Characterization of rabbit liver P450IIE1 synthesized in transformed yeast cells

cDNA for chimeric protein, P450(3P4), consisting of the amino-terminal 43 residues (the membrane-anchor region) of rabbit P450IIC14 and the remaining 447 residues of rabbit P450IIE1 was constructed, then cloned into expression vector pAAH5, and expressed in Saccharomyces cerevisiae AH22 cells under the control of yeast ADH1 promoter. P450(3P4) thus synthesized in the transformed yeast cells was partially purified, and its spectral and catalytic properties were examined. In the oxidized state P450(3P4) exhibited a high-spin type absorption spectrum even in the absence of a substrate. The reduced CO complex of the P450 showed a Soret absorption maximum at 452 nm. P450(3P4) catalyzed aniline p-hydroxylation, N-nitrosodimethylamine demethylation, benzphetamine N-demethylation, and laurate and caprate (omega-1)-hydroxylation in the reconstituted system containing the P450 and NADPH-P450 reductase. These results indicate that P450(3P4) preparation obtained from the transformed yeast cells has spectral and catalytic characteristics identical with those of P450IIE1 purified from rabbit liver microsomes, confirming the substrate specificity reported of P450IIE1.     

Acetaminophen activation by human liver cytochromes P450IIE1 and P450IA2

Acetaminophen (APAP), a widely used over-the-counter analgesic, is known to cause hepatotoxicity when ingested in large quantities in both animals and man, especially when administered after chronic ethanol consumption. Hepatotoxicity stems from APAP activation by microsomal P450 monooxygenases to a reactive metabolite that binds to tissue macromolecules, thereby initiating cellular necrosis. Alcohol consumption also causes the induction of P450IIE1, a liver microsomal enzyme that in reconstitution studies has proven to be an effective catalyst of APAP oxidation. Thus, elevated microsomal P450IIE1 levels could explain not only the known increase in APAP bioactivating activity of liver microsomes after prolonged ethanol ingestion but also the enhanced susceptibility to APAP toxicity. We therefore examined the role of P450IIE1 in human liver microsomal APAP activation. Liver microsomes from seven non-alcoholic subjects were found to convert 1 mM APAP to a reactive intermediate (detected as an APAP-cysteine conjugate by high-pressure liquid chromatography) at a rate of 0.25 +/- 0.1 nmol conjugate formed/min/nmol microsomal P450 (mean +/- SD), whereas at 10 mM, this rate increased to 0.73 +/- 0.2 nmol product/min/nmol P450. In a reconstituted system, purified human liver P450IIE1 catalyzed APAP activation at rates threefold higher than those obtained with microsomes whereas two other human P450s, P450IIC8 and P450IIC9, exhibited negligible APAP-oxidizing activity. Monospecific antibodies (IgG) directed against human P450IIE1 inhibited APAP activation in each of the human samples, with anti-P450IIE1 IgG-mediated inhibition averaging 52% (range = 30-78%) of the rates determined in the presence of control IgG. The ability of anti-P450IIE1 IgG to inhibit only one-half of the total APAP activation by microsomes suggests, however, that other P450 isozymes besides P450IIE1 contribute to bioactivation of this compound in human liver. Of the other purified P450 isozymes examined, a beta-naphthoflavone (BNF)-inducible hamster liver P450 promoted APAP activation at rates even higher than those obtained with human P450IIE1. The extensive APAP-oxidizing capacity of this hamster P450, designated P450IA2 based upon its similarity to rat P450d and rabbit form 4 in terms of NH2-terminal amino acid sequence, spectral characteristics, immunochemical properties, and inducibility by BNF, agrees with previous reports concerning the APAP substrate specificity of the rat and rabbit P450IA2 proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of primary structures deduced from cDNA nucleotide sequences for various forms of liver microsomal cytochrome P-450 from phenobarbital-treated rabbits

cDNA clones, termed pHP2, b32-3, b43, and b43-1, encoding cytochromes P-450 that are expressed in the liver of phenobarbital- (PB-) treated rabbits were isolated, and their nucleotide sequences were determined. pHP2 cDNA contains an open reading frame for a 490-residue protein and is a full-length counterpart of pP-450PBc2 [Leighton, J. K., Debrunner-Vossbrinck, B. A., & Kemper, B. (1984) Biochemistry 23, 204-210]. The b32-3 insert has a sequence for a protein whose primary structure is 91% similar to that of progesterone 21-hydroxylase P-450 1, though this cDNA lacks the sequence encoding the amino-terminal 110 residues. The overlapping clones b43 and b43-1 together encode an ethanol-inducible form of cytochrome P-450, though the amino-terminal five or more residues are missing in the composite b43/b43-1 sequence. Northern blot analysis showed that the b43/b43-1 protein is more strongly inducible by polycyclic aromatic hydrocarbons and isosafrole than by PB, in contrast to the case of the HP2 and b32-3 proteins. A comparison of the primary structures of eight forms of cytochrome P-450, including the HP2, b32-3, and b43/b43-1 proteins, that are expressed in the liver of PB-treated rabbits showed that 149 out of 487-492 amino acid residues are conserved in these cytochromes P-450. The eight forms can be assigned to three rabbit cytochrome P-450 gene subfamilies, P450IIB, P450IIC, and P450IIE. It was also shown that the members of the rabbit P450IIC subfamily can be further classified into three subgroups on the basis of their sequence similarity.     

A hypervariable region of P450IIC5 confers progesterone 21-hydroxylase activity to P450IIC1

Cytochrome P450IIC5 is a hepatic progesterone 21-hydroxylase while the 95% identical P450IIC4 has a greater than 10-fold higher Km for progesterone 21-hydroxylation and the 74% identical P450IIC1 does not hydroxylate progesterone at detectable rates. Previous work demonstrated that the apparent Km of P450IIC4 for progesterone 21-hydroxylation can be markedly improved by replacing a valine at position 113 with an alanine which is present at this position in P450IIC5. In the present studies, a single point mutation in cytochrome P450IIC1 that changed valine at position 113 to alanine conferred progesterone 21-hydroxylase activity to this enzyme. Although the catalytic activity was less than that of P450IIC5, these results indicate the residue 113 plays a critical role in the determination of the substrate/product selectivity in subfamily IIC P450s. By alignment with the sequence of P450cam, the segment of the polypeptide, residues 95-123, containing residue 113 corresponds to a substrate-contacting loop in the bacterial enzyme. The region containing residue 113, which is highly variable among family II P450s, may also be a substrate-contacting loop in the mammalian cytochromes P450. The exchange of this hypervariable region of cytochrome P450IIC1, residues 95-123, with that of P450IIC5 enhanced the 21-hydroxylase activity of the cells transfected with this chimera to levels similar to those of cells transfected with the plasmid encoding P450IIC5. Kinetic analysis of microsomes isolated from the transfected cells showed that the apparent Km for progesterone 21-hydroxylation of the chimera was indistinguishable from that of P450IIC5.(ABSTRACT TRUNCATED AT 250 WORDS)     

A cDNA encoding a rat mitochondrial cytochrome P450 catalyzing both the 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3: gonadotropic regulation of the cognate mRNA in ovaries

A cDNA expression library prepared from rat liver RNA was screened with a polyclonal antibody specific for mitochondrial vitamin D3 25-hydroxylase and a cDNA for rabbit liver mitochondrial cytochrome P450c26 (CYP 26), yielding cDNA clones with identical sequences. The deduced amino acid sequence derived from a 1.9-kb full-length cDNA was 73% identical to that of rabbit cytochrome P450c26. A monoclonal antibody was used to demonstrate that the product of the 1.9-kb cDNA clone was targeted to the mitochondrial compartment when expressed in COS cells. Mitochondrial membranes containing the expressed protein showed both vitamin D3 25-hydroxylase and cholesterol 26-hydroxylase activities when reconstituted with ferredoxin reductase and ferredoxin, demonstrating that the same P450, designated as P450c26/25, can catalyze both reactions. Northern blot analysis revealed that the P450c26/25 cDNA hybridizes with a 2.4-kb RNA from rat liver and unstimulated ovaries. Treatment of rats with pregnant mare's serum gonadotropin resulted in a fivefold increase in the 2.4-kb mRNA as well as the appearance of a 2.1-kb mRNA species in the ovaries. Our findings document the presence of a regulated bifunctional mitochondrial cytochrome P450 capable of catalyzing the 25-hydroxylation of vitamin D3 and the 26-hydroxylation of cholesterol.     

Sequence and gene expression of rabbit cytochrome P450 IIC16: comparison to highly related family members.

Cytochrome P450s are heme containing proteins which evolved from an ancestral gene(s) to form a large superfamily of enzymes. We have isolated a unique cDNA from the rabbit P450 IIC subfamily, IIC16, which is 2028 bp in length. Nucleotide sequence determination indicated an ATG start codon 66 bp from the 5' end of the molecule, and an open reading frame coding for a protein of 487 amino acids. P450 IIC16 protein is greater than or equal to 90% identical in sequence to rabbit P450 IIC4, IIC5, and to the partial sequence available for IIC15. Northern and slot blot experiments demonstrated that the P450 IIC16 gene is expressed constitutively in liver, lung, testes, and kidney, and is inducible by phenobarbital in each tissue with the exception of the kidney, where mRNA levels are repressed. Alignment analysis of eight rabbit P450 IIC proteins revealed conserved and variable regions common to all IIC enzymes, and specific areas are suggested which may be important with respect to structure and function.     

Metabolism of retinol and retinoic acid by human liver cytochrome P450IIC8

Liver microsomes obtained from nine subjects were found to metabolize retinol to polar metabolites, including 4-hydroxyretinol. In a reconstituted monooxygenase system containing human liver P450IIC8, retinol was converted to 4-hydroxyretinol and other polar metabolites, with a Km of 0.071 mM and a Vmax of 1.73 nmol/min/nmol P450. Neither P450IIC9 nor P450IIE1, two other purified human P450s, displayed significant retinol hydroxylase activity. Immunoblots performed with a monospecific antibody directed against human P450IIC8 revealed that appreciable amounts of this enzyme were present in human liver microsomes. The same antibody significantly inhibited retinol metabolism in liver microsomes and in the system reconstituted with P450IIC8. The system reconstituted with P450IIC8 also converted retinoic acid to polar metabolites. Thus, this study shows, for the first time, metabolism of two physiologic substrates by a human liver cytochrome P450 related to a group of "constitutive" rodent P450s believed to participate in the metabolism of endogenous compounds. Through its involvement in vitamin A metabolism, P450IIC8 may participate in maintaining the balance between those vitamin A concentrations that promote cellular integrity (and oppose the development of cancer) and those concentrations that cause cellular toxicity.     

Purification and characterization of two unique forms of cytochrome P-450 from rabbit nasal microsomes

Two forms of cytochrome P-450, designated P-450NMa and P-450NMb, were purified to electrophoretic homogeneity from rabbit nasal microsomes. The purified cytochromes, which contained 14-16 nmol of P-450/mg of protein, exhibited apparent monomeric molecular weights of 49,500 and 51,000, respectively. As indicated by several criteria, including the amino acid composition, absorption spectra, and peptide maps, the two nasal forms of P-450 are distinct from each other. Furthermore, as judged by the NH2-terminal amino acid sequences, they are distinct from all other P-450 cytochromes described to date. In the ferric form, P-450NMa is in the low-spin state, whereas P-450NMb is predominantly in the high-spin state. When reconstituted with NADPH-cytochrome P-450 reductase and phospholipid, P-450NMa is very active in the oxidation of ethanol as well as several nasal procarcinogens, including the N-deethylation of N-nitrosodiethylamine, the O-deethylation of phenacetin, and the N-demethylation of hexamethyl-phosphoramide. P-450NMb also metabolizes these substrates, but at lower rates. Both nasal forms are also active with testosterone, with P-450NMa oxidizing the substrate in the 17-position to give androstenedione and P-450NMb catalyzing hydroxylation in the 15 alpha-, 16 alpha-, and 19-positions. The two cytochromes represent the major portion of the total P-450 in nasal microsomes, but the corresponding forms could not be detected in hepatic microsomes.     

Gene expression and cDNA cloning identified a major basic protein constituent of bovine seminal plasma as bovine monocyte-chemoattractant protein-1 (MCP-1).

P6 is one of the major basic proteins of bovine seminal plasma. Using cell-free translation of poly(A)+RNA from bovine seminal vesicle tissue and monospecific anti-P6-IgGs, we show that P6 is a secretory product of the seminal vesicles. Immunohistochemical experiments supported this finding. Immunoscreening of a lambda gt11 cDNA library derived from seminal vesicle poly(A)+RNA furnished a number of positive cDNA clones, from which clone pH42 was characterized by sequencing. The partial amino acid sequence of a CNBr-fragment of P6 permitted identification of the reading frame of clone pH42 encoding the precursor protein of P6. The P6 precursor contains a signal peptide of 23 amino acids followed by the mature P6 sequence of 76 amino acid residues. The cDNA sequence of pH42 was 80% homologous with that of the human monocyte-chemoattractant protein-1 (hMCP-1). The respective amino acid sequences for the precursor molecules are 72% identical. Northern analysis of seminal vesicle poly(A)+RNA using pH42 as probe probe identified a 0.9-kb P6 mRNA. Stimulation of P6 mRNA expression by phytohemagglutinin in bovine peripheral mononuclear leukocytes suggests that P6 is identical to bovine MCP-1.     

Topological analysis of the active sites of cytochromes P450IIB4 (rabbit), P450IIB10 (mouse), and P450IIB11 (dog) by in situ rearrangement of phenyl-iron complexes.

The reaction of phenyldiazene with purified, phenobarbital-inducible rabbit cytochrome P450IIB4, mouse cytochrome P450IIB10, and dog cytochrome P450IIB11 yields complexes with absorbance maxima at 480 nm. Treatment of the cytochrome P450 complexes with K3Fe(CN)6 results in disappearance of the 480-nm absorption. Extraction of the prosthetic group from the proteins after these reactions yields the two isomers of N-phenylprotoporphyrin IX with the N-phenyl group on pyrrole rings A and D as the major products and the regioisomer with the N-phenyl on pyrrole ring C as a minor product. The A:C:D arylated pyrrole ring ratio is 3:2:3 for rabbit P450IIB4, 3:1:3 for mouse P450IIB10, and 4:1:2 for dog P450IIB11. Formation of the A and D regioisomers is consistent with the results obtained previously for rat isozymes IA1, IIB1, IIB2, and IIE1, but the rabbit, mouse, and dog P450IIB enzymes differ from the four rat enzymes in that a substantial amount of the isomer with the N-phenyl on pyrrole ring C is also formed. The results indicate that the region over pyrrole ring B is masked by protein residues in all the active sites and suggest that the region over pyrrole ring C is more hindered by protein residues in the rat than in the rabbit, mouse, or dog enzymes so far examined.     

Purification and immunological characterization of a novel cytochrome P450 from C3H/10T1/2 cells

The major polycyclic aromatic hydrocarbon-metabolizing cytochrome P450 in the mouse embryo fibroblast-derived C3H/10T1/2 CL8 cell line (P450-EF) has been partially purified from benz[a]anthracene (BA)-induced 10T1/2 cells (40 pmol P450/mg). The purification of P450-EF was carried out by sequential chromatography of solubilized microsomes over hydrophobic aminohexyl-Sepharose 4B, anion exchange DE-52 cellulose, and cation exchange carboxymethyl trisacryl columns. The final preparation (1700 pmol/mg) appeared as a single major 55-kDa band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Reconstitution of detergent-free partially purified P450-EF yielded a relatively high turnover for 7,12-dimethylbenz[a]anthracene (DMBA) metabolism (5.4 nmol/nmol/min). Polyclonal antibodies to purified P450-EF (anti-P450-EF), raised in, respectively, rabbit and chicken, detected a single 55-kDa band in 10T1/2 cell microsomes that was highly inducible by BA (approximately 20-fold) and TCDD (approximately 5-fold). Rabbit anti-P450-EF was much more effective than the corresponding chicken antibody at binding denatured P450-EF protein on Western blots. Conversely, only the chicken antibody was effective at inhibiting DMBA metabolism catalyzed by microsomal P450-EF. This antibody did not inhibit P450IA1-mediated DMBA metabolism. Rabbit anti-P450-EF recognized very weakly (less than 1% of homologous protein response) pure P450IA1, IIB1, IIC7, IIE1, and IIIA1 proteins on Western blots but exhibited substantial cross-reactivity (approximately 10%) with pure P450IIA1 and very strong cross-reactivity (approximately 75%) with a hormonally regulated rat adrenal P450. Polyclonal antibodies to several major P450 subfamilies either did not recognize P450-EF (anti-P450IA, IIB, and IIC) or recognized it very weakly (anti-P450IIA1). P450-EF is probably distantly related to the P450IIA subfamily and may belong to a new P450 subfamily.     

Post-translational reduction of cytochrome P450IIE by CCl4, its substrate

The molecular mechanism of cytochrome P450IIE reduction by CCl4 was reexamined by measuring its enzyme activity, immunoreactive protein contents, and mRNA levels. Aniline hydroxylase and the amounts of immunoreactive P450IIE were rapidly decreased in a time-dependent manner after a single dose of CCl4. No changes were observed in the amounts of immunoreactive P450IIC and P450IA despite significant decreases decrease in their catalytic activities. However, the decreases in P450IIE enzyme activity and immunoreactive protein by CCl4 were not accompanied by a decline in its mRNA level. The data thus suggested a post-translational reduction of P450IIE by CCl4, probably due to specific destruction of the P450IIE protein by its own substrate rather than heme moiety.     

Expression of human liver cytochrome P450 IIIA4 in yeast. A functional model for the hepatic enzyme

Cytochrome P-450 (P450) NF, a member of the P450 IIIA subfamily, is the major contributor to the oxidation of the calcium-channel blocker nifedipine in human liver microsomes. A cDNA clone designated NF25 encoding for human P450 NF was isolated from a bacteriophage lambda gt11 expression library [Beaune, P. H., Umbenhauer, D. R., Bork, R. W., Lloyd, R. S. & Guengerich, F. P. (1986) Proc. Natl Acad. Sci. USA 83, 8064-8068]. We have expressed NF25 cDNA in Saccharomyces cerevisiae using an expression vector constructed from pYeDP1/8-2 [Cullin, C. & Pompon, D. (1988) Gene 65, 203-217]. Yeast transformed with the plasmid containing the NF25 sequence (pVNF25) showed a ferrous-CO spectrum typical of cytochrome P-450. Microsomal preparations contained a protein with an apparent molecular mass identical to that of P450-5 (a form isolated from human liver indistinguishable from P450 NF) that was not present in microsomes from control yeast (transformed with pYeDP1/8-2 alone), as revealed by immunoblotting with anti-P450-5 antibodies. On the other hand, antibodies raised in rabbits against human liver P450 IIC8-10 and rat liver P450 IA1 and P450 IIE1 did not recognize yeast-expressed P450 NF25. The P450 NF25 content in microsomes was about 90 pmol/mg protein. Microsomal, yeast-expressed P450 NF25 exhibited a high affinity for different substrates including macrolide antibiotics, dihydroergotamine and miconazole as shown by difference visible spectroscopy. Microsomal suspensions containing P450 NF25 were also able to catalyze several oxidation reactions that were expected from the activities of the protein isolated from human liver, including nifedipine 1,4-oxidation, quinidine 3-hydroxylation and N-oxygenation, and N-demethylation of the macrolide antibiotics erythromycin and troleandomycin. The yeast endogenous NADPH-cytochrome P-450 reductase thus couples efficiently with the heterologous P450 NF25 though its level is far lower than that of its ortholog in human liver. Indeed addition of rabbit liver NADPH-cytochrome P-450 reductase increased the oxidation rates. Rabbit liver cytochrome b5 also caused a marked enhancement of catalytic activities, as had been noted previously for this particular P450 enzyme in a reconstituted system involving the protein purified from human liver. Furthermore, the level of the yeast endogenous cytochrome P-450 (lanosterol 14-demethylase) has been found to be negligible compared to the heterologously expressed cytochrome P-450 (30 times less). Thus, yeast microsomes containing P450 NF25 constitute by themselves a good functional model for studying the binding capacities and catalytic activities of this individual form of human hepatic cytochrome P-450.     

Cloning and characterization of the cDNAs for human and rabbit interleukin-1 precursor.

DNA sequence complementary to the mRNA for rabbit interleukin-1 precursor (preIL-1) has been cloned from the cDNA library constructed using partially purified poly(A)+RNA from induced rabbit alveolar macrophages by mRNA hybridization-translation assay. By using this cDNA as a probe, human IL-1 cDNA was isolated from the cDNA library prepared using poly(A)+RNA from induced HL-60 cells, a human monocyte-like cell line. The amino acid sequences of the human and rabbit preIL-1 deduced from the cDNA sequences reveal their primary structures which consists of 271 and 267 amino acid residues, respectively. The amino acid sequence is 64% conserved between human and rabbit. The difference in number of amino acid residues results from the carboxy-terminal extention of 4 amino acid residues in human preIL-1. Expression of the cloned human cDNA in E. coli yielded biologically active IL-1.     

An inhibitory monoclonal antibody binds in close proximity to a determinant for substrate binding in cytochrome P450IIC5.

We used the expression of chimeric proteins and point mutants to identify amino acids of the hepatic progesterone 21-hydroxylase P450IIC5 which are part of an epitope recognized by an inhibitory monoclonal antibody and which affect substrate binding. Three amino acids of P450IIC5 at positions 113, 115, and 118 were introduced into P450IIC4, which is 95% identical to P450IIC5. The resultant chimeric protein acquired binding of the monoclonal antibody 1F11, which is highly specific and inhibitory for P450IIC5. Point mutants in P450IIC4 showed that two of the three changes, T115S and N118K, contribute to the epitope recognized by this antibody. The T115S mutant bound the antibody weakly (Kd greater than 30 nM) whereas the N118K mutant bound the antibody as tightly as P450IIC5 (Kd less than or equal to 0.7 nM). Thus, residues 115 and 118 are located on the surface of these enzymes, and the Lys/Asn difference at amino acid 118 is largely responsible for the high degree of discrimination which this antibody exhibits between P450IIC5 and P450IIC4. The valine to alanine mutation at position 113 conferred to P450IIC4 a lower apparent Km for progesterone 21-hydroxylation. Because antibody binding was not affected by this mutation, it is tempting to speculate that this residue is buried in the protein where it exerts its effect on the catalytic activity by interaction with the substrate or alters the positions of residues of the active site. The close proximity of the epitope at positions 115 and 118 to Ala113 suggests that the inhibitory monoclonal antibody interferes with substrate binding.     

Differences in deoxyribonuclease I hypersensitive sites in phenobarbital-inducible and constitutive rabbit P450IIC genes.

DNase I hypersensitivity of nuclear chromatin near the rabbit cytochrome P450IIC genes was investigated by indirect end-labeling genomic Southern analysis. Major DNase I hypersensitive sites were observed in proximal (-200 base pairs) and distal (-2000 to -2200 base pairs) regions of the genes. The presence of the proximal site correlated well with the expression states of the individual genes. In contrast, the distal site was present in DNA from both liver and kidney nuclei and in untreated and phenobarbital-treated animals irrespective of the expression state of the genes. However, the distal site was present only in genes that respond to phenobarbital (cytochromes P450IIC1 and P450IIC2) and was not detected in the constitutive cytochrome P450IIC3 gene. The nucleotide sequences of 500 base pairs in the distal site regions of cytochromes P450IIC1 and P450IIC2 were 67% similar, and hepatocyte nuclear factor 1 like motifs were conserved in these two sequences. These results are consistent with the hypothesis that distal regions cooperate with the proximal promoter regions in the regulation of cytochrome P450IIC gene expression.     

Alcohol-inducible cytochrome P-450IIE1 lacking the hydrophobic NH2-terminal segment retains catalytic activity and is membrane-bound when expressed in Escherichia coli

We have expressed in Escherichia coli a cDNA encoding rabbit liver cytochrome P-450IIE1, the ethanol-inducible P-450. The expressed P-450 is located primarily in the bacterial inner cell membrane and comprises 3% of the E. coli total membrane protein. The partially purified cytochrome exhibits a reduced CO difference spectrum with a maximum at 452 nm, characteristic of P-450IIE1, and solubilized membranes or partially purified P-450 preparations reconstituted with NADPH-cytochrome P-450 reductase and phosphatidylcholine catalyze the deethylation of N-nitrosodiethylamine with a turnover number equal to that of purified liver P-450IIE1 (approximately 4.5 nmol/min/nmol of P-450). A modified IIE1 cDNA that encodes a protein lacking amino acids 3-29, a proposed membrane anchor for cytochrome P-450, was also expressed in E. coli and, unexpectedly, the shortened protein was also found to be predominantly located in the bacterial inner membrane rather than the cytosol. Like the full-length protein, this truncated cytochrome has a reduced CO difference spectrum characteristic of P-450IIE1 and is fully active in the deethylation of N-nitrosodiethylamine. These results demonstrate that the NH2-terminal hydrophobic segment is not solely responsible for attachment to the membrane and evidently is not required for proper protein folding or catalytic activity.