The cytochromes in microsomal fractions of germinating mung beans.

Detailed studies of microsomal cytochromes from mung-bean radicles showed the presence of cytochrome P-420, particularly in dark-grown seedlings, accompanied by smaller quantities of cytochrome P-450. Similar proportions of cytochrome P-420 to cytochrome P-450 were found spectrophotometrically in vivo with whole radicles and hypocotyls. Assayed in vitro, maximum concentrations of both cytochromes were attained after 4 days of growth, before undergoing rapid degradation. Illumination of seedlings stabilized cytochrome P-450 and decreased the amount of cytochrome P-420. Three b cytochromes were present in the microsomal fraction, namely cytochromes b-562.5 (Em + 105 +/- 23 mV), b-560.5 (Em + 49 +/- 13 mV) and b5 (Em - 45 +/- 14 mV), all at pH 7.0. Of the b cytochromes, cytochrome b5 alone undergoes a rapid degradation after day 4, Changes in cytochrome b concentrations were confined to the microsomal fraction: mitochondrial b cytochrome concentrations were unaltered with age. Protohaem degradation (of exogenous methaemalbumin) was detected in microsomal fractions of mung beans. The rates of degradation were highest in extracts of young tissue and declined after day 4. The degradation mechanism and products did not resemble those of mammalian haem oxygenase.     

The effect of exogenous δ-aminolaevulinate on rat liver haem and cytochromes

The activity of delta-aminolaevulinate synthetase is generally regarded as rate-limiting for hepatic haem biosynthesis. It has been suggested that cytochrome synthesis may also be regulated by changes in delta-aminolaevulinate synthetase activity. This hypothesis was studied by injecting product, delta-aminolaevulinate, into adult rats over a 4-240h period. The concentrations of hepatic mitochondrial cytochromes a, b, c and c(1) were unchanged by treatment with delta-aminolaevulinate, allylisopropylacetamide or phenobarbital. In control animals, total microsomal haem content equalled the sum of cytochromes b(5) plus P-450. After delta-aminolaevulinate administration the total amount of microsomal haem, measured as the pyridine haemochromogen, exceeded these components, indicating the formation of a ;free' haem pool. Haem synthesis does not appear rate-limiting for hepatic cytochrome synthesis in the adult rat.     

Site-Directed Mutagenesis of Cytochrome P450scc. II. Effect of Replacement of the Arg425 and Arg426 Residues on the Structural and Functional Properties of the Cytochrome P450scc

Cytochrome P450-dependent monooxygenases, in spite of their wide distribution, can be simply divided into a few groups differing in the location of the electron transfer chain and their composition. The two main groups of cytochrome P450-dependent monooxygenases are the mitochondrial and microsomal enzymes. While in two-component microsomal cytochrome P450-dependent monooxygenases electrons are supplied to cytochrome P450 by a flavoprotein (NADPH-cytochrome P450 reductase), in three-component mitochondrial monooxygenases the electrons are supplied to cytochrome P450 by a low molecular weight protein (ferredoxin). The interaction of cytochrome P450 with NADPH-cytochrome P450 reductase and ferredoxin is the subject of intensive studies. Using chemical modification, chemical cross-linking, and sitedirected mutagenesis, we identified surface exposed positively charged residues of cytochrome P450scc which might be important for interaction with adrenodoxin. Theoretical analysis of the distribution of surface electrostatic potential in cytochrome P450 indicates that in contrast to microsomal monooxygenases, cytochromes P450 of mitochondrial type, and cholesterol side-chain cleavage cytochrome P450 (P450scc) in part, carry on the proximal surface an evidently positively charged site that is formed by residues Arg425 and Arg426. In the present work, to estimate the functional role of Arg425 and Arg426 of cytochrome P450scc, we used site-directed mutagenesis to replace these residues with glutamine. The results indicate that residues Arg425 and Arg426 are involved in the formation of a heme-binding center and electrostatic interaction of cytochrome P450scc with its physiological electron-transfer partner, adrenodoxin.     

Evidence that in chick embryos destruction of hepatic microsomal cytochrome P-450 haem is a general mechanism of induction of delta-aminolaevulinate synthase by porphyria-causing drugs.

A variety of prophyrinogenic compounds were tested for their effect in ovo on chick-embryo liver microsomal cytochrome P-450 haem concentration and mitochondrial delta-aminolaevulinate synthase activity. With all drugs tested, there was a 30--50% decrease in cytochrome P-450 haem concentration within 1 h of treatment, and this was closely followed by an increase in delta-aminolaevulinate synthase activity. The relationship was independent of the extent of enzyme induction and is consistent with the proposal that drug-mediated destruction of cytochrome P-450 haem is the primary mechanism of induction of delta-aminolaevulinate synthase. After induction, synthesis of delta-aminolaevulinate synthase could be maintained by inhibiting further haem synthesis. These studies suggest that induction of porphyria is a combination of two distinct processes: (a) induction of delta-aminolaevulinate synthase synthesis by destruction of cytochrome P-450 haem and consequent depletion of cellular free haem; (b) maintenance of continued delta-aminolaevulinate synthase synthesis by preventing replenishment of cellular haem either by inhibiting haem synthesis and/or by promoting continuous removal of newly synthesized haem.     

Thermodynamic properties of oxidation-reduction reactions of bacterial, microsomal, and mitochondrial cytochromes P-450: an entropy-enthalpy compensation effect

An optically transparent thin-layer electrode cell with a very small volume was used for determination of the formal reduction potentials of bacterial, microsomal, and mitochondrial cytochromes P-450. At an extrapolated zero concentration of dye, the bacterial cytochrome from Pseudomonas putida catalyzing the hydroxylation of camphor and the adrenal mitochondrial cytochrome catalyzing the cholesterol side-chain cleavage reaction had formal reduction potentials of -168 and -285 mV (pH 7.5 and 25 degrees C), respectively. The oxidation-reduction potentials for the rabbit liver microsomal cytochrome P-450 induced by 3-methylcholanthrene and the mitochondrial cytochrome for steroid 11 beta-hydroxylation were found as -360 and -286 mV, respectively. Potential measurements at different temperatures allowed documentation of the standard thermodynamic parameters for cytochrome P-450 reduction for the first time. All cytochromes tested were found to have a relatively large negative entropy change upon reduction. The extent of these changes is comparable to that observed for the ferric-ferrous couple of cytochrome c. An entropy-enthalpy compensation effect was observed among the four cytochromes P-450 examined although the correlation is weaker than that observed with cytochrome c isolated from various sources.     

Membrane topology of the mammalian P450 cytochromes.

The membrane topology of the mammalian P450 cytochromes has been studied intensively by computational approaches, proteolysis, chemical modification, genetic engineering, and immunochemistry. Initial results for the cytochromes of the endoplasmic reticulum appeared to indicate a polytopic, four to eight transmembrane anchor model with an active site buried in the membrane. However, recent findings show that the microsomal P450s are bound to the endoplasmic reticulum by only one or two transmembrane peptides located at the NH2-terminal end, and that the active site is part of a large cytoplasmic domain that may have one or two additional peripheral membrane contacts. The membrane-bound state is viewed as rather rigid, and the plane of the heme lies between perpendicular and parallel to the plane of the endoplasmic reticulum. The mitochondrial P450 cytochromes lack a hydrophobic NH2 terminus in the mature form, and thus differ from the microsomal isozymes in this significant way. However, although the exact topology of cytochrome P450 in the inner mitochondrial membrane remains to be elucidated, certain features are clearly comparable to those of microsomal P450. Therefore, the membrane topology of the P450 gene superfamily may follow a similar pattern.     

Mammalian cytochromes P450—Importance of tissue specificity

Mammals express multiple cytochromes P450 simultaneously in a variety of tissues, including the liver, kidney, lung, adrenal, gonads, brain, and most others. For cytochromes P450 that are expressed in many tissues or cell types, the tissue/cell type-specific expression might be associated with their special physiological roles. Several cytochrome P450 enzymes are found not only in different cell types and tissues, but also in different subcellular compartments. Generally, all mammalian cytochrome P450 enzymes are membrane bound. The two major groups are represented by microsomal cytochromes P450 that reside in the endoplasmic reticulum, and mitochondrial cytochromes P450, that reside in the inner mitochondrial membrane. However, the outer nuclear membrane, different Golgi compartments, peroxisomes and the plasma membrane are also sites where cytochromes P450 were observed. For example, CYP51 is an ER enzyme in majority of tissues but in male germ cells it trafficks through the Golgi to acrosome, where it is stabilized for several weeks. Surprisingly, in brains of heme synthesis deficient mice, a soluble form of CYP1A1 was detected whose activity has been restored by the addition of heme. In the majority of cases each cytochrome P450 enzyme resides in a single subcellular compartment in a certain cell, however, examples of simultaneous localization in different subcellular compartments have also been described, such as endoplasmic reticulum, Golgi and plasma membrane for CYP2E1. This review will focus on the physiological importance of mammalian cytochrome P450 expression and localization in different tissues or cell types and subcellular compartments.     

Hepatic mitochondrial cytochrome P-450 system. Distinctive features of cytochrome P-450 involved in the activation of aflatoxin B1 and benzo(a)pyrene

Rat liver mitoplasts containing less than 1% microsomal contamination contain cytochrome P-450 at 25% of the microsomal level and retain the capacity for monooxygenase activation of structurally different carcinogens such as aflatoxin B1 (AFB1), benzo(a)pyrene (BaP), and dimethylnitrosamine. Both phenobarbital (PB) and 3-methylcholanthrene (3-MC) induce the level of mitochondrial cytochrome P-450 by 2.0- to 2.5-fold above the level of control mitoplasts. The enzyme activities for AFB1 (3-fold) and BaP (16-fold) metabolism were selectively induced by PB and 3-MC, respectively. Furthermore, the metabolism of AFB1 and BaP by intact mitochondria was supported by Krebs cycle substrates but not by NADPH. Both PB and 3-MC administration cause a shift in the CO difference spectrum of mitoplasts (control, 448 nm; PB, 451 nm; and 3-MC, 446 nm) suggesting that they induce two different forms of mitochondrial cytochromes P-450. Mitoplasts solubilized with cholate and fractionated with polyethylene glycol exhibit only marginal monooxygenase activities. The activity, however, was restored to preparations from both PB-induced and 3-MC-induced mitochondrial enzymes (AFB1 activation, ethylmorphine, and benzphetamine deamination and BaP metabolism) by addition of purified rat liver cytochrome P-450 reductase, and beef adrenodoxin and adrenodoxin reductase. The latter proteins failed to reconstitute activity to purified microsomal cytochromes P-450b and P-450c that were fully active with P-450 reductase. Monospecific rabbit antibodies against cytochrome P-450b and P-450c inhibited both P-450 reductase and adrenodoxin-supported activities to similar extents. Anti-P-450b and anti-P-450c provided Ouchterlony precipitin bands against PB- and 3-MC induced mitoplasts, respectively. We conclude that liver mitoplasts contain cytochrome P-450 that is closely similar to the corresponding microsomal cytochrome P-450 but can be distinguished by a capacity to interact with adrenodoxin. These inducible cytochromes P-450 are of mitochondrial origin since their levels in purified mitoplasts are over 10 times greater than can arise from the highest possible microsomal contamination.     

Mammalian cytochromes P450--importance of tissue specificity

Mammals express multiple cytochromes P450 simultaneously in a variety of tissues, including the liver, kidney, lung, adrenal, gonads, brain, and most others. For cytochromes P450 that are expressed in many tissues or cell types, the tissue/cell type-specific expression might be associated with their special physiological roles. Several cytochrome P450 enzymes are found not only in different cell types and tissues, but also in different subcellular compartments. Generally, all mammalian cytochrome P450 enzymes are membrane bound. The two major groups are represented by microsomal cytochromes P450 that reside in the endoplasmic reticulum, and mitochondrial cytochromes P450, that reside in the inner mitochondrial membrane. However, the outer nuclear membrane, different Golgi compartments, peroxisomes and the plasma membrane are also sites where cytochromes P450 were observed. For example, CYP51 is an ER enzyme in majority of tissues but in male germ cells it trafficks through the Golgi to acrosome, where it is stabilized for several weeks. Surprisingly, in brains of heme synthesis deficient mice, a soluble form of CYP1A1 was detected whose activity has been restored by the addition of heme. In the majority of cases each cytochrome P450 enzyme resides in a single subcellular compartment in a certain cell, however, examples of simultaneous localization in different subcellular compartments have also been described, such as endoplasmic reticulum, Golgi and plasma membrane for CYP2E1. This review will focus on the physiological importance of mammalian cytochrome P450 expression and localization in different tissues or cell types and subcellular compartments.     

Composition of the inner mitochondrial membrane of porcine corpus luteum

An inner mitochondrial membrane fraction was prepared from porcine corpus luteum. The concentrations of the respiratory cytochromes, cytochrome P-450scc, cholesterol, ubiquinone, cardiolipin and the total phospholipids were measured. The fatty acid compositions of cardiolipin and the total phospholipid fraction were determined. Comparative data from porcine heart and liver were obtained using the same methods. Differences in both the concentration and the fatty acid composition of the phospholipids were observed between the tissues. It appeared that the phospholipid bilayer was expanded relative to haem a in luteal mitochondria. It is proposed that in the ovary this expansion may be necessary to accommodate cytochrome P-450scc and its substrate, cholesterol.     

Characterization of an NADH-dependent haem-degrading system in ox heart mitochondria.

We report the identification of an NADH-dependent haem-degrading system in ox heart mitochondria. The activity was localized to the mitochondrial inner membrane, specifically associated with complex I (NADH:ubiquinone oxidoreductase). The mitochondrial NADH-dependent haem-degradation activity was highly effective and displayed a rate nearly 60% higher than that of the microsomal activity. The following observations suggested the enzymic nature of the activity: (i) haem degradation by complex I did not proceed upon exposure to elevated temperature and extremes of pH; (ii) it displayed substrate specificity; (iii) it was inhibited by a substrate analogue; and (iv) it showed a cofactor requirement. Moreover, the activity was distinctly different from the ascorbate-mediated haem-degradation activity. Also, complex I differed from the microsomal NADPH:cytochrome c (P-450) reductase inasmuch as the formation of an effective interaction with the microsomal haem oxygenase could not be detected. Addition of purified haem oxygenase to complex I neither influenced the rate of haem degradation nor resulted in the formation of biliverdin IX alpha. In contrast, addition of haem oxygenase to NADPH:cytochrome c (P-450) reductase enhanced the rate of haem degradation by nearly 8-fold, and more than 60% of the degraded haem could be accounted for as biliverdin IX alpha. The haem-degrading activity of complex I appeared to involve the activity of H2O2, as the reaction was inhibited by nearly 90% by catalase, and propentdyopents were detected as reaction products. Intact haemoproteins such as cytochrome c and myoglobin were not effective substrates. However, the haem undecapeptide of cytochrome c was degraded at a rate equal to that observed for haem. Haematohaem was degraded at a rate 50% lower than that observed for haem. It is suggested that the NADH-dependent haem-degradation system may have a biological role in the regulation of the concentration of respiratory haemoproteins and the disposition of the aberrant forms of the mitochondrial haemoproteins.     

Stimulation of liver 5-aminolaevulinate synthetase by drugs and its relevance to drug-induced accumulation of cytochrome P-450. Studies with phenylbutazone and 3,5-diethoxycarbonyl-1,4-dihydrocollidine

The relevance of the stimulation of 5-aminolaevulinate synthetase to the accumulation of cytochrome P-450 after administration of drugs was examined in rats treated with phenylbutazone and with 3,5-diethoxycarbonyl-1,4-dihydrocollidine. 3,5-Diethoxycarbonyl-1,4-dihydrocollidine alone stimulated 5-aminolaevulinate synthetase without increasing the concentration of cytochrome P-450, whereas phenylbutazone alone increased the microsomal cytochrome P-450 without significantly affecting the activity of the enzyme. When the two drugs were given together both effects were found. It is concluded that if an increased amount of 5-aminolaevulinate and haem must be made to provide for the accumulation of cytochrome P-450, it need only be a small amount. It is also concluded from these findings that stimulation of the drug-metabolizing system on the one hand and marked enhancement of 5-aminolaevulinate synthetase activity and porphyria on the other are likely to result from different actions of the drugs. Evidence is presented suggesting that porphyrogenic drugs stimulate markedly the activity of 5-aminolaevulinate synthetase by lowering the concentration of haem in the liver, thereby decreasing the normal feedback control. With 3,5-diethoxycarbonyl-1,4-dihydrocollidine a rapid inhibition of mitochondrial ferrochelatase and of liver haem synthesis may be the primary mechanism involved.     

Loss of haem in rat liver caused by the porphyrogenic agent 2-allyl-2-isopropylacetamide

1. The effect of a single dose of 2-allyl-2-isopropylacetamide on the cytochrome P-450 concentration in rat liver microsomal fraction was studied. The drug caused a rapid loss of cytochrome P-450 followed by a gradual increase to above the normal concentration. 2. The loss of cytochrome P-450 was accompanied by a loss of microsomal haem and by a brown-green discoloration of the microsomal fraction suggesting that a change in the chemical constitution of the lost haem had taken place. Direct evidence for this was obtained by prelabelling the liver haems with radioactive 5-aminolaevulate: the drug caused a loss of radioactivity from the haem with an increase of radioactivity in a fraction containing certain un-identified green pigments. 3. Evidence was obtained by a dual-isotopic procedure that rapidly turning-over haem(s) may be preferentially affected. 4. The loss of cytochrome P-450 as well as the loss of microsomal haem and the discoloration of the microsomal fraction were more intense in animals pretreated with phenobarbitone and were much less evident when compound SKF 525-A (2-diethylaminoethyl 3,3-diphenylpropylacetate) was given before 2-allyl-2-isopropylacetamide, suggesting that the activity of the drug-metabolizing enzymes may be involved in these effects. 5. The relevance of the destruction of liver haem to the increased activity of 5-aminolaevulate synthetase caused by 2-allyl-2-isopropylacetamide is discussed.     

Parallel changes in catabolite repression of haem biosynthesis and cytochromes in repression-resistant mutants of Saccharomyces cerevisiae

Effects of three mutant genes, CAT1-2d, cat2-1 and hex2-3, on catabolite repression of mitochondrial cytochromes and the first two enzymes of haem biosynthesis were compared. The CAT1-2d mutation gave no resistance to glucose, whereas cat2-1 endowed both cytochromes and 5-aminolaevulinate dehydratase with resistance, but did not alter the effect of glucose on 5-aminolaevulinate synthase. The hex2-3 mutation caused repression resistance of cytochromes and of the two haem biosynthetic enzymes. hex2-3 strains also accumulated intracellular 5-aminolaevulinate. Co-inheritance of the latter traits, sensitivity to maltose inhibition and ability to grow on raffinose in the presence of 2-deoxyglucose, demonstrated that the pleiotropic phenotype is a function of the single gene hex2-3. Revertants which grew on maltose regained sensitivity to deoxyglucose and exhibited normal sensitivity of cytochromes and haem biosynthesis enzymes to repression. Addition of the hex1-18 mutation, which renders cytochromes resistant to repression, to a cat2-1 strain did not produce the same effect on 5-aminolaevulinate synthase as hex2-3. It is concluded that repression patterns of haem and cytochrome biosynthesis are substantially affected by hex2-3 and cat2-1 but not by CAT1-2d.     

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.     

Antibodies targeted against hypervariable and constant regions of cytochromes P450IIB1 and P450IIB2

Fusion proteins constructed between beta-galactosidase and six different segments of either cytochrome P450IIB1 or cytochrome P450IIB2 (ranging from 18 to 33 amino acids in length) were expressed in Escherichia coli. Rabbit antibodies raised against these fusion proteins were first adsorbed through a beta-galactosidase column and then immunopurified on a second column containing the corresponding fusion protein. With the exception of the antibodies directed against the hydrophobic amino-terminal segment of cytochrome P450IIB1, all the antipeptide antibodies recognized the major phenobarbital-inducible cytochromes P450IIB1 and -IIB2 on immunoblots of liver microsomal proteins. Two of the antibodies were raised against regions where cytochromes P450IIB1 and -IIB2 differ in primary structure, and were differentially reactive toward these two highly homologous cytochromes. Several of the antipeptide antibodies were also reactive with a third phenobarbital-inducible microsomal protein expressed in livers of some individual Sprague-Dawley rats which was shown to be more highly related to P450IIB1 than P450IIB2. This P450IIB1-related P450, designated P450IIB1*, was purified to apparent homogeneity and shown to hydroxylate the steroid hormones testosterone and androstenedione with the well-defined regiospecificity and high catalytic activity characteristic of P450IIB1. A fourth microsomal protein detected using the antipeptide antibodies appeared to be more highly related to P450IIB2. Because the segments on the P450 molecules recognized by these antipeptide antibodies are known, it is possible to predict where P450IIB1* and the P450IIB2-related protein differ from cytochromes P450IIB2 and -IIB1, respectively. These studies demonstrate the utility of site-specific anti-P450 antibodies raised to fusion peptides for studies on the expression of structurally related P450s and polymorphic variants within the cytochrome P450 gene superfamily.     

Phenylhydrazine-mediated induction of haem oxygenase activity in rat liver and kidney and development of hyperbilirubinaemia. Inhibition by zinc-protoporphyrin.

Phenylhydrazine was found to be a potent inducer of microsomal haem oxygenase activity in rat liver and kidney, but not in spleen. The phenylhydrazine-mediated increase in haem oxygenase activity was time-dependent. Maximum activity was attained 12h after treatment in the liver, and 24h after treatment in the kidney. The increases in the activity of haem oxygenase in the liver and the kidney could be inhibited by cycloheximide. Furthermore, the increases could not be elicited by the treatment of microsomal preparations in vitro with phenylhydrazine. In consonance with the increased haem oxygenase activity, a marked increase (16-fold) was observed in the serum total bilirubin concentration in phenylhydrazine-treated rats. The mechanism of haem degradation promoted by phenylhydrazine in vivo appears to differ from that in vitro; only in the former case is bilirubin formed as the end-product of haem degradation. When rats were given zinc-protoporphyrin (40 mumol/kg) 12h before and after phenylhydrazine treatment, the phenylhydrazine-mediated increases in haem oxygenase activity in the liver and the kidney were effectively blocked. Treatment of rats in vivo with the metalloporphyrin also inhibited the activity of splenic haem oxygenase, and promoted a major decrease in the serum bilirubin levels. In phenylhydrazine-treated animals, the microsomal content of cytochrome P-450 was significantly decreased in the absence of a decrease in the microsomal haem concentration. The decrease in cytochrome P-450 content was accompanied by an increased absorption in the 420nm region of the reduced CO-difference spectrum, suggesting the conversion of the cytochrome to an inactive form. The marked depletion of cellular glutathione levels suggests that this conversion may be related to the action of active intermediates and free radicals formed in the course of the interaction of phenylhydrazine with the haem moiety of cytochrome P-450.     

Hydroxylations in biosynthesis of bile acids. Isolation of a cytochrome P-450 from rabbit liver mitochondria catalyzing 26-hydroxylation of C27-steroids

A cytochrome P-450 catalyzing 26-hydroxylation of C27-steroids was purified from liver mitochondria of untreated rabbits. The enzyme fraction contained 10 nmol of cytochrome P-450/mg of protein and showed only one protein band with a minimum Mr = 53,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified mitochondrial cytochrome P-450 showed apparent molecular weight similar to microsomal cytochromes P-450LM4 but differed in spectral and catalytic properties from these microsomal isozymes. The purified cytochrome P-450 catalyzed 26-hydroxylation of cholesterol, 5-cholestene-3 beta,7 alpha-diol, 7 alpha-hydroxy-4-cholesten-3-one, 5 beta-cholestane-3 alpha,7 alpha-diol, and 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol up to 1000 times more efficiently than the mitochondria. The cytochrome P-450 required both ferredoxin and ferredoxin reductase for catalytic activity. Microsomal NADPH-cytochrome P-450 reductase could not replace ferredoxin and ferredoxin reductase. The cytochrome P-450 was inactive in 7 alpha-, 12 alpha- and 25-hydroxylations of C27-steroids. The results suggest that mitochondrial 26-hydroxylation of various C27-steroids is catalyzed by the same species of cytochrome P-450.     

Mitochondrial targeted cytochrome P450 2E1 (P450 MT5) contains an intact N terminus and requires mitochondrial specific electron transfer proteins for activity

Hepatic mitochondria contain an inducible cytochrome P450, referred to as P450 MT5, which cross-reacts with antibodies to microsomal cytochrome P450 2E1. In the present study, we purified, partially sequenced, and determined enzymatic properties of the rat liver mitochondrial form. The mitochondrial cytochrome P450 2E1 was purified from pyrazole-induced rat livers using a combination of hydrophobic and ion-exchange chromatography. Mass spectrometry analysis of tryptic fragments of the purified protein further ascertained its identity. N-terminal sequencing of the purified protein showed that its N terminus is identical to that of the microsomal cytochrome P450 2E1. In reconstitution experiments, the mitochondrial cytochrome P450 2E1 displayed the same catalytic activity as the microsomal counterpart, although the activity of the mitochondrial enzyme was supported exclusively by adrenodoxin and adrenodoxin reductase. Mass spectrometry analysis of tryptic fragments and also immunoblot analysis of proteins with anti-serine phosphate antibody demonstrated that the mitochondrial cytochrome P450 2E1 is phosphorylated at a higher level compared with the microsomal counterpart. A different conformational state of the mitochondrial targeted cytochrome P450 2E1 (P450 MT5) is likely to be responsible for its observed preference for adrenodoxin and adrenodoxin reductase electron transfer proteins.     

Occurrence of cytochrome P450 in continuous cultures of Saccharomyces cerevisiae

Summary Cytochrome P450 of Saccharomyces cerevisiae is an inducible enzyme system. Hitherto, its induction was related to semi-anaerobic culture conditions and high glucose concentrations in the growth medium respectively. Since glucose and oxygen are main regulatory effectors in this yeast, the relationship between the occurrence of cytochrome P450 and these two effectors was established in continuous culture. At glucose-derepressed conditions it was not possible to induce the formation of cytochrome P450 by oxygen limitation alone. The oxygen supply had to be decreased to a level where glucose repression also became active. At glucose-repressed conditions cytochrome P450 was obtained in good yield (3 to 5 pmol per mg dry cell weight) below a dissolved oxygen tension of appproximately 15%. There was a correlation between the content of mitochondrial cytochromes and that of cytochrome P450. The presence of mitochondrial cytochromes was reciprocal with cytochrome P450 when its content was increased by lowering the dissolved oxygen tension.