microsomal system (with respect to the absolute concentration of both the individual cytochrome P-450 isozyme and NADPH-cytochrome P-450 reductase), their catalytic activity was actually less (69-81%) than that of the microsomal isozymes. These results established that cytochromes P-450a, P-450b, and P-450c were not inhibited by each other, nor by any of the other isozymes in the liver microsomal preparation. Incorporation of purified NADPH-cytochrome P-450 reductase into liver microsomes from Aroclor 1254-induced rats stimulated the catalytic activity of cytochromes P-450a, P-450b, and P-450c. Similarly, purified cytochromes P-450a, P-450b, and P-450c expressed increased catalytic activity in a reconstituted system only when the ratio of NADPH-cytochrome P-450 reductase to cytochrome P-450 exceeded that normally found in liver microsomes. These results indicate that the inhibitory cytochrome P-450 isozyme:isozyme interactions described for warfarin hydroxylation were not observed when testosterone was the substrate. In addition to establishing that inhibitory interactions between different cytochrome P-450 isozymes is not a general phenomenon, the results of the present study support a simple mass action model for the interaction between membrane-bound or purified cytochrome P-450 and NADPH-cytochrome P-450 reductase during the hydroxylation of testosterone.     

Hepatic mitochondrial cytochrome P-450 system. Purification and characterization of two distinct forms of mitochondrial cytochrome P-450 from beta-naphthoflavone-induced rat liver

We have purified two distinct isoforms of mitochondrial cytochrome P-450 from beta-naphthoflavone (beta-NF)-induced rat liver to greater than 85% homogeneity and characterized their molecular and catalytic properties. One of these isoforms showing an apparent molecular mass of 52 kDa is termed P-450mt1 and the second isoform with 54-kDa molecular mass is termed P-450mt2. Cytochrome P-450mt2 comigrates with similarly induced microsomal P-450c (the major beta-NF-inducible form) on sodium dodecyl sulfate-polyacrylamide gels and cross-reacts with polyclonal antibody monospecific for cytochrome P-450c. Cytochrome P-450mt2, however, represents a distinct molecular species since it failed to react with a monoclonal antibody to P-450c and produced V8 protease fingerprints different from P-450c. Cytochrome P-450mt1, on the other hand, did not show any immunochemical homology with P-450c or P-450mt2 as well as partially purified P-450 from control mitochondria. Electrophoretic comparisons and Western blot analysis show that both P-450mt1 and P-450mt2 are induced forms not present in detectable levels in control liver mitochondria. A distinctive property of mitochondrial P-450mt1 and P-450mt2 was that their catalytic activities could be reconstituted with both NADPH-cytochrome P-450 reductase as well as mitochondrial specific ferredoxin and ferredoxin reductase electron transfer systems, while P-450c showed exclusive requirement for NADPH-cytochrome P-450 reductase. Cytochromes P-450mt1 and P-450mt2 were able to metabolize xenobiotics like benzo(a)pyrene and dimethyl benzanthracene at rates only one-tenth with cytochrome P-450c. Furthermore, P-450mt1, P-450mt2, as well as partially purified P-450 from control liver, but not P-450c, showed varying activities for 25- and 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3. These results provide evidence for the presence of at least two distinct forms of beta-NF-inducible cytochrome P-450 in rat hepatic mitochondria.     

Subfractionation of rat liver microsomes by immunoprecipitation and immunoadsorption methods.

Rabbit antisera were prepared against cytochrome b5 and NADPH-cytochrome c reductase [EC 1.6.2.4] purified from rat liver microsomes, and utilized in examining the distribution of these and other membrane-bound enzymes among the vesicles of rat liver microsomal preparations by immunoprecipitation and immunoadsorption methods. Smooth microsomes with an average vesicular size of 200 nm (diameter) and sonicated smooth microsomes with an average diameter of 40-60 nm were used in subfractionation experiments. Immunoprecipitation of microsomal vesicles with anti-cytochrome b5 immunoglobulin failed to show any separation of the microsomes into fractions having different enzyme compositions. Cytochrome b5 was apparently distributed among all vesicles even when sonicated microsomes were used. When the antibody against NADPH-cytochrome c reductase was used, however, immunoadsorption of microsomes on Sepharose-bound antibody produced some separation of NADPH-cytochrome c reductase and cytochrome P-450 from NADH-cytochrome b5 reductase and cytochrome b5. The separation was more pronounced when sonicated microsomes were used. These results indicate microheterogeneity of the microsomal membrane, and suggest the clustering of NADPH-cytochrome c reductase and cytochrome P-450 molecules in the membrane.     

Studies on the microsomal mixed function oxidase system: redox properties of detergent-solubilized NADPH-cytochrome P-450 reductase.

Hepatic microsomal NADPH-cytochrome P-450 reductase was solubilized from rabbit liver microsomes in the presence of detergents and purified to homogeneity by column chromatography. The purified reductase had a molecular weight of 78 000 and contained 1 mol each of FAD and FMN per mol of enzyme. On reduction with NADPH in the presence of molecular oxygen, an 02-stable semiquinone containing one flavin free radical per two flavins was formed, in agreement with previous work on purified trypsin-solubilized reductase. The reduction of oxidized enzyme by NADPH, and autoxidation of NADPH-reduced enzyme by air, proceeded by both one-electron equivalent and two-electron equivalent mechanisms. The reductase reduced cytochrome P-450 (from phenobarbital-treated rabbits) and cytochrome P-448 (from 3-methylcholanthrene-treated rabbits). The rate of reduction of cytochrome P-450 increased in the presence of a substrate, benzphetamine, but that of cytochrome P-448 did not.     

Hydroxylation of prostaglandin A1 by the microsomes of rabbit intestinal mucosa

Microsomes from rabbit small intestine mucosa were found to catalyze the hydroxylation of PGA1 in the presence of NADPH. The major product was identified as 20-hydroxy PGA1 by using high performance liquid chromatography and gas chromatography-mass spectrometry, and the minor product was assumed to be 19-hydroxy PGA1. The ratio of the former product to the latter was about 24.1. The specific PGA1 omega-hydroxylase activity of small intestine microsomes was comparable to that of liver microsomes, and was significantly higher than those of microsomes from other tissues such as kidney cortex and lung. Microsomes from rabbit colon mucosa also catalyzed the hydroxylation of PGA1 in the presence of NADPH, with the ratio of omega- to (omega-1)-hydroxy PGA1 formed being 33.0. The PGA1 hydroxylase activities of the microsomes from both small intestine and colon were inhibited markedly by carbon monoxide, indicating the participation of cytochrome P-450. A cytochrome P-450 was solubilized from small intestine microsomes, and purified to a specific content of 10.5 nmol of cytochrome P-450/mg of protein. This cytochrome hydroxylated PGA1 at the omega-position with a turnover rate of 38.2 nmol/min/nmol of cytochrome P-450 in the reconstituted system containing cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5 and phosphatidylcholine. It is suggested that this cytochrome P-450 is specialized for the omega-hydroxylation of PGA1 in small intestine microsomes.     

Involvement of FMN and phenobarbital cytochrome P-450 in stimulating a one-electron reductive denitrosation of 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea catalyzed by NADPH-cytochrome P-450 reductase

Purified hepatic NADPH-cytochrome P-450 reductase, which was reconstituted with dilauroylphosphatidylcholine, catalyzed a one-electron reductive denitrosation of 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)-1-nitrosourea ([14C]CCNU) to give 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)urea at the expense of NADPH. Ambient oxygen or anoxic conditions did not alter the rates of [14C]CCNU denitrosation catalyzed by NADPH-cytochrome P-450 reductase with NADPH. Electron equivalents for reduction could be supplied by NADPH or sodium dithionite. However, the turnover number with NADPH was slightly greater than with sodium dithionite. Enzymatic denitrosation with sodium dithionite or NADPH was observed in anaerobic incubation mixtures which contained NADPH-cytochrome P-450 reductase with or without cytochrome P-450 purified from livers of phenobarbital (PB)-treated rats; PB cytochrome P-450 alone did not support catalysis. PB cytochrome P-450 stimulated reductase activity at molar concentrations approximately equal to or less than NADPH-cytochrome P-450 reductase concentration, but PB cytochrome P-450 concentrations greater than NADPH-cytochrome P-450 reductase inhibited catalytic denitrosation. Cytochrome c, FMN, and riboflavin demonstrated different degrees of stimulation of NADPH-cytochrome P-450 reductase-dependent denitrosation. Of the flavins tested, FMN demonstrated greater stimulation than riboflavin and FAD had no observable effect. A 3-fold stimulation by FMN was not observed in the absence of NADPH-cytochrome P-450 reductase. These studies provided evidence which establish NADPH-cytochrome P-450 reductase rather than PB cytochrome P-450 as the enzyme in the hepatic endoplasmic reticulum responsible for CCNU reductive metabolism.