Mouse liver cytochrome P-450 (P-450IIIAM1): its cDNA cloning and inducibility by dexamethasone.

A full-length cDNA complementary to mouse liver mRNA coding for one of the cytochromes P-450 (P-450) in the P-450IIIA family, namely P-450IIIM1, was isolated and completely sequenced. The sequence of this cDNA clone, pMDex13, revealed that it encoded a polypeptide of 504 deduced amino acid residues (Mr = 57,853). The deduced amino acid sequence showed 87.3 and 84.9% identity with rat P-450IIIA1 and P-450IIIA2, respectively. The NH2-terminal 24 amino acid sequences of P-450IIIAM1 were completely identical with purified mouse P-450UT protein. RNA blot analysis showed that mRNA content of hepatic P-450IIIAM1 was remarkably increased by treatment of mice with dexamethasone.     

Characterization of a phenobarbital-inducible dog liver cytochrome P450 structurally related to rat and human enzymes of the P450IIIA (steroid-inducible) gene subfamily

A cytochrome P450 called PBD-1 isolated from liver microsomes of an adult male Beagle dog treated with phenobarbital (PB) is structurally and functionally similar to members of the P450IIIA gene subfamily in rat and human liver microsomes. The sequence of the first 28 amino-terminal residues of PBD-1 is identical in 15 and 20 positions, respectively, to the P450IIIA forms P450p from rat and P450NF (and HLp) from human. Upon immunoblot analysis, anti-PBD-1 IgG recognizes PCNa (P450p) and PCNb (PB/PCN-E) from rat, P450NF from human, and two proteins in liver microsomes from both untreated and PB-treated dogs. Similarly, anti-PCNb IgG cross-reacts with PBD-1 and with at least one protein in microsomes from untreated dogs and two proteins in microsomes from PB-treated dogs. P450IIIA-form marker steroid 6 beta-hydroxylase activities increase 2.5-fold upon PB-treatment of dogs and are selectively inhibited by anti-PBD-1 IgG. NADPH-dependent triacetyloleandomycin (TAO) complex formation and erythromycin demethylase, also marker activities for P450IIIA forms from rats and humans, increase 4- and 5-fold in dog liver microsomes upon PB treatment, whereas immunochemically reactive PBD-1 is induced 3-fold. In microsomes from PB-treated dogs, 5 mg anti-PBD-1 IgG/nmol P450 inhibits greater than 75 and 50% of TAO complex formation and erythromycin demethylase activity, respectively. TAO complex formation is not inhibited by chloramphenicol, a selective inhibitor of the major PB-inducible dog liver cytochrome P450, PBD-2. These data suggest that PBD-1 or another immunochemically related form is responsible for a major portion of macrolide antibiotic metabolism by microsomes from PB-treated dogs and for steroid 6 beta-hydroxylation by microsomes from both untreated and PB-treated dogs. Major species differences were noted, however, in the apparent Km for 6 beta-hydroxylation of androstenedione by liver microsomes from untreated rats (24 microM), humans (380 microM), and untreated dogs (4700 microM).     

Molecular cloning of monkey liver cytochrome P-450 cDNAs: similarity of the primary sequences to human cytochromes P-450.

Three cDNAs coding for monkey cytochrome P-450 (P450) 2C, 2E and 3A (MKmp13, MKj1 and MKnf2, respectively) were isolated from a lambda gt11 cDNA library of a liver from a 3-methylcholanthrene (3MC)-treated crab-eating monkey, using cDNA fragments for human P450 2C, 2E and 3A as respective probes. MKmp13 and MKnf2 were 1901 and 2032 bp long, containing entire coding regions for polypeptides of 490 and 503 residues, respectively. The deduced N-terminal amino acid sequences of MKmp13 and MKnf2 were identical with those of P450-MK1 and P450-MK2, which had been purified from liver microsomes of untreated and polychlorinated biphenyl (PCB)-treated crab-eating monkeys, respectively. MKj1 was 1508 bp long, encoding a polypeptide of 449 residues, which is presumed to lack N-terminal 45 residues as compared with the sequence for human P450 2E1. Northern blot analysis indicated that monkey P450 2C, 2E and 3A mRNAs were expressed constitutively in monkey livers. P450 2E and 3A mRNAs were induced by both 3MC and PCB, while P450 2C mRNA was induced only by PCB. The deduced amino acid sequences of four monkey cytochrome P-450 cDNAs, including P450 1A1 (MKah1) which we isolated previously, were more than 92% identical with those of corresponding human cytochrome P-450 cDNAs.     

Identification of a new P450 expressed in human lung: complete cDNA sequence, cDNA-directed expression, and chromosome mapping

A cDNA coding for a P450 expressed in human lung was isolated from a lambda gt11 library constructed from human lung mRNA using a cDNA probe to rat P450 IVA1. The cDNA-deduced amino acid sequence of this P450, designated IVB1, consisted of 511 amino acids and had a calculated molecular weight of 59,558. The IVB1 amino acid sequence bore 51%, 53%, and 52% similarities to rat IVA1, IVA2, and rabbit P450p-2, respectively. Comparison of the primary amino acid sequence of human IVB1 with rat IVA and rabbit p-2 P450 sequences revealed a region of absolute sequence identity of 17 amino acids between residues 304 and 320. However, the functional significance of this conserved sequence is unknown. Human IVB1 also appears to be related to P450 isozyme 5 that has been extensively characterized in rabbits. The IVB1 cDNA was inserted into a vaccinia virus expression vector and the enzyme expressed in human cell lines. The expressed enzyme had an absorption spectrum with a lambda max at 450 nm when reduced and complexed with carbon monoxide, typical of other cytochrome P450s. Unlike rabbit P450 isozyme 5, however, human IVB1 was unable to activate the promutagen 2-aminofluorene. Human lung microsomal P450s were also unable to metabolize this compound despite the presence of IVB1 mRNA in three out of four human lungs analyzed. In contrast to its expression in lung, IVB1 mRNA was undetectable in livers from 14 individuals, including those from which the lungs were derived. IVB1-related mRNA was also expressed in rat lung and was undetectable in untreated rat liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino-terminal sequence and structure of monoclonal antibody immunopurified cytochromes P-450

Six hepatic cytochromes P-450 were isolated from 3-methylcholanthrene-treated animals by immunopurification with monoclonal antibodies. The purified cytochromes P-450 include 57- and 56-kDa polypeptides from Sprague-Dawley rats, 57- and 56-kDa polypeptides from C57BL/6 mice, a 56-kDa polypeptide from DBA/2 mice, and a 53-kDa polypeptide from guinea pigs. These isozymes were structurally compared by peptide mapping using both sodium dodecyl sulfate--polyacrylamide gel electrophoresis and high-pressure liquid chromatography and by amino acid and NH2-terminal sequence analyses. The 57-kDa polypeptides from rats and mice have similar but nonidentical peptide maps and amino acid compositions and are about 80% homologous in their NH2-terminal amino acid sequence. The 56-kDa polypeptides from rats and both mice strains have very similar peptide maps and amino acid compositions and identical NH2-terminal sequences. The NH2-terminal sequence of the mice 56-kDa polypeptides corresponds to that reported for the mouse P1-450 isozyme except that we identified two additional residues, proline and serine, at the NH2 terminus in the 57-kDa polypeptide from C57BL/6 mice that were not deduced from the cDNA sequence of the mouse P1-450 isozyme. The guinea pig 53-kDa polypeptide has a distinct peptide map relative to the other polypeptides studied and an NH2-terminal sequence with only partial homology to the 56- and 57-kDa polypeptides from rats and mice. This report shows the varying degree of structural relatedness among the isozymes examined and demonstrates the suitability and advantage of immunopurified cytochromes P-450 for sequencing and structural studies.     

半滑舌鳎脑芳香化酶基因cDNA克隆及表达分析

为研究脑型芳香化酶(P450aromB)在半滑舌鳎性别分化中的作用,采用同源克隆策略,从半滑舌鳎脑分离了2184bp长的脑型芳香化酶的全长cDNA,该基因编码498个氨基酸。氨基酸序列和系统发育分析表明,P450aromB属于脑型P450arom,P450aromB的氨基酸序列与其他鱼类脑型P450arom的同源性较高(48.3%-66.1%),与性腺型P450arom的同源性较低(34.2%-49.9%),与自身的性腺型芳香化酶同源性为45.1%。RT-PCR分析表明:P450aromB mRNA的表达具有明显组织特异性,P450aromB只在性腺、脑、鳃和皮肤中表达,且脑中表达量远高于性腺,而在雌雄鱼的其他组织中都不表达。经过甲基睾酮浸浴处理和高温诱导半滑舌鳎由雌性性反转为雄性后,脑中P450aromB的表达量降低,这些结果表明P450aromB参与了半滑舌鳎的性腺分化和性别决定过程。     

NH2-terminal sequence analyses of four rat hepatic microsomal cytochromes P-450

Cytochromes P-450f, P-450g, P-450h, and P-450i are four hepatic microsomal hemoproteins that have been purified from adult rats. Whereas cytochromes P-450g and P-450h appear to be male-specific hemoproteins, cytochrome P-450i is apparently a female-specific enzyme purified from untreated adult female rats. Cytochrome P-450f has been purified from adult male and female rats with equivalent recoveries. Amino-terminal sequence analyses of the first 15-20 amino acid residues of each of these cytochromes P-450 has been accomplished in the current investigation. Each protein possesses a hydrophobic leader sequence consisting of 65-87% hydrophobic amino acids, and only one charged amino acid (Asp) in the amino-terminal region. Although differences in the amino-terminal sequences of cytochromes P-450f, P-450g, P-450h, and P-450i are identified, these hemoproteins all begin with Met-Asp, and marked structural homology is observed among certain of these enzymes. Cytochromes P-450g and P-450h, two male-specific proteins, have 11-12/15 identical residues with cytochrome P-450i, a female-specific isozyme. Cytochromes P-450f and P-450h have 16/20 identical amino-terminal residues. Only limited sequence homology is observed between the amino-terminal sequences of cytochromes P-450f-i compared to rat liver cytochromes P-450a-e. The results demonstrate that cytochromes P-450f, P-450g, P-450h, and P-450i are isozymic to each other and five additional rat hepatic microsomal cytochrome P-450 isozymes (P-450a-e).     

Isolation and amino acid sequence of insulins and C-peptides of European bison (Bison bonasus) and fox (Alopex lagopus)

Insulins and C-peptides were extracted and purified from bison and fox pancreatic glands. The insulins were reduced and pyridylethylated, and the derived A- and B-chains separated by HPLC. Amino acid sequence determinations of the pyridylethylated A- and B-chains proved bisontine insulin to be identical to bovine insulin and fox insulin to be identical to dog and porcine insulin. Bisontine C-peptide proved to be identical to bovine C-peptide. The isolated fox C-peptide comprises 23 amino acid residues and probably represents a major tryptic fragment of a larger C-peptide. The fox C-peptide fragment is identical to the dog C-peptide (9-31) except for residue 3 (residue 11 in the dog C-peptide), which is aspartic acid as compared with glutamic acid in the dog C-peptide.     

A constitutive form of guinea pig liver cytochrome P450 closely related to phenobarbital inducible P450b(e)

In order to provide evidence that a cytochrome P450 belonging to the IIB subfamily is expressed as a constitutive form in the guinea pig, we tried to purify an isozyme from liver microsomes of untreated guinea pigs by assessing its reactivity with anti-P450b antibody in the present study. One form of cytochrome P450, named P450GP-1, was obtained. The minimum molecular weight of this isozyme was estimated to be 52,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino terminal sequence up to the 33rd amino acid of P450GP-1 was determined. As expected, comparison of the amino acid sequence with those of cytochrome P450 isozymes from other species reported so far indicated that P450GP-1 was highly homologous to P450s categorized in the IIB subfamily; that is, 67% similarity to rat P450b, 82% to rabbit LM2, 76% to dog PBD-2, 70% to mouse pf 3/46, and 73% to human IIB1. On the other hand, P450GP-1 showed only low similarity, less than 41%, to other cytochrome P450s of the II subfamily and those of the I, III, and IV families. Affinity of P450GP-1 to anti-P450b immunoglobulin G was confirmed to be comparable with that of a principal antigen, P450b. Immunoblot analysis revealed that P450GP-1 in the guinea pig liver microsomes was induced by phenobarbital treatment, but the increase was not as large as in the rat. P450GP-1 efficiently catalyzed benzphetamine N-demethylation, strychnine 2-hydroxylation, and testosterone 16 beta-hydroxylation, all of which are also catalyzed by P450b. Based on these results, it was strongly suggested that the IIB-type of cytochrome P450 in guinea pigs, at least one of them, is a constitutive form which is moderately induced by phenobarbital.     

Molecular cloning and expression of cDNAS encoding rat aldosterone synthase: variants of cytochrome P-450(11 beta)

Two distinct forms of cDNA encoding rat aldosterone synthase were cloned from an adrenal capsular tissue cDNA library. The deduced amino acid sequences showed that one of the enzymes (P-450(11 beta),aldo-1) had a long extension peptide composed of 34 amino acid residues while the other (P-450(11 beta),aldo-2) had an extension peptide identical to that of rat P-450(11 beta). Glu at the 320th position of P-450(11 beta),aldo-1 was replaced with Lys in P-450(11 beta),aldo-2. The amino acid sequence of the aldosterone synthase was highly homologous (81%) to rat P-450(11 beta). Constructed expression vector containing the cDNA for extension peptide of P-450(11 beta) and the mature protein of P-450(11 beta),aldo-1 was transfected into COS-7 cells. The cells converted 11-deoxycorticosterone into corticosterone, 18-hydroxycorticosterone, and aldosterone.     

Cytochrome P450 IIIA1 (P450p) requires cytochrome b5 and phospholipid with unsaturated fatty acids.

In contrast to other P450 enzymes purified from rat liver microsomes, purified P450 IIIA1 (P450p) is catalytically inactive when reconstituted with NADPH-cytochrome P450 reductase and the synthetic lipid, dilauroylphosphatidylcholine. However, purified P450 IIIA1 catalyzes the oxidation of testosterone when reconstituted with NADPH-cytochrome P450 reductase, cytochrome b5, an extract of microsomal lipid, and detergent (Emulgen 911). The present study demonstrates that the microsomal lipid extract can be replaced with one of several naturally occurring phospholipids, but not with cholesterol, sphingosine, sphingomyelin, ceramide, cerebroside, or cardiolipin. The ratio of the testosterone metabolites formed by purified P450 IIIA1 (i.e., 2 beta-, 6 beta-, and 15 beta-hydroxytestosterone) was influenced by the type of phospholipid added to the reconstitution system. The ability to replace microsomal lipid extract with several different phospholipids suggests that the nature of the polar group (i.e., choline, serine, ethanolamine, or inositol) is not critical for P450 IIIA1 activity, which implies that P450 IIIA1 activity is highly dependent on the fatty acid component of these lipids. To test this possibility, P450 IIIA1 was reconstituted with a series of synthetic phosphatidylcholines. Those phosphatidylcholines containing saturated fatty acids were unable to support testosterone oxidation by purified P450 IIIA1, regardless of the acyl chain length (C6 to C18). In contrast, several unsaturated phosphatidylcholines supported testosterone oxidation by purified P450 IIIA1, and in this regard dioleoylphosphatidylcholine (PC(18:1)2) was as effective as microsomal lipid extract and naturally occurring phosphatidylcholine or phosphatidylserine. These results confirmed that P450 IIIA1 activity is highly dependent on the fatty acid component of phospholipids. A second series of experiments was undertaken to determine whether microsomal P450 IIIA1, like the purified enzyme, is dependent on cytochrome b5. A polyclonal antibody against purified cytochrome b5 was raised in rabbits and was purified by affinity chromatography. Anti-cytochrome b5 caused a approximately 60% inhibition of testosterone 2 beta-, 6 beta-, and 15 beta-hydroxylation by purified P450 IIIA1 and inhibited these same reactions by approximately 70% when added to liver microsomes from dexamethasone-induced female rats. Overall, these results suggest that testosterone oxidation by microsomal cytochrome P450 IIIA1 requires cytochrome b5 and phospholipid containing unsaturated fatty acids.     

Conservation in the CYP51 family. Role of the B' helix/BC loop and helices F and G in enzymatic function

CYP51 (sterol 14 alpha-demethylase) is an essential enzyme in sterol biosynthetic pathways and the only P450 gene family having catalytically identical orthologues in different biological kingdoms. The proteins have low sequence similarity across phyla, and the whole family contains about 40 completely conserved amino acid residues. Fifteen of these residues lie in the secondary structural elements predicted to form potential substrate recognition sites within the P450 structural fold. The role of 10 of these residues, in the B' helix/BC loop, helices F and G, has been studied by site-directed mutagenesis using as a template the soluble sterol 14 alpha-demethylase of known structure, CYP51 from Mycobacterium tuberculosis (MT) and the human orthologue. Single amino acid substitutions of seven residues (Y76, F83, G84, D90, L172, G175, and R194) result in loss of the ability of the mutant MTCYP51 to metabolize lanosterol. Residual activity of D195A is very low, V87A is not expressed as a P450, and A197G has almost 1 order of magnitude increased activity. After purification, all of the mutants show normal spectral properties, heme incorporation, and the ability to be reduced enzymatically and to interact with azole inhibitors. Profound influence on the catalytic activity correlates well with the spectral response to substrate binding, effect of substrate stabilization on the reduced state of the P450, and substrate-enhanced efficiency of enzymatic reduction. Mutagenesis of corresponding residues in human CYP51 implies that the conserved amino acids might be essential for the evolutionary conservation of sterol 14 alpha-demethylation from bacteria to mammals.     

Role of C-terminal sequence of cytochrome P450scc in folding and functional activity

To elucidate the role of Arg472 and C-terminal sequence of the mature form of cytochrome P450scc, a mitochondrial cytochrome P450, in the present work we have performed sequential removal of the C-terminal amino acid residues of the hemeprotein and evaluated their functional role in folding and catalysis. The removal of 2, 4, 7, or 9 amino acid residues (cytochrome P450scc mutants Delta2, Delta4, Delta7, and Delta9) does not significantly affect the physicochemical properties of the truncated forms of cytochrome P450scc, but results in significant increase in the expression level of the hemeprotein in Escherichia coli (Delta4 cytochrome P450scc mutant). However, removal of 10 C-terminal amino acid residues (Delta10 cytochrome P450scc) of mature form of cytochrome P450scc (replacement of codon for Arg472 for stop-codon) is followed by loss of the ability for correct folding in E. coli. Based on these data, it is concluded that the C-terminal amino acid residues of cytochrome P450scc (DeltaArg472-Ala481) play an important role in correct recombinant protein folding and heme binding by cytochrome P450scc during its expression in E. coli, while folding of mitochondrial cytochrome P450scc during its heterologous expression in bacterial cells is more similar to the folding of prokaryotic soluble cytochrome P450's than to microsomal cytochrome P450's.     

A new family of cytochrome P450 genes (CYP41) from the cattle tick, Boophilus microplus.

We isolated and sequenced a cytochrome P450 (CYP) gene that is sufficiently different from other CYP genes that a new CYP family, CYP41 was created. CYP41 encodes a protein of 518 residues and is most similar to genes from the family CYP3; it is 36% identical to CYP3A2 and 34% identical to CYP3A28. We hypothesise that CYP41 encodes an enzyme that metabolizes xenobiotic compounds i.e. compounds that are foreign to the cattle tick. The phylogenetic position of CYP41 could not be resolved because of the high level of sequence divergence at both the nucleotide and amino acid levels.     

Molecular cloning and pharmacological characterization of the canine B1 and B2 bradykinin receptors

The dog is a valuable animal model in the study of the physiological role of both the B1 and B2 bradykinin receptors. To more thoroughly characterize the pharmacological properties of the canine kinin receptors we isolated the cDNA sequence encoding the B1 and B2 bradykinin receptor subtypes and overexpressed them in Chinese hamster ovary (CHO) cells. The cDNA sequence of the canine B1 bradykinin receptor encodes a protein comprised of 350 amino acids that is 76% identical to the human B1 bradykinin receptor. The cDNA sequence of the canine B2 bradykinin receptor encodes a protein of 392 amino acids that is 81% identical to the human B2 bradykinin receptor. The amino acid sequence of the canine B1 and B2 receptors are 35% identical. Pharmacological studies of the cloned receptors revealed that the agonist affinity of the dog B1 receptor is similar to the rodent B1 receptors, and differs from the human form in that there is no preference for the presence of the N-terminal Lys residue of [des-Arg10]Lys-bradykinin. Significantly, the B1 receptor antagonist [des-Arg9,Leu8]BK behaves as partial agonist on the cloned dog B1 receptor. The dog B2 receptor exhibits the 'classical' pharmacological properties of this receptor subtype.     

热带假丝酵母1230细胞色素P450基因CYPA14与CYPA16的克隆和表达

细胞色素P450(CYP)是一种单加氧酶,在热带假丝酵母(Candidatropicalis)ω-氧化过程中发挥关键作用。通过对来源不同的P450基因进行同源性分析,首先克隆到热带假丝酵母1230中P450基因的部分序列,再利用基因组步行法克隆其未知序列,结果分别获得了两个P450同工酶基因CYPA14和CYPA16的完整序列。经PCR方法证实,二者在染色体上的位置相邻,其读码框分别编码522和540个氨基酸残基的肽链。经NCBIBLAST搜索比较后发现,二者与热带假丝酵母ATCC20336中的P450成员CYP52A14和CYP52A16分别编码的序列几乎完全一致,与热带假丝酵母ATCC750中的P450成员CYP52A2和CYP52A1也具有较高的相似性。同时,对经诱变后的几株二元酸生产菌株的CYPA14与CYPA16也进行了克隆和序列比较,发现部分序列中的个别氨基酸残基发生了突变。CYPA14和CYPA16均在酿酒酵母中获得了有效表达,其中CYPA16的P450表达含量高于CYPA14,后者有部分表达产物发生了变性。     

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.