Search PubMed⌕ Search

Biomedical subjects

F J Gonzalez

Publications and source records attributed to F J Gonzalez.

At least 343 records · Page 19Linked to original sources
PubMed1989

Novel exogenous heme-dependent expression of mammalian cytochrome P450 using baculovirus.

Rat P450 IIA1 is a membrane-bound hemoprotein that catalyzes the 7 alpha- and 6 alpha-hydroxylation of testosterone. A recombinant baculovirus, containing the cDNA encoding IIA1, was constructed and used to infect Spodoptera frugiperda cells. Infected cells contained up to 10% IIA1 protein as quantified by Western blot analysis; however, only a small percentage of this protein contained heme and was catalytically active. Addition of hemin to the culture medium during the course of viral infection resulted in a marked sixfold increase in both testosterone hydroxylase activity and heme-containing IIA1 protein. When the exogenously added protoheme was substituted with modified heme molecules containing hydrogen or ethyl groups at the 2 and 4 positions of the protoporphyrin IX, enzymes with only marginal changes in catalytic properties were produced. These data indicate that baculovirus can be used as a system to produce high levels of mammalian cytochrome P450s containing custom modified heme moieties.

Animals↗
PubMed1989

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 Acid Sequence↗
PubMed1989

cDNA cloning and sequence and cDNA-directed expression of human P450 IIB1: identification of a normal and two variant cDNAs derived from the CYP2B locus on chromosome 19 and differential expression of the IIB mRNAs in human liver.

A cDNA designated hIIB1, representing the entire coding sequence of a P450 in the IIB gene family, was isolated from a human liver lambda gt11 library by using the rat IIB1 cDNA as a probe. The hIIB1 protein, deduced from the cDNA sequence, contained 491 amino acids, had a calculated molecular weight of 56,286, and displayed 76% amino acid similarity with the rat IIB1 protein. Expression of this cDNA, using the vaccinia virus system, yielded a P450 that had a reduced CO-binding spectrum with an absorption maximum of 452 nm. The expressed human enzyme was able to catalyze the deethylation of 7-ethoxy-coumarin. Total RNA from 13 livers was probed for levels of hIIB mRNA. Two livers had high levels, four contained moderate levels, and eight contained very low, or no detectable, mRNA. These data suggest either that defective hIIB1 genes exist in humans or that the hIIB1 gene is regulated and variably induced in our liver specimens. To search for mutant mRNA transcripts, libraries were constructed from livers expressing low levels of hIIB1 mRNA. A cDNA, designated hIIB2, was isolated that was identical with the hIIB1 cDNA except for the presence of an unusual alteration of the DNA near its 5' end corresponding to the putative exon 4. This alteration was caused by a deletion of 29 bp and an insertion of 44 bp of nonhomologous DNA. This sequence replacement occurs at the junction of the third and fourth exons as predicted from the structure of the rat IIB1 gene, suggesting that a faulty splice might have given rise to the variant hIIb2 transcript.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1989

The CYP2D gene subfamily: analysis of the molecular basis of the debrisoquine 4-hydroxylase deficiency in DA rats.

The DA rat has been proposed as an animal model for the human debrisoquine 4-hydroxylase/bufuralol 1'-hydroxylase genetic deficiency. To determine the mechanism of this deficiency, we isolated and sequenced five cDNAs in the CYP2D gene subfamily including a new IID1 allele and two cDNAs of novel P450s, designated IID3 and IID5. IID3 and IID5 cDNA-deduced amino acid sequences contained 500 and 504 residues with calculated molecular weights of 56,683 and 57,081, respectively. IID5 displayed 20 amino acid differences with the IID1, yet bore only 72% and 76% similarity to IID2 and IID3. Despite an overall nucleotide similarity of 80-98% between the 4 cDNAs, a region of 134 nucleotides of sequence exists that contains only 1 base difference. This region is probably the result of gene conversion events between the P450 IID genes. Although all IID cDNAs were expressed into immunodetectable proteins using the COS cell SV40-based expression system, only IID1 could effectively catalyze the oxidation of the prototype substrate bufuralol. Expression of a cDNA isolated in an earlier study [Gonzalez, F. J., Matsunaga, T., Nagata, K., Meyer, U. A., Nebert, D. W., Pastewka, J., Kozak, C. A., Gillette, J., Gelboin, H. V., & Hardwick, J. P. (1987) DNA 6, 149-161], previously called db1 and now designated IID1v, produced a protein with a drastically reduced activity as compared to cDNA-expressed IID1 despite only four amino acid differences between the two cDNA-deduced protein sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1989

Identification of a novel P450 expressed in rat lung: cDNA cloning and sequence, chromosome mapping, and induction by 3-methylcholanthrene.

A novel P450 cDNA was isolated from a rat lung lambda gt11 library by hybridization with the rat P450 IIB1 cDNA probe. The cDNA-deduced amino acid sequence of this clone was 71% and 73% similar to rat IIA1 and IIA2 P450s; it was, therefore, designated IIA3 as the third member of the rat IIA subfamily. IIA3 demonstrates only 55% amino acid similarity with IIB1. Interestingly, this P450 also shared 85% and 94% amino acid similarities with human IIA3 and a mouse testosterone 15 alpha-hydroxylase P450, respectively, indicating that these P450s are orthologous counterparts to rat IIA3. Chromosome mapping, using mouse-hamster somatic cell hybrids, revealed that the IIA3 gene is localized on mouse chromosome 7. The IIA3 mRNA was detected in rat lung, and its level was induced 3-fold by treatment of rats with 3-methylcholanthrene. No IIA3 mRNA was seen in the liver, kidney, or intestine, even after long exposure of Northern blot filters to X-ray film. In contrast, the orthologous mouse and human IIA3 genes are expressed in liver.

Amino Acid Sequence↗
PubMed1989

Induction of rat hepatic N-nitrosodimethylamine demethylase by acetone is due to protein stabilization.

The N-nitrosodimethylamine demethylase (P450I-IE1) is induced severalfold in liver by giving rats ethanol, acetone, pyrazole, and other related small molecular weight compounds. This induction is not the result of an increase in IIE1 mRNA, but could be due to either an increase in translation rate or a decrease in protein degradation. To determine the mechanism of induction, we measured IIE1 synthesis and degradation rates in untreated and acetone-treated rats. This was accomplished by immunopurification of radiolabeled IIE1 protein using a specific monoclonal antibody subsequent to in vivo labeling of total cellular protein with either NaH14CO3 or [3H]leucine. We found that in rats fed acetone, the rate of IIE1 synthesis was not changed; however, IIE1 degradation was markedly altered. In untreated rats, IIE1 protein was degraded via a biphasic pathway consisting of both a rapid and slow component with approximate half-lives of 7 and 37 h, respectively. However, in acetone-treated rats, only a monophasic curve with a half-life of 37 h was observed. The abolition of the rapid degradation component of the IIE1 turnover cycle indicates that induction of IIE1 by acetone is primarily due to specific stabilization of IIE1 protein. Since acetone is also metabolized by IIE1, we believe that this may be a substrate-induced enzyme stabilization.

Acetone↗
PubMed1989

Mutagen activation by cDNA-expressed P(1)450, P(3)450, and P450a.

cDNAs for rodent P(1)450, P(3)450, and P450a were expressed in the modified vaccinia virus-T7 RNA polymerase system. Each P450 exhibited its appropriate molecular weight and characteristic enzyme activity. Aryl hydrocarbon hydroxylase activity was catalyzed by P(1)450, acetanilide hydroxylase by P(3)450, and testosterone 7 alpha-hydroxylase by P450a. Ethoxycoumarin deethylase was exhibited by both P(1)450 and P(3)450. Each expressed P450 was also analyzed for its ability to activate 19 carcinogens of diverse classes to their mutagenic forms. Most notable was the activation of several polycyclic aromatic hydrocarbons by P1 and the activation of acetylaminofluorene, 4-aminobiphenyl, and several heterocyclic amine food pyrolysate products by P(3)450. P450a, in contrast, showed slight mutagen activation only toward N-hydroxy-2-acetyl aminofluorene. The vaccinia virus-T7 RNA polymerase system described here can express cDNAs for diverse forms of P450, each of which can then be characterized for substrate and product specificity and for mutagen activation.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1989

Human cDNA-expressed cytochrome P450 IA2: mutagen activation and substrate specificity.

The vaccinia virus cDNA expression system was used to produce human cytochrome P450 IA2 in a hepatoma cell line that is devoid of significant basal levels of P450. The expressed enzyme yielded a reduced carbon monoxide-bound difference spectrum with a lambda max of 449 nm. Catalytic activities and mutagen activation ability of the human enzyme were assessed and directly compared with results obtained with the orthologous mouse IA2, which was also expressed using vaccinia virus. Both the human and mouse enzymes were able to catalyze efficiently the p-hydroxylation of aniline. Mouse IA2 also catalyzed ethoxyresorufin O-deethylation, and its activity was sevenfold greater than expressed human IA2. The mouse and human enzymes also activated several promutagens and procarcinogens. Mouse IA2 was five- to sevenfold more active than the human enzyme for activation of the procarcinogens 2-acetylaminofluorene and benzo[a]pyrene-trans-7,8-dihydrodiol and the promutagens Glu-P-2 and Trp-P-1. Comparable activities were observed with 2-aminoanthracene, 2-aminofluorene, and Glu-P-1. These data demonstrate the utility of cDNA expression for examining the activities of human P450s and further suggest potentially important differences in catalytic activities of orthologous P450s found in different species.

Animals↗
PubMed1989

Lidocaine metabolism in human liver microsomes by cytochrome P450IIIA4.

The metabolism of lidocaine to its major metabolite monoethylglycinexylidide (MEGX) was studied in human liver microsomes of 13 kidney transplant donors and of one patient with liver cirrhosis. Interindividual variation in metabolite formation was considerable. Biphasic kinetics indicated the involvement of at least two distinct enzymatic activities. With use of a series of antisera that recognize different human cytochrome P450 isozymes, we were able to identify an enzyme of the P450III gene family as one of two enzymes. By expressing human P450IIIA4 complementary deoxyribonucleic acid (cDNA) in HepG2 cells, we directly demonstrated lidocaine-deethylase activity for this P450 isozyme. These data suggest that P450IIIA4 is at least in part responsible for microsomal MEGX formation.

Cells, Cultured↗
PubMed1989

Marked increases in hepatic NAD(P)H:oxidoreductase gene transcription and mRNA levels correlated with a mouse chromosome 7 deletion.

The NAD(P)H:menadione oxidoreductase gene (Nmo-1) codes for a quinone reductase (also called DT diaphorase; EC 1.6.99.2) believed to play a central role in protection against oxidative stress. We have studied mice with a radiation-induced chromosomal deletion involving the albino locus (c) on chromosome 7 and found that Nmo-1 mRNA levels and the rate of Nmo-1 gene transcription are markedly increased (greater than 100-fold and greater than 12-fold, respectively) in the untreated c14CoS/c14CoS deletion homozygote, compared with the untreated Cch/Cch wild-type and the Cch/C14CoS heterozygote. These data suggest that a gene located on chromosome 7 encodes a trans-acting regulatory factor that might be a negative effector of the Nmo-1 gene, which we show here is located on chromosome 8 approximately 1.4 centimorgans (about 1000 kilobase pairs) from the Es-2 gene. Conversely, there are no detectable basal levels of cytochrome P1450 (Cyp1a1 gene) or cytochrome P3450 (Cyp1a2 gene) mRNA, indicating that the regulation of basal expression of the Cyp1a1 and Cyp1a2 genes is distinct from that of the Nmo-1 gene. Moreover, the Cyp1a1 and Cyp1a2 genes and the Nmo-1 gene are induced by tetrachlorodibenzo-p-dioxin in the cch/cch, cch/c14CoS, and c14CoS/c14CoS mice. The mechanism of tetrachlorodibenzo-p-dioxin inducibility of the Cyp1a1, Cyp1a2, and Nmo-1 genes is, therefore, independent of the mechanism of Nmo-1 gene activation in untreated c14CoS/c14CoS mice.

Aging↗
PubMed1989

The rat clofibrate-inducible CYP4A gene subfamily. I. Complete intron and exon sequence of the CYP4A1 and CYP4A2 genes, unique exon organization, and identification of a conserved 19-bp upstream element.

The P450 CYP4A1 and CYP4A2 genes were isolated from a rat genomic library constructed in the vector lambda EMBL3 and their complete sequences were determined. The CYP4A1 and CYP4A2 genes spanned 14,144 and 10,576 bp and contained 13 and 12 exons, respectively. The CYP4A1 gene contained an additional intron that splits the exon corresponding to exon 12 of the CYP4A2 gene, resulting in a noncoding 13th exon in CYP4A1. The exon numbers of these genes were distinct among known P450 genes, and yet several intron-exon junctions along the P450 amino acid coding region were conserved with P450 genes in the CYP2, CYP11, and CYP21 gene families. On the basis of these data, the number of exons in the putative ancestral P450 gene was estimated. The evolutionary implications of this finding are discussed. No consensus TATA sequence was found upstream of either gene's transcription start site. Comparison of the CYP4A1 and CYP4A2 promoters with other genes that lack TATA boxes did not reveal any strong consensus sequence in their immediate upstream regions. However, a conserved 19-bp sequence was located at the positions of 42 and 48 bp upstream from the CYP4A1 and CYP4A2 genes' start sites, respectively. The CYP4A2 gene also contained two 378-bp direct repeats upstream from the start site; these repeats are derived from portions of the long interspersed middle repetitive element present in high copy numbers in the rat genome.

Amino Acid Sequence↗
PubMed1989

The rat clofibrate-inducible CYP4A subfamily. II. cDNA sequence of IVA3, mapping of the Cyp4a locus to mouse chromosome 4, and coordinate and tissue-specific regulation of the CYP4A genes.

A novel P450 cDNA, designated IVA3, was isolated from a lambda gt11 cDNA library from clofibrate-treated rat liver by screening with the lauric acid omega-hydroxylase, IVA1, cDNA probe. This cDNA encoded a protein with 507 amino acids and a calculated Mr of 58,239. The IVA3 cDNA shared 65% and 97% nucleotide and 72% and 96% DNA-deduced amino acid sequence similarities with IVA1 and IVA2, respectively. The CYP4A gene family, designated the Cyp4a locus, was mapped to mouse chromosome 4 using a panel of mouse-hamster somatic cell hybrids. Levels of the IVA mRNAs were analyzed in rat tissues and cell cultures after treatment with the hypolipidemic drug clofibrate. The IVA1, IVA2, and IVA3 mRNAs were present at very low levels in the livers of untreated rats and markedly induced in rats treated with clofibrate. Dose-response and time-course studies revealed that all three genes were regulated coordinately in liver. In contrast to the coordinate induction in liver, only the CYP4A3 gene was induced in the rat hepatoma cell line McA-RH7777. In the kidney, IVA1 and IVA3 mRNAs were present at low levels and were induced by clofibrate in a manner similar to that in liver. In contrast, the IVA2 mRNA was expressed in the kidney of untreated rats at a level similar to that of the maximally induced IVA2 mRNA in liver. These data indicate that the CYP4A1, CYP4A2, and CYP4A3 genes are regulated coordinately in liver while only CYP4A2 is activated constitutively in kidney.

Amino Acid Sequence↗
PubMed1989

Structure and regulation of P-450s in the rat P450IIA gene subfamily.

The rat P450IIA subfamily was characterized at the protein, cDNA, and gene level. The purified IIA1 and IIA2 P-450s displayed distinct positional specificities toward hydroxylation of the prototype substrate testosterone. The IIA1 and IIA2 genes were also regulated differently during development; IIA1 was expressed within 1 week after birth in both males and females, and was specifically suppressed in males at puberty, while IIA2 was not expressed in females and was activated at puberty in males. The cDNA-deduced amino acid sequences of these enzymes showed 88% similarity with interspersed regions of high and low similarity indicative of former gene conversion events. Both the IIA1 and IIA2 genes contained nine exons.

Aging↗
PubMed1989

Human NADPH-P450 oxidoreductase: complementary DNA cloning, sequence and vaccinia virus-mediated expression and localization of the CYPOR gene to chromosome 7.

The cDNA containing the full coding sequence of human NADPH-P450 oxidoreductase was isolated and completely sequenced. The cDNA contained 2398 base pairs, including 9 and 358 base pairs of 5' and 3' noncoding sequences, respectively. The human NADPH-P450 oxidoreductase protein deduced from the cDNA has 677 amino acids, with a calculated molecular weight of 76,656. The cDNA nucleotide and deduced amino acid sequences displayed 83 and 92% similarities, respectively, with those of the rat NADPH-P450 oxidoreductase. By use of somatic cell hybrids, the NADPH-P450 oxidoreductase gene was regionally localized to human chromosome 7 (7p15-q35). The levels of NADPH-P450 oxidoreductase protein and mRNA were analyzed in 13 human liver specimens and less than 3-fold variation was found among the different livers. The NADPH-P450 oxidoreductase cDNA was inserted into vaccinia virus and expressed in cell culture. The cDNA-expressed enzyme was active in reducing the electron acceptor cytochrome c. In addition, the NADPH-P450 oxidoreductase stimulated the enzymatic activity of vaccinia virus-expressed human P3(450) when both recombinant viruses were used to coinfect human cells in culture. An approximate equal mole level of NADPH-P450 oxidoreductase and P3(450) was required to achieve maximal activity for both ethoxycoumarin O-deethylase and aryl hydrocarbon hydroxylase.

Animals↗
PubMed1989

The human debrisoquine 4-hydroxylase (CYP2D) locus: sequence and identification of the polymorphic CYP2D6 gene, a related gene, and a pseudogene.

The debrisoquine-4-hydroxylase polymorphism is a genetic variation in oxidative drug metabolism characterized by two phenotypes, the extensive metabolizer (EM) and poor metabolizer (PM). Of the Caucasian populations of Europe and North America, 5%-10% are of the PM phenotype and are unable to metabolize debrisoquine and numerous other drugs. The defect is caused by several mutant alleles of the CYP2D6 gene, two of which are detected in about 70% of PMs. We have constructed a genomic library from lymphocyte DNA of an EM positively identified by pedigree analysis to be homozygous for the normal CYP2D6 allele. The normal CYP2D6 gene was isolated; was completely sequenced, including 1,531 and 3,522 bp of 5' and 3' flanking DNA, respectively; and was found to contain nine exons within 4,378 bp. Two other genes, designated CYP2D7 and CYP2D8P, were also cloned and sequenced. CYP2D8P contains several gene-disrupting insertions, deletions, and termination codons within its exons, indicating that this is a pseudogene. CYP2D7, which is just downstream of CYP2D8P, is apparently normal, except for the presence, in the first exon, of an insertion that disrupts the reading frame. A hypothesis is presented that the presence of a pseudogene within the CYP2D subfamily transfers detrimental mutations via gene conversions into the CYP2D6 gene, thus accounting for the high frequency of mutations observed in the CYP2D6 gene in humans.

Amino Acid Sequence↗
PubMed1989

Oxidation of midazolam and triazolam by human liver cytochrome P450IIIA4.

The metabolism of midazolam and triazolam to their 1'-hydroxy and 4-hydroxy metabolites was studied in microsomes of 15 human livers. The formation of both metabolites was inhibited by more than 90% by an antiserum directed against a pregnenolone 16 alpha-carbonitrile-inducible cytochrome P450 (P450PCN1) of rat liver. Moreover, midazolam hydroxylase activity was immunoprecipitated from solubilized human microsomes with polyclonal antibodies against rat P450PCN1 and the closely related human isozyme P450NF. A close correlation was observed between the amount of protein detected in immunoblots with these antibodies and the midazolam or triazolam hydrxylase activity. The formation of both metabolites of midazolam was inhibited by triacetyloleandomycin, a known inhibitor of cytochromes P450 of the IIIA family. Direct evidence that P450IIIA4 catalyzes the metabolism of midazolam was provided through the use of cDNA-directed expression. Monkey COS cells transfected with human P450PCN1 cDNA were able to catalyze both the 1'- and the 4-hydroxylation of midazolam. We conclude that the metabolism of midazolam and triazolam in human liver is predominantly mediated by cytochrome P450IIIA4. Two of 15 human livers expressed a second immunoreactive microsomal protein of higher apparent Mr and were more active in midazolam 1'-hydroxylation. Our data also provide evidence that the marked interindividual variation in the response to these widely used benzodiazepine drugs is due to variable hepatic metabolism.

Animals↗
PubMed1988

Human ethanol-inducible P450IIE1: complete gene sequence, promoter characterization, chromosome mapping, and cDNA-directed expression.

The human P450IIE1 gene, coding for an ethanol-inducible nitrosamine-metabolizing P-450, was isolated from a lambda EMBL3 genomic library and completely sequenced. The human gene spanned 11,413 base pairs and contained nine exons and a typical TATA box. Upstream and downstream DNAs of 2788 and 559 base pairs were also sequenced and compared to the rat gene. Significant areas of sequence similarity were observed within 140 base pairs upstream of the transcription start site in the rat and human genes. Human DNA 539 base pairs upstream of the transcription start site was inserted into the expression vector pSVOAL delta 5', and luciferase activity was detected when the constructs were introduced into a rat hepatoma cell line. The activity was over 100-fold lower than that of pRSVL, a Rous sarcoma virus LTR-driven luciferase gene. By use of panels of rodent-human cell hybrids, the gene was mapped to chromosome 10 (CYP2E locus). A full-length cDNA, constructed with the first exon of the genomic clone and a partial cDNA clone, was expressed in COS cells and found to code for N-nitrosodimethylamine demethylase activity.

Animals↗