A previously reported cDNA clone [pP450(1)] coding for a phenobarbital-inducible cytochrome P-450 variant of rat liver microsomal membranes, designated P-450e(U.C.), was used as a specific hybridization probe to screen a human liver cDNA library. Restriction mapping showed that two of the colonies isolated contained plasmids coding for overlapping regions of the same cDNA sequence. The clone [pHP450(1)] having the longer cDNA insert (1.25 kilobase pairs) was sequenced. The homology between the rat and human cDNAs is 62% in their coding regions but is only random (24%) in the 3'-noncoding nucleotides. The amino acid sequence deduced from the human cDNA is 50% identical to that of P-450e(U.C.). The homology increases to 72% if conservative changes in amino acid residues are permitted. The hydropathy profile of the polypeptide encoded by pHP450(1) is almost identical to that of P-450e(U.C.). Regions known to be highly conserved in cytochrome P-450 isozymes isolated from rat, rabbit, and mouse were found to be conserved in the amino acid sequence derived from pHP450(1). Analysis by Southern blotting indicated that the human cytochrome P-450 encoded by pHP450(1) is part of a multigene family. The cytochrome P-450-mediated mixed-function monooxygenases of liver microsomal membranes are of central importance in the metabolism of steroid hormones and xenobiotics, including many drugs, carcinogens, and environmental pollutants (1, 2). These enzyme systems are also present in many other tissues and in a wide range of organisms (3). Each individual organism usually possesses a number of different cytochromes P-450 (4, 5), many of which are selectively inducible by a variety of foreign compounds. The spectrum of isozymes induced varies with the nature of the xenobiotic to which an organism is exposed (6, 7). In previous studies, we isolated and sequenced a cDNA clone [pP450(1)]* that codes for a phenobarbital-inducible cytochrome P-450 variant of rat liver microsomal membranes, designated P-450e(U.C.) (8, 9). cDNA clones coding for several cytochromes P-450 of rat, rabbit, mouse, cow, and chicken have been isolated by ourselves and others (10-18). They have already provided valuable information on the amino acid sequences of some cytochromes P-450 from different species and on the molecular biology of the induction processes. Individual human variations are well known in the metabolism of some foreign compounds such as therapeutic drugs (19-22). This metabolic variability may be due, in part, to genetically determined factors relating to the structure and expression of cytochrome P-450 isozymes. The existence of multiple forms of cytochrome P-450 in humans has been directly demonstrated by the isolation, from human liver microsomes, of several cytochrome P-450 isozymes that vary in their catalytic activity toward drugs and carcinogens (23). The amounts of some of the isozymes were found to vary greatly between individuals. Virtually nothing is known about the structure of human cytochromes P-450 nor is there any information available on the organization, expression, and control of the genes that code for these proteins. The isolation and characterization of DNA clones coding for human cytochrome P-450 isozymes will facilitate elucidation of the structure of these proteins and is essential for investigation of the molecular biological basis of genetically determined variation in the metabolism of foreign compounds. In this paper, we describe the use of a cloned sequence coding for a rat liver cytochrome P-450 as a molecular hybridization probe for the isolation of a cDNA clone coding for a human cytochrome P-450. The nucleotide sequence of the human cDNA and the amino acid sequence deduced from it is reported and compared with those of cytochrome P-450 variants from other organisms. MATERIALS AND METHODS Screening of the Adult Human Liver cDNA Library. The cDNA library used was provided by Derek Woods of the Harvard University Hospital Medical School, and its construction has been described (24). Bacteria were plated on two 22 x 22 cm nitrocellulose filters (Millipore Triton Free HATF) at a density of -50,000 colonies per filter and grown at 37°C on L-agar plates containing tetracycline at 12.5 ,ug/ml. When the colonies had reached a diameter of -0.5 mm, they were replica-plated twice. The colonies on the master filters were grown to a diameter of 2 mm on plates containing 25% glycerol (25) and stored at -20°C. After growth of the colonies on the duplicate replica filters to a diameter of =1 mm plasmids were amplified by chloramphenicol treatment. Colony DNA was fixed to the filters (26) and bacterial debris was removed (27). The filters were prehybridized overnight at 30°C in 50% (vol/vol) formamide/0.9 M NaCI/50 mM sodium phosphate, pH 7.7/5 mM EDTA/5 x standard Denhardt's solution (28)/0.1% NaDodSO4/denatured salmon sperm DNA (200 ,ug/ml)/ poly(A) (10 ,g/ml). The buffer was then replaced with 50% formamide/0.9 M NaCl/50 mM sodium phosphate, pH 7.7/5 mM EDTA/2 x standard Denhardt's solution/0.1% NaDodS04/denatured salmon sperm DNA (100 ,g/ml)/Escherichia coli DNA (5 ,ug/ml)/pAT153 (10 ,g/ml)/poly(A) (10 ,ug/ml)/9% (wt/vol) dextran sulfate (50 1td/cm2 of filter) and incubation was continued for 5 hr. The 32P-labeled insert of pP450(1) (ref. 9) was added to a final concentration of 5 ng/ml and the filters were hybridized for 40 hr at 30°C. The filters were washed with gentle agitation for four 15-min periods with 1 M NaCl at room temperature then for two 1-hr periods with 1 M NaCl/0.1% NaDodSO4 at 50°C (29, 30). *pP450(1) refers to the first recombinant plasmid that our group identified as containing a cDNA insert coding for a rat cytochrome P-450. pHP450(1) and pHP450(2) refer to the first and second recombinant plasmids, respectively, that were identified as containing a cDNA insert coding for a human cytochrome P-450. 983 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 984 Biochemistry: Phillips et al. Bacterial colonies in areas corresponding to strong positive signals were picked from the master filters and screened as above at a colony density of -300 per 88-mm-diameter nitrocellulose filter. Positive colonies were picked into microtiter trays, grown at 370C, transferred in an ordered array to nitrocellulose filters (9), and rescreened as above. Preparation of Radiolabeled Nucleic Acid Probes. Plasmids pP450(1) and pHP450(1) were isolated by alkaline lysis (31) and purified by centrifugation to equilibrium in a CsCl gradient (26). The cDNA insert of pP450(1) was excised by digestion with BamHI and isolated by electrophoresis in a gel of 0.8% agarose (low-melting; Seakem). The insert was recovered by chromatography on an NACS-52 column (Bethesda Research Laboratories) according to the manufacturer's recommendations and precipitated by addition of ethanol. DNAs were labeled by nick-translation (26) to a specific radioactivity of =108 dpm/,ug with [a-32P]dATP (specific radioactivity, 400-800 Ci/mmol; 1 Ci = 37 GBq; Amersham International). Southern Blotting. Plasmid DNA. Plasmid DNA was isolated (31) and cDNA inserts were excised by digestion with the appropriate restriction endonuclease. DNA was electrophoresed in a 1.5% agarose gel and blotted onto a nitrocellulose filter (0.45 ,Am; Schleicher & Schuell) (32). The filter was prehybridized at 30'C for 2 hr. The solution was replaced with hybridization buffer and incubation was continued at 30'C for 1 hr. Prehybridization and hybridization buffers were as described above for colony hybridizations. The radioactive probe was added to a final concentration of 1 ng/ml and the filter was hybridized at 30'C for 40 hr. The filter was washed under low-stringency conditions (see below). Genomic DNA. High molecular weight genomic DNA was isolated from rat liver or human leukocytes (33) and digested for 5 hr with EcoRI (New England Biolabs) (4 units/,g of DNA) under conditions recommended by the supplier. Digested DNA was electrophoresed in an 0.8% agarose gel at 1.5 V/cm for 16 hr. DNA was denatured and blotted onto a nitrocellulose filter (32). Filters were prehybridized and hybridized as described for plasmid DNA Southern blots but with the following modifications. E. coli DNA and pAT153

An amplification assay for the measurement of alkaline phosphatase has been combined with a luminescent end point using the luminol-peroxidase system to produce the first enzyme-amplified chemiluminescent assay based on the principle of prosthetogenesis. This assay is both quantitative and extremely sensitive. When the assay was used to detect alkaline phosphatase in solution, the detection limit was 0.4 amol in a 5-min assay. The interassay variance ranged from 4 to 20% and 7 to 19% across the dynamic range of the assay for a chemiluminescent assay and an enhanced chemiluminescent assay, respectively, employing two different preparations of luminol.

Nonradioactive immunoassays incorporating an element of amplification in their detection system require the use of components that are highly purified. Flavin adenine dinucleotide-3′-phosphate (FADP) is the primary substrate used in such an amplification assay. For incorporation into a simple, single-pot assay system, the concentration of contaminating flavin adenine dinucleotide (a prosthetic group for the enzyme d-aminoacid oxidase used in the amplification cascade assay) in this primary substrate must be minimized to achieve maximum sensitivity. Production of the substrate to a high degree of purity has been achieved using apo-glucose oxidase to specifically remove contaminating flavin adenine dinucleotide from solution and hydrolysis of a cyclic intermediate as a final production protocol by ribonuclease T2 to give the product in high yield. The use of continuous ultrafiltration reactors at each stage is described and compared to a final production step utilizing immobilized ribonuclease T2. These reactors allow large volumes of material to be handled and assist in the scale-up of these processes. The suitability of each protocol is assessed for the commercial production of FADP.

The coding sequence for the human pancreatic ribonuclease (HP-RNase) gene has been obtained from genomic DNA extracted from buccal epithelial cells. In order to direct the expression of the recombinant human pancreatic enzyme to the periplasmic space of E.coli, the bovine pancreatic RNase signal sequence has been fused 5' to the human gene. Initial attempts to express the recombinant enzyme were not successful, consequently site-directed mutagenesis tehniques were used to genetically engineer the HP-RNase gene to enable expression in E.coli. The resultant engineered enzyme shows similar kinetic characteristics to the homologous bovine enzyme.

In addition to hydrolysing RNA, bovine pancreatic ribonuclease splits esters of pyrimidine nucleoside 3'-phosphates, including dinucleotides. For a series of 3':5'-linked dinucleotides of general structure CpN, where N is a 5' linked nucleoside, kcat for the release of N varies enormously with the precise structure of N. Structural studies have been interpreted to indicate that the group N interacts with a subsite, B2, on the enzyme that comprises Gln69, Asn71 and Glu111. We report studies by site-directed mutagenesis that indicate that Gln69 is not involved in productive interactions with any of the dinucleotide substrates and that Asn71 is an important component of subsite B2 for all dinucleotide substrates tested. Glu111 appears to be functionally involved in catalysis for dinucleotide substrates solely when N is guanosine.

A mathematical model describing the behaviour of a new class of prosthetogenic enzyme amplification assays is described. The predictions of the model are favourably compared with an enzyme amplification assay for alkaline phosphatase. The model is used to kinetically characterise and optimise the enzyme amplification assay.

Summary  Dissociation of FAD from D-aminoacid oxidase occurred most rapidly at pH 6.0 in the presence of 1 M KBr. Diafiltration of 0.6 g of enzyme under these conditions yielded apoenzyme containing 1.3% of residual holoenzyme activity, which was subsequently reduced to less than 0.01% by chromatography on Blue Sepharose and ion exchange, giving material containing <1 ppb of contaminating phosphatase and nucleotidase.

The coding sequence for the bovine pancreatic ribonuclease (RNase) precursor has been cloned and produced in Escherichia coli using the polymerase chain reaction (PCR) technique. A PCR amplification has been carried out utilizing as template the recombinant plasmid, pQR138, which contains the coding sequence for the RNase precursor, and primers that allow for the addition of new sequences at the 5' and 3' ends of the coding sequence. The resultant fragment contains two coding sequences, one for a hexapeptide and the other for pre-RNase. This fragment has been cloned into the expression vector, pKK223.3, under the control of the tac promoter, to form a two-cistron vector. Upon induction with IPTG, E. coli cells harboring this construct generate a bicistronic mRNA which upon translation produces a hexapeptide and pre-RNase. The RNase precursor is efficiently translocated into the periplasmic space of E. coli. Upon translocation, the signal sequence is removed generating mature RNase. Formation of the disulfide bridges in RNase is facilitated by the oxidative environment of the periplasm and a fully active protein is obtained. RNase produced in E. coli has been purified to homogeneity by cation-exchange chromatography, and the removal of the signal sequence has been verified by N-terminal sequencing. The total process from inoculation of media to obtaining pure and fully active recombinant RNase is achieved in 48 h.

A simple to use, robust, quantitative, and extremely sensitive colorimetric assay for alkaline phosphatase (EC 3.1.3.1), designed to be used as a detection system in diagnostic assays employing antibodies or gene probes, is described. This technology is based on the novel principle of prosthetogenesis, according to which a purpose-designed substrate (a prosthetogen) for a primary analyte-linked enzyme label is hydrolyzed to produce a prosthetic group for a detector enzyme system. The prosthetogen employed here is a derivative of FAD which is phosphorylated at the 3'-position of the ribose ring (FADP), the label enzyme is alkaline phosphatase, and the detector is a D-amino-acid oxidase/horseradish peroxidase-coupled system. Essentially each turnover of every molecule of alkaline phosphatase produces a molecule of D-amino-acid oxidase for detection. Thus enormous amplification of the initial signal is achieved in short time periods because of the relatively high turnover number of alkaline phosphatase for FADP. The system can be formatted as a stable, preformed, freeze-dried preparation containing all analytical components, which is reconstituted simply by addition of buffer solution. This methodology can quantitate less than 0.1 amol of alkaline phosphatase in 30 min at 25 degrees C using microtiter plates.

An assay for alkaline phosphatase is described which is based on the hydrolysis of riboflavin phosphates (5'FMN or 4'FMN) to produce riboflavin. This is converted to 5'FMN using riboflavin kinase, and then assayed using the bacterial bioluminescent system from Vibrio harveyi or V. fischeri. The most sensitive assay is obtained using 4'FMN, which can measure less than 20 amol after a 1-hour incubation.

Alkaline phosphatase hydrolyzes riboflavin 4′-phosphate to produce riboflavin. This is converted to riboflavin 5′-phosphate, using riboflavin kinase, which reconstitutes apoglycolate oxidase to give hologlycolate oxidase. This enzyme catalyzes the oxidation of glycolate with simultaneous production of hydrogen peroxide which is detected via the formation of a colored product through the action of peroxidase. The system allows the detection of 4 amol after a 2-h incubation.

A highly sensitive flavin adenine dinucleotide-3'-phosphate (FADP)-based enzyme amplification cascade has been developed for determining alkaline phosphatase (ALP; EC 3.1.3.1). The cascade detects ALP via the dephosphorylation of the novel substrate FADP to produce the cofactor FAD, which binds stoichiometrically to inactive apo D-amino acid oxidase (D-AAO). The resulting active holo D-AAO oxidizes D-proline to produce hydrogen peroxide, which is quantified by the horseradish peroxidase-mediated conversion of 3,5-dichloro-2-hydroxybenzenesulfonic acid and 4-aminoantipyrine to a colored product. The FADP-based enzyme amplification cascade has been used in a novel releasable linker immunoassay (RELIA) to quantify thyrotropin (TSH). In the assay, TSH is first captured onto antibody-coated chromium dioxide particles. After formation of an antibody-TSH sandwich with a dethiobiotinylated second antibody, the complex is reacted with a streptavidin-ALP conjugate. Biotin is then used to release the conjugate into solution, and ALP is quantified in an automated version of the FADP-based amplification cascade on the aca discrete clinical analyzer (Du Pont). The sensitivity of the colorimetric RELIA assay for TSH (less than 0.1 milli-int. unit/L) is comparable with that of fluorometric assays. This technology provides a way to adapt to the aca high-sensitivity immunoassays for a wide range of analytes via colorimetric detection.

We have investigated the ability of camphor, menthol, pinene, limonene and myrcene to induce in rats members of a cytochrome P-450 sub-family termed PB P-450. These proteins have recently been designated as members of the P450IIB sub-family. None of these naturally occurring terpenoids significantly changed the total content of cytochromes P-450 or cytochrome b5. Radioimmunoassay results showed that PB P-450 was induced 6-fold by camphor and to a lesser extent by menthol and pinene. The induction was confirmed by Western blotting. It was shown by nucleic acid hybridization that induction of PB P-450 by terpenoids was mediated by an increase in the amount of the corresponding mRNA. Analysis of the denaturation of mRNA-cDNA hybrids demonstrated that the mRNA induced by the terpenoids was encoded by a member of the P450IIB sub-family. None of the terpenoids had an effect on the amount of mRNA coding for P450IA2 (a cytochrome P-450 inducible by beta-naphthoflavone and isosafrole). The results indicate that cytochromes P-450 induced by a synthetic compound, phenobarbital, may have originally evolved in response to terpenoid compounds normally present in the environment.

We have previously shown that the 43-fold induction by phenobarbital of the major phenobarbital-inducible cytochrome P-450 of rat liver microsomal membranes (PB P-450) is mediated by a 20-fold increase in the amount of its mRNA in the cytoplasm. Here we demonstrate that the induction of the mRNA can be almost entirely accounted for by an increase in the rate of transcription of genes coding for PB P-450, and involves little or no change in the rates of processing, transport or degradation of the mRNA. Phenobarbital treatment resulted in no amplification or rearrangement of PB P-450 genes.

A previously reported cDNA clone [pP450(1)] coding for a phenobarbital-inducible cytochrome P-450 variant of rat liver microsomal membranes, designated P-450e(U.C.), was used as a specific hybridization probe to screen a human liver cDNA library. Restriction mapping showed that two of the colonies isolated contained plasmids coding for overlapping regions of the same cDNA sequence. The clone [pHP450(1)] having the longer cDNA insert (1.25 kilobase pairs) was sequenced. The homology between the rat and human cDNAs is 62% in their coding regions but is only random (24%) in the 3'-noncoding nucleotides. The amino acid sequence deduced from the human cDNA is 50% identical to that of P-450e(U.C.). The homology increases to 72% if conservative changes in amino acid residues are permitted. The hydropathy profile of the polypeptide encoded by pHP450(1) is almost identical to that of P-450e(U.C.). Regions known to be highly conserved in cytochrome P-450 isozymes isolated from rat, rabbit, and mouse were found to be conserved in the amino acid sequence derived from pHP450(1). Analysis by Southern blotting indicated that the human cytochrome P-450 encoded by pHP450(1) is part of a multigene family.

Conference Article| August 01 1984 Microheterogeneity in a cytochrome P-450 multigene family ALAN ASHWORTH; ALAN ASHWORTH 1Department of Biochemistry, University College London, Gower Street, London WC1E 6BT, U.K. Search for other works by this author on: This Site PubMed Google Scholar ELIZABETH SHEPHARD; ELIZABETH SHEPHARD 1Department of Biochemistry, University College London, Gower Street, London WC1E 6BT, U.K. Search for other works by this author on: This Site PubMed Google Scholar BRIAN RABIN; BRIAN RABIN 1Department of Biochemistry, University College London, Gower Street, London WC1E 6BT, U.K. Search for other works by this author on: This Site PubMed Google Scholar IAN PHILLIPS IAN PHILLIPS 1Department of Biochemistry, University College London, Gower Street, London WC1E 6BT, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1984) 12 (4): 669–670. https://doi.org/10.1042/bst0120669 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation ALAN ASHWORTH, ELIZABETH SHEPHARD, BRIAN RABIN, IAN PHILLIPS; Microheterogeneity in a cytochrome P-450 multigene family. Biochem Soc Trans 1 August 1984; 12 (4): 669–670. doi: https://doi.org/10.1042/bst0120669 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: cDNA, DNA complementary to mRNA, NF, β-naphthoflavone, PB, phenobarbital, PB P-50, the major phenobarbital-inducible cytochrome P-50 of Mr 52000, and its immunochemically indistinguishable microheterogeneous variants, P-450e(U.C), a microheterogeneous variant of cytochrome P-450e present in Sprague-Dawley rats of the University College London animal facility, SSPE, 0.18m-NaCl, 10mm-sodium phosphate (pH7.7), 1mm-EDTA This content is only available as a PDF. © 1984 Biochemical Society1984 Article PDF first page preview Close Modal You do not currently have access to this content.

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 β-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.

NADPH-cytochrome c (P-450) reductase from liver microsomes of phenobarbital-treated rats has been purified in a single step by affinity chromatography on agarose-hexane-adenosine 2',5'-diphosphate. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme assay, and radioimmunoassay the protein obtained by this single step procedure is as pure as that isolated by multicolumn procedures.