Cloning and sequence analysis of a rat liver cDNA coding for a phenobarbital-inducible microheterogenous cytochrome P-450 variant: regulation of its messenger level by xenobiotics

A rat liver cDNA library was prepared from total polyribosomal poly(A)+ RNA extracted from phenobarbital-treated animals. A cDNA clone coding for a phenobarbital-inducible cytochrome P-450 (PB P-450) was identified by differential colony hybridization to cDNAs synthesized from liver poly(A)+RNAs isolated from phenobarbital-treated rats for positive selection and cDNAs from either untreated rats or beta-naphthoflavone-treated rats as negative controls, followed by hybrid-selected translation and analysis of the translation products by immunoprecipitation. As the cloning and screening strategies involve no prior enrichment for specific mRNAs, they also permit the identification of sequences coding for phenobarbital-induced proteins other than cytochromes P-450. This relatively straightforward approach is generally applicable to the molecular cloning of sequences coding for other inducible cytochromes P-450. Nucleic acid sequencing data indicated that the cloned PB P-450 cDNA codes for a cytochrome P-450 variant [designated P-450e(U.C.)] that is very similar, but not identical, to P-450e. Sequence analysis of the section of cDNA specifying the 3'-non-coding region of the mRNA revealed that it lacked the usual poly(A) addition site signal sequence but contained three inverted repeat structures. Solution hybridization analysis demonstrated that PB P-450 mRNA is increased 20-fold by phenobarbital treatment and decreased 3-fold by beta-naphthoflavone treatment.     

Detection of phenobarbital-induced cytochrome P-450 in rat hepatic microsomes using an enzyme-linked immunosorbent assay

The major phenobarbital-inducible form of cytochrome P-450 (cytochrome P-450 PB) was purified to homogeneity from rat liver microsomes and rabbit antibodies prepared against the purified enzyme. Using these antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed for the detection of cytochrome P-450 PB in microsomes which was sensitive at the nanogram level. The content of cytochrome P-450 PB was determined in hepatic microsomes from rats treated with various xenobiotics. Phenobarbital and Aroclor 1254 pretreatments resulted in several-fold increases in immunoreactive cytochrome P-450 PB over control levels. ELISA measurements of cytochrome P-450 PB were also carried out over a 48-h time course of phenobarbital induction in liver microsomes. Significant increases over control levels were seen at 16 h and beyond. Measurements of ELISA-detectable cytochrome P-450 PB were made in microsomes following the administration of CCl4 to phenobarbital-pretreated rats. Immunoreactive cytochrome P-450 PB was observed to decrease less rapidly than the spectrally detectable enzyme in the microsomal membranes. Inhibition of heme synthesis was carried out by the administration of 3-amino-1,2,4-triazole (AT) to rats. Concomitant pretreatment with phenobarbital and AT resulted in levels of ELISA-detectable cytochrome P-450 PB which were significantly increased over control levels, while spectrally detectable levels of total holoenzyme remained unchanged. These results support the idea that this cytochrome P-450 may exist, at least partly, in the microsomal membrane in an inactive or apoprotein form.     

Expression of cytochrome P-450 isozymes in the liver of hypophysectomized rats. Evidence for different regulation mechanisms concerning P450IIB and P450IIIA subfamilies

1. Monooxygenase activities have been examined in rat liver to determine the effects of castration and hypophysectomy on cytochrome P-450 species. In adult males, hypophysectomy caused a decrease of total P-450 concentration, aniline hydroxylase, benzopyrene hydroxylase, benzphetamine demethylase, testosterone hydroxylase and imipramine hydroxylase and demethylase activities. The treatment of hypophysectomized animals with human growth hormone or testosterone did not restore the full activity. 2. When probed with antibodies, microsomes from hypophysectomized males and females exhibited an intense reaction with a polyclonal anti-(phenobarbital-induced P-450) which was not observed with a monoclonal antibody of anti-(phenobarbital-induced P-450). 3. These microsomal preparations also reacted with an antibody raised against a developmentally regulated P-450. No sex difference could be detected with this antibody. Furthermore, administration of human growth hormone to hypophysectomized males prevented this immunoreaction. 4. Total RNA has been prepared from the same liver; when probed with cDNAs, no changes occurred in the content in P-450 b/e, PB 24 (a constitutive member of the phenobarbital subfamily) and phenobarbital-inducible mRNA for UDP-glucuronosyltransferase. 5. In contrast, P-450 mRNA induced by pregnenolone 16 alpha-carbonitrile was modulated by hormonal manipulations: lower in females and castrated males than in intact males, increased in both sexes after hypophysectomy. Treatment of hypophysectomized males with human growth hormone abolished this rise in pregnenolone-16 alpha-carbonitrile-induced P-450 mRNA accumulation. Data collected in this study support the assumption that hypophysectomy acts differently on the regulation of various P-450 isozymes and that this regulation clearly does not involve the phenobarbital subfamily of P-450s.     

Differential induction and tissue-specific expression of closely related members of the phenobarbital-inducible rabbit cytochrome P-450 gene family

We have examined the tissue-specific expression of three rabbit genes that are closely related members of a subfamily of the phenobarbital-inducible cytochrome P-450 gene family. Analysis of the levels of mRNA in liver revealed that (a) cytochrome P-450PBc1 mRNA was not detectable in livers from control animals but was present in livers from animals treated with phenobarbital, (b) cytochrome P-450PBc2 was present in control tissue and was increased by about 3-fold 24 h after phenobarbital treatment, and (c) the levels of cytochrome P-450PBc3 mRNA was the same in livers from control and treated animals. In the kidney, only P-450PBc2 mRNA was detected at a level 15% of that in the liver, and the levels increased about 3-fold after phenobarbital treatment. None of the mRNAs was detected in lung tissue. Multiple species of RNA were observed that hybridized to probes for cytochrome P-450PBc1 and P-450PBc2 cDNAs by Northern blot analysis ranging in size from 2300 to 4000 nucleotides. Differential sites for polyadenylation probably cause the heterogeneity in size. A single species of RNA of 2200 nucleotides that hybridized to cytochrome P-450PBc3 cDNA probes was observed. These data demonstrate that three closely related cytochrome P-450 genes are differentially responsive to phenobarbital treatment and that they exhibit different tissue-specific patterns of expression.     

Multiple constitutive but phenobarbital-inducible rat hepatic nuclear RNA species homologous to a novel cytochrome P-450 cDNA

A cDNA clone, pPB8, representing partial information for a phenobarbital-inducible rat hepatic cytochrome P-450, immunochemically related to cytochrome P-450b and/or P-450e, hybridized to multiple hepatic nuclear RNA species. In addition to the 3.7 +/- 0.2 kb mRNA encoding this novel cytochrome P-450 isozyme, pPB8 hybridized to nuclear RNAs of 4.9 +/- 0.3, 5.4, 5.7 +/- 0.2, and 6.3 +/- 0.1 kb. These nuclear RNAs were constitutively expressed and were inducible to various extents by phenobarbital administration. The time course of induction of these nuclear RNA components suggested product-precursor relationships. A "full-length" cDNA clone, pPB8/7, synthesized from poly(A)+ RNA homologous to pPB8, detected two mRNA species of 4.6 and 1.8 kb. The 4.6 kb nuclear RNA was inducible by 3-methylcholanthrene, Aroclor 1254, and phenobarbital, while the 1.8 kb nuclear RNA was not appreciably affected. It is suggested that pPB8 and pPB8/7 were synthesized from distinct mRNAs that share homology in their 3' regions.     

Phenobarbital induction of cytochromes P-450. High-level long-term responsiveness of primary rat hepatocyte cultures to drug induction, and glucocorticoid dependence of the phenobarbital response.

The induction of hepatic cytochromes P-450 by phenobarbital (PB) was studied in rat hepatocytes cultured for up to 5 weeks on Vitrogen-coated plates in serum-free modified Chee's medium then exposed to PB (0.75 mM) for an additional 4 days. Immunoblotting analysis indicated that P-450 forms PB4 (IIB1) and PB5 (IIB2) were induced dramatically (greater than 50-fold increase), up to levels nearly as high as those achieved in PB-induced rat liver in vivo. The newly synthesized cytochrome P-450 was enzymically active, as shown by the major induction of the P-450 PB4-dependent steroid 16 beta-hydroxylase and pentoxyresorufin O-dealkylase activities in the PB-induced hepatocyte microsomes (up to 90-fold increase). PB induction of these P-450s was markedly enhanced by the presence of dexamethasone (50 nM-1 microM), which alone was not an affective inducing agent, and was inhibited by greater than 90% by 10% fetal bovine serum. The PB response was also inhibited (greater than 85%) by growth hormone (250 ng/ml), indicating that this hormone probably acts directly on the hepatocyte when it antagonizes the induction of P-450 PB4 in intact rats. In untreated hepatocytes, P-450 RLM2 (IIA2), P-450 3 (IIA1) and NADPH P-450 reductase levels were substantially maintained in the cultures for 10-20 days. The latter two enzymes were also inducible by PB to an extent (3-4 fold elevation) that is comparable with that observed in the liver in vivo. Moreover, P-450c (IA1) and P-450 3 (IIA1) were highly inducible by 3-methylcholanthrene (5 microM; 48 h exposure) even after 3 weeks in culture. In contrast, the male-specific pituitary-regulated P-450 form 2c (IIC11) was rapidly lost upon culturing the hepatocytes, suggesting that supplementation of appropriate hormonal factors may be necessary for its expression. The present hepatocyte culture system exhibits a responsiveness to drug inducers that is qualitatively and quantitatively comparable with that observed in vivo, and should prove valuable for more detailed investigations of the molecular and mechanistic basis of the response to PB and its modulation by endogenous hormones.     

Induction of drug metabolizing enzymes in human liver cell line Hep G2

Human cytochrome P-450, UDP-glucuronosyltransferase and sulphotransferase activities have been measured in the cell line Hep G2 following treatment of cells with 3-methylcholanthrene or phenobarbital. 3-Methylcholanthrene treatment caused a 20-30-fold increase in the O-deethylation of 7-ethoxycoumarin. The glucuronidation and sulphation of the product 7-hydroxycoumarin were increased 36 and 7 fold, respectively. In comparison, phenobarbital treatment did not increase these activities significantly. However, phenobarbital-inducible proteins were identified on "Western blots' using antibodies to a rat liver phenobarbital inducible P-450 form. The molecular masses of the proteins did not coincide with those expected for cytochromes P-450. However, characteristic of P-450 forms, the synthesis of these proteins was suppressed by 3-methylcholanthrene treatment. The Hep G2 cell line represents a potentially useful model for studying the regulation of human P-450 genes.     

Immunochemical similarity of P-450 HFLa, a form of cytochrome P-450 in human fetal livers, to a form of rat liver cytochrome P-450 inducible by macrolide antibiotics

A protein immunochemically related to P-450 HFLa, a form of cytochrome P-450 purified from human fetal livers, was detected in rat liver microsomes. The content of the immunoreactive protein in rat liver microsomes was increased by treatments with phenobarbital, pregnenolone 16 alpha-carbonitrile (PCN), erythromycin, erythromycin estolate, and oleandomycin but not with 3-methylcholanthrene, imidazole, ethanol, isosafrole, josamycin, midecamycin, or miocamycin. The activity of erythromycin N-demethylase correlated with the content of the immunoreactive protein in rat liver microsomes (r = 0.72). In addition, anti-P-450 HFLa IgG inhibited erythromycin N-demethylase in liver microsomes from erythromycin- or oleandomycin-pretreated rats. Furthermore, the content of the immunoreactive protein highly correlated with that of P-450 PB-1, which is distinct from Waxman's terminology, and is one of the forms of PCN-inducible cytochrome P-450s (r = 0.95). From these results and the results reported so far, it seems possible that P-450 HFLa is one of the forms of cytochrome P-450 inducible by glucocorticoids.     

Regulation of gene expression in adult rat hepatocytes cultured on a basement membrane matrix

Freshly isolated adult rat hepatocytes, when cultured on type I collagen (commercially available as Vitrogen), assume a polygonal shape, form a stable monolayer within 24 hours, but lose the capacity to express some liver-specific functions over time in culture. We incubated hepatocytes in a serum-free medium on a reconstituted basement membrane gel, "matrigel" (prepared from an extract of extracellular matrix of the murine Engelbreth-Holm-Swarm sarcoma), and observed that the cells adhered firmly, remained rounded as single cells or clusters, and maintained liver-specific gene expression for more than 1 week in vitro. Hepatocytes on matrigel secreted substantially higher amounts of albumin, transferrin, haptoglobin, and hemopexin, Northern blot analyses of extracted cellular RNA, expressed increased amounts of mRNA for the liver-specific protein albumin (as compared with cells on vitrogen). In cultures treated with phenobarbital, cytochrome P-450b, and cytochrome P-450e, mRNAs and proteins were barely detectable in cells on Vitrogen but were induced to levels similar to those in the liver in vivo in matrigel cultures. Likewise, the use of matrigel greatly enhanced the induction of mRNA and protein for P-450c by 3-methylcholanthrene and for P-450p by steroidal and nonsteroidal inducers. However, neither substratum permitted induction of P-450d by 3-methylcholanthrene, suggesting that the effects of matrigel are selective even for expression in liver of members of the superfamily of cytochrome P-450 genes. Within 5 days in cultures on Vitrogen, hepatocytes expressed detectable amounts of fetal liver aldolase activity and also mRNA for vimentin and type I collagen, each considered a phenotypic change reflecting hepatocyte "dedifferentiation." None of these was present in cells on matrigel. Responsiveness to mitogenic stimuli, as judged by incorporation of 3H-thymidine into DNA, was also decreased in hepatocytes cultured on matrigel. Finally, there was a remarkable increase in the levels of both matrices during the first 2 days in culture. However, the continuously cytoskeleton mRNA over time in culture than did the rounded cells on matrigel. We conclude that hepatocytes cultured on matrigel, as opposed to the standard collagen, exhibit remarkably enhanced expression of many liver-specific functions.     

Phenobarbital-type induction of cytochrome P-450 in liver microsomes by perfluorodecalin

Cytochrome P-450 induction in hepatic microsomes after injections of rats with a fluorocarbon emulsion containing perfluorodecalin was studied in comparison with phenobarbital and methylcholanthrene type inductions. It was shown that perfluorodecalin injection as well as the phenobarbital one cause an increase in the cytochrome P-450 content, NADPH-cytochrome c reductase activity, the rates of benzphetamine N-demethylation and aldrin epoxidation in the microsomes. Using the Ouchterlony double immunodiffusion test with antibodies against cytochrome P-450b, an immunological identity of cytochrome P-450 isoforms during perfluorodecalin and phenobarbital inductions was shown. Upon "rocket" immunoelectrophoresis the recovery of cytochrome P-450 which is immunologically indistinguishable from cytochrome P-450b was approximately 72% in perfluorodecalin-induced microsomes. The activity of benzphetamine demethylase and aldrin epoxidase was inhibited by antibodies against cytochrome P-450b. These results suggest that in rat hepatic microsomes perfluorodecalin induces the cytochrome P-450 isoform whose immunological properties and substrate specificity correspond to those of phenobarbital-type cytochrome P-450.     

Regulation of cytochrome P-450p by phenobarbital and phenobarbital-like inducers in adult rat hepatocytes in primary monolayer culture and in vivo

Treatment of rats with phenobarbital increases the hepatic concentration of P-450p, a form of cytochrome P-450 believed to be controlled primarily by a mechanism that stereospecifically recognizes glucocorticoids like dexamethasone and anti-glucocorticoids like pregnenolone-16 alpha-carbonitrile [Schuetz, E.G., & Guzelian, P.S. (1984) J. Biol. Chem. 259, 2007]. To test the possibility that phenobarbital induces P-450p indirectly by increasing the availability of endogenous glucocorticoids in the liver, we added phenobarbital and phenobarbital-like inducers to primary monolayer cultures of adult rat hepatocytes incubated in serum-free medium without glucocorticoids and found stimulated de novo synthesis of P-450p measured as increased incorporation of [3H]leucine into immunoprecipitable P-450p protein. With some of the inducers, notably the organochlorine pesticides chlordane and trans-nonachlor, there was a greater accumulation of P-450p measured on quantitative immunoblots than could be accounted for by the increase in P-450p synthesis. "Pulse-chase" experiments confirmed that these compounds significantly lengthen the half-life of P-450p up to 60 h as compared to the values in control (11 h) or dexamethasone-treated (10 h) cultures. Treatment of rats with chlordane, trans-nonachlor, or other cyclodiene organochlorine pesticides confirmed that these agents increase the concentration of P-450p in liver microsomes analyzed on immunoblots of two-dimensional electrophoretic gels. The time courses of induction in trans-nonachlor-treated rats of P-450p protein and of P-450PB proteins induced by phenobarbital were similar as were the amounts of P-450PB mRNA and P-450p mRNA measured by hybridization to cloned cDNA probes. However, analysis of structure-activity relationships among polychlorinated biphenyls revealed that isomers with two ortho chlorinated positions maximally induced P-450PB whereas isomers with three and four ortho chlorines maximally induced P-450p in rats and in hepatocyte culture, respectively. We conclude that P-450p is induced by the phenobarbital class of inducers through direct contact with the hepatocytes involving decreased degradation of the protein and stimulation of its synthesis in a manner similar but not identical with that of P-450PB.     

Isolation and sequence analysis of three cloned cDNAs for rabbit liver proteins that are related to rabbit cytochrome P-450 (form 2), the major phenobarbital-inducible form

We have isolated from rabbit liver three cDNA clones of 1400-1800 base pairs that hybridize selectively to RNA from animals treated with phenobarbital. The nucleotide sequences of the cDNAs have been determined. In the protein coding region the nucleotide sequences of two of the cDNAs are 88% homologous, and the third cDNA is about 72-74% homologous to the other two. All three are 55-60% homologous to rat liver cytochrome P-450b cDNA. The amino acid sequences derived from the cDNA sequences are about 50% homologous to those of rat liver cytochrome P-450b and rabbit liver cytochrome P-450 (form 2). The degree of homology differs substantially in different regions of the protein. The hydrophobicity profiles of these five mammalian cytochromes P-450 are very similar and contain up to eight regions of hydrophobicity that are long enough to span a membrane. These results indicate that these three cDNAs code for rabbit liver cytochromes P-450 which are different from any rabbit liver cytochrome P-450 for which amino acid sequence information is published. These cDNAs are part of a family of genes that are related to rabbit liver cytochrome P-450 (form 2) and rat liver cytochrome P-450b which are the major phenobarbital-inducible forms. The divergence of amino acid sequence between the rat and rabbit forms and the divergence of nucleotide sequences of silent sites in the two most closely related rabbit forms suggest that cytochromes P-450 have a relatively high rate of amino acid divergence compared to many other vertebrate proteins.     

Induction and repression of the major phenobarbital-induced cytochrome P-450 measured by radioimmunoassay.

Two independent radioimmunoassay techniques for the major phenobarbital-inducible cytochrome P-450 (PB P-450) of rat liver microsomal membranes are described. The first technique employs as the source of radiolabelled antigen the products of translation in vitro labelled with [35S]methionine. The second technique employs purified antigen labelled with 125I and is quicker, less expensive and more precise. Both assays are highly specific for PB P-450 and can detect quantities of this variant as small as 1 ng. This is several orders of magnitude more sensitive than any method described previously for the quantification of cytochromes P-450, and consequently the technique is particularly well suited for the quantification of so-called constitutive cytochrome P-450 variants that are present in very low amounts. The results of the radioimmunoassays demonstrate that the apparent 2.6-fold induction of total cytochromes P-450 after phenobarbital treatment is due to a 43-fold increase in Pb P-450. Although beta-naphthoflavone increases the total content of cytochrome P-450 of microsomal membranes 1.4-fold, it actually causes a 55% decrease in the amount of PB P-450. Thus different xenobiotics can have differential effects on the expression of the genes for specific cytochrome P-450 variants.     

Regulation of cytochrome P-450b and P-450e mRNA expression in the developing rat. Hybridization to synthetic oligodeoxyribonucleotide probes

We conducted solution hybridization and Northern blot experiments utilizing synthetic 18'-mer oligodeoxyribonucleotides complementary to two major rat hepatic phenobarbital-inducible cytochrome P-450s, P-450b and P-450e, to assess their mRNA expression during rat development. At all ages studied, with one exception (i.e. in day 22 neonates), P-450b mRNA was not detected in control animals. However, traces of P-450e message were observed in control animals on day 22 and persisted to adulthood. Phenobarbital pretreatment caused marked increases in hepatic mRNA for both P-450s as early as 22 days after conception. No increases were observed in RNA isolated from phenobarbital-pretreated day 10 or 19 rats. In general, the inducible levels of P-450b and P-450e mRNA increased as a function of age. The age-dependent increase in responsiveness to phenobarbital was associated with an age-dependent decrease in the ratio of P-450b to P-450e mRNA levels. The levels of P-450b/P-450e varied from a ratio of 19 at day 22 of development to a ratio of 5 at day 62 of development. Maximal levels of phenobarbital-induced hepatic RNA for both isozymes occurred 24 days after birth (day 46 of development), at which time P-450b and P-450e mRNAs accumulated to levels 2.4- and 1.8-fold greater, respectively, than levels found in comparably induced adult rat liver. Northern blot analyses indicated that the major mRNA species hybridizing to either the P-450b or P-450e oligomers in all age groups studied was approximately 1.8 kilobases.     

Isolation and characterization of cDNA clones for cytochromes P-450 immunochemically related to rat hepatic P-450 form PB-1

Rat hepatic cytochrome P-450 PB-1 is a prominent constitutive P-450 form whose levels increase approximately 2-3 fold upon phenobarbital administration. Antibodies raised against this protein recognized two major proteins in immunoblots of rat liver microsomal proteins and precipitated comparable amounts of two electrophoretically separable hepatic mRNA translation products. The levels of the two mRNAs encoding these polypeptides were increased substantially upon phenobarbital administration. The anti-PB-1 antibodies were used to screen a cDNA library, and two distinct cDNA clones, pTF-1 and pTF-2, were isolated. These clones contain inserts of 1227 and 410 base pairs, respectively, and show 80% nucleic acid sequence homology in their region of overlap. The DNA sequences of these clones show 54% sequence homology to the corresponding portions of the mRNA encoding P-450 PB-4, a major phenobarbital-inducible form of rat liver P-450, and can be optimally aligned with the PB-4 sequence without introducing insertions or deletions. The level of hepatic mRNA which hybridizes to clone pTF-2 increases approximately 2-4-fold after phenobarbital treatment, whereas mRNA which hybridizes to pTF-1 does not change in concentration after this treatment. mRNA, which hybridizes to pTF-1, is, however, 4-fold more abundant in livers of female rats than in livers of male rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

The expression and metabolic activity of cytochrome P-450 isozymes in control and phenobarbital-induced primary cultures of rat hepatocytes

The expression and activity of the phenobarbital (PB)-inducible P-450 isozymes, P-450b and P-450e, and the major 3-methylcholanthrene (MC)-inducible form, P-450c, were studied in primary cultures of adult rat hepatocytes in T1, Leibovitz L-15 (L-15), and a modification of Waymouth 752/1 (Way) media. P-450 isozymes in initially isolated hepatocytes and control and PB-treated cultures were quantitated by Western blot analysis, and activity was determined with 7,12-dimethylbenz[a]anthracene (DMBA) as substrate. Data from the Western blot analysis correlated well with the metabolic activity toward DMBA. P-450b was consistently induced by PB in hepatocytes in T1 and to a lesser extent in Way. P-450e protein was constitutive in initially isolated cells, expressed in control cultures at a reduced level, and increased or maintained by PB in all three media. DMBA metabolite formation associated with P-450b and P-450e activity was induced by PB in hepatocytes in T1 and Way and was inhibited by antibodies to P-450b. P-450c was only infrequently expressed in freshly prepared hepatocytes, but was detected in all control and PB-treated cultures although at a much higher level in T1. Thus, the amounts of P-450 isozymes, their inducibility by PB, and their activity toward DMBA were found to be dependent on the medium. We have demonstrated enzyme induction and increased activity of the major PB-inducible isozymes in hepatocytes in T1; these are also associated with a change in the control of P-450c expression leading to enhanced constitutive expression and inducibility by phenobarbital.     

Secondary structure prediction of liver microsomal cytochrome P-450; proposed model of spatial arrangement in a membrane

The secondary structure of rabbit liver microsomal cytochrome P-450 LM2, rat liver microsomal cytochromes P-450b and P-450e (phenobarbital-inducible), and rat liver microsomal cytochromes P-450c, P-450d (3-methylcholanthrene-inducible) was predicted by a combination of methods (i) identifying the transmembrane parts of integral membrane proteins, and (ii) statistically predicting the secondary structure of globular proteins. The results are similar for all phenobarbital-inducible enzymes and make it possible to construct two structural models with seven or four transmembrane alpha-helices. The cytochromes of the second group obviously form a second structural family with four membrane-spanning alpha-helices. In both cases, a large ectodomain with several consecutive alpha-helices, which may provide the heme-binding pocket, is exposed out of the membrane.