A comparison of levels of glutathione transferases, cytochromes P450 and acetyltransferases in human livers.
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Acetyltransferases↗
Biomedical subjects
Y Yamazoe
Publications and source records attributed to Y Yamazoe.
An arylamine acetyltransferase (AT-I) from Syrian golden hamster liver: cloning, complete nucleotide sequence, and expression in mammalian cells.
A cDNA clone (designated hamAT101) encoding an arylamine acetyltransferase, AT-1, was isolated from a hamster liver lambda gt11 cDNA library using a specific polyclonal antibody raised against AT-1. The cloned cDNA insert consisted of 1181 nucleotides, including an open reading frame of 870 nucleotides encoding 290 amino acid (Mr 33,503). The isolated cDNA displayed high sequence similarity to those of chicken, rabbit, and human acetyltransferases. In Northern blots, the hamAT101 cDNA probe hybridized to an RNA band of 18S in the livers of both slow and rapid acetylator phenotypes. To confirm that hamAT101 cDNA encodes the monomorphic but not the polymorphic protein, the isolated cDNA was expressed in monkey kidney cells (COS-1 cells) using the vector p91023(B). A protein with a molecular weight similar to that of AT-1 was detected upon Western blotting in the 9000 x g supernatant from the transfected cells. The activity toward four different substrates of the 9000 x g supernatant was also examined. In agreement with the results of purified AT-1, the cDNA-expressed protein exhibited a high capacity for N-acetylation of 4-aminoazobenzene and 2-aminofluorene, and O-acetylation of 2-hydroxyamino-6-methyldipyrido [1,2-a:3',2'-d] imidazole, whereas no activity was found for the N-acetylation of p-aminobenzoic acid. These results, in addition to the RNA blot hybridization, indicate that hamAT101 encodes the hamster acetyltransferase AT-1.
Amino Acid Sequence↗
Characterization of human P450IIC isozymes by using yeast expression system.
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Base Sequence↗
Cytochrome P-450 human-2 (P-450IIC9) in mephenytoin hydroxylation polymorphism in human livers: differences in substrate and stereoselectivities among microheterogeneous P-450IIC species expressed in yeasts.
The cDNA of a P-450 human-2 and the two other closely related cDNAs, MP-8 (two deduced amino acids substituted) and lambda hPA6 (two deduced amino acids deleted) were expressed in Saccharomyces cerevisiae cells, and their catalytic and chemical properties were compared to identify which cDNA encodes a major S-mephenytoin 4'-hydroxylase in human livers. In immunoblots, P-450 human-2 cDNA-derived protein in yeasts was stained at the position identical with P-450 human-2 purified from liver and a major protein in microsomes of 19 Japanese livers. MP-8- and lambda hPA6-derived proteins were immunostained at positions near, but distinct from P-450 human-2, and were not detected in those 19 livers. All three proteins expressed in yeasts catalyzed hydroxylation of mephenytoin, hexobarbital, benzo[a]pyrene and tolbutamide, although the rates of the hydroxylation of most of the drugs by P-450 human-2 were higher than those of the two others. In addition, these expressed proteins showed clear differences in the hydroxylation of chiral substrates: P-450 human-2 catalyzed the hydroxylation of S-mephenytoin five times faster than that of the R-enantiomer. Similar high enantioselectivities were also observed on the hydroxylation of R- and S-hexobarbital. However, MP-8- and lambda hPA6-derived proteins catalyzed hydroxylation of these two drugs with less or almost no stereoselectivity. These results indicate that only a few amino acid alterations cause dramatic changes in both the chemical and catalytic properties of P-450 human-2.
Base Sequence↗
Purification of hepatic N-hydroxyarylamine sulfotransferases and their regulation by growth hormone and thyroid hormone in rats.
From liver cytosols of male Sprague-Dawley rats, two N-hydroxyarylamine sulfotransferases (HASTs) which sulfate N-hydroxy-2-acetylaminofluorene and a phenolsulfotransferase (PST-I) have been purified about 290-, 690-, and 210-fold, respectively, by the use of DEAE-anion exchange, Blue-Sepharose CL-6B, DEAE-HPLC, and ATP-agarose affinity chromatography. All three enzymes showed almost the same molecular weight of 33 kDa on SDS-polyacrylamide gel electrophoresis. In Western blots using antibody raised against HAST I, the two HASTs, but not PST-I, were detected. The content of total HAST in male rats was estimated as 4-5 micrograms/mg cytosolic protein, about 4 times higher than that in female rats. Direct comparison of the DEAE-HPLC elution profiles of cytosol showed that HAST I and HAST II were male-dominant and male-specific, respectively, in their expression in the livers. Hypophysectomy decreased the level of HAST by 60% in male rats, but had no obvious effect in female rats. Intermittent injection of growth hormone to mimic the male secretory pattern raised the content in hypophysectomized rats of both sexes close to that in intact male rats, while the continuous infusion of growth hormone to mimic the female secretory pattern showed limited effects. In addition, the administration of triiodothyronine stimulated HAST in hypophysectomized rats of both sexes, and the extent of stimulation was nearly the same as observed in the male-type growth hormone treatment. PST-I, in contrast to HAST, showed no clear sex-related difference in hepatic content, and was not apparently affected by growth hormone or triiodothyronine.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Regulations of male-dominant P-450Md mRNA in rat liver by hormonal factors and xenobiotics.
Male-dominant P-450Md mRNA was undetectable in the livers of newborn rats. In female rat livers, the mRNA appeared at 17 days of age and then decreased to very low levels in the adult periods. The level of P-450Md mRNA in female rats was increased by phenobarbital or dexamethasone treatment, whereas the level in the males was depressed by methylcholanthrene. Hypophysectomy decreased the level of P-450Md mRNA in male rat livers, and continuous infusion or twice-daily injections of growth hormone to hypophysectomized rats caused further suppression or clear restoration, respectively, of the mRNA level.
Animals↗
Difference in the susceptibility of two phenobarbital-inducible forms, P450IIB1 and P450IIB2, to thyroid hormone- and growth hormone-induced suppression in rat liver: phenobarbital-inducible P450IIB2 suppression by thyroid hormone acting directly, but not through the pituitary system.
Suppression of two major phenobarbital-inducible cytochrome P-450s, P450IIB1 and P450IIB2, by thyroid hormone was studied and compared with growth hormone (GH)-induced suppression in rats in vivo and hepatocytes in primary culture in vitro. Treatment of adult male rats with 50 micrograms/kg triiodothyronine (T3) reduced the constitutively expressed amounts of P450IIB1 (up to 1 pmol/mg of protein) and P450IIB2 (2-5 pmol/mg of protein) to 42% and 3% of their levels in nontreated controls. Thyroidectomy increased the hepatic contents of P450IIB2 (to levels of 50-80 pmol/mg of protein) and, to a lesser extent, P450IIB1 (1-5 pmol/mg of protein) in male and female rats. Supplement of T3 to thyroidectomized rats reversed the increased contents to levels similar to those observed in normal rats. Hypophysectomy also increased both P450IIB1 and P450IIB2 protein, and their levels in both sexes were similar to that of P450IIB2 in thyroidectomized rats. Treatment of hypophysectomized rats with T3 as well as human GH suppressed hepatic contents of P450IIB1 and P450IIB2. In a hepatocyte culture including 2 mM phenobarbital, T3 and GH suppressed both P450IIB1 and P450IIB2. Other thyroid hormone derivatives, including thyroxine, D-T3, and reversed T3, also showed suppressive effects, in parallel with the potencies for their stimulatory action that have been reported. These results indicate that thyroid hormone may suppress both P450IIB1 and P450IIB2 by a direct effect on the liver, but not by an indirect effect through the modulation of pituitary GH synthesis. The high susceptibility of hepatic P450IIB2 to thyroid hormone-induced suppression also indicates that constitutive and phenobarbital-induced levels of P450IIB2 are suppressively regulated preferentially by thyroid hormone, in contrast to the high susceptibility to GH of P450IIB1 in rat liver. In addition, a difference in the suppressive mechanisms of thyroid hormone and GH was suggested by the difference in susceptibility to cycloheximide.
Animals↗
cDNA and deduced amino acid sequences of a male dominant P-450Md mRNA in rats.
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Amino Acid Sequence↗
Nucleotide sequence of a full-length cDNA (PST-1) for aryl sulfotransferase from rat liver.
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Amino Acid Sequence↗
cDNA cloning of the hydroxysteroid sulfotransferase STa sharing a strong homology in amino acid sequence with the senescence marker protein SMP-2 in rat livers.
A cDNA encoding hydroxysteroid sulfotransferase a (STa), which catalyzes activation of carcinogenic polycyclic hydroxymethyl-arenes, was isolated from a lambda gtll cDNA expression library constructed from poly(A)+RNA of a female Sprague-Dawley (SD) rat liver. The cDNA, designated as ST-40, consisted of 1,015 base pairs which had an open reading frame of 852 base pairs encoding the entire rat STa subunit of 284 amino acids. The nucleotide base sequence of the ST-40 cDNA shared a strong homology of 94.4% with that of ST-20 cDNA encoding a hydroxysteroid ST which had been reported by us. The deduced amino acid sequence of STa had a homology of 73.7% with that of an SD rat liver senescence marker protein (SMP-2) consisting of 282 amino acid residues. However, STa was found to share a much stronger homology of 92% on the average with SMP-2 in their four specific regions corresponding to about 60% of the total sequences, indicating SMP-2 to be an isozyme of hydroxysteroid ST.
Amino Acid Sequence↗
Effect of growth hormone on rat hepatic cytochrome P-450f mRNA: a new mode of regulation.
Growth hormone (GH) is known to be involved in the control of rat hepatic drug and steroid metabolism through its action on cytochrome P-450s. To examine the role of GH in the regulation of cytochrome P-450f (P-450f), a full-length cDNA clone corresponding to P-450f was isolated and the 3'-non-coding region was utilized for Northern and slot-blot analyses. P-450f mRNA levels were low in neonates, increased after age 4 weeks in male and female rats, and were approximately 3 times higher in the liver of adult female rats than male rats. Hypophysectomy caused a significant decrease in P-450f mRNA levels in male and female rats. Intermittent injection with human growth hormone (hGH) to mimic the male secretory pattern of GH caused a 9-fold increase in P-450f mRNA in hypophysectomized male rats to levels near male control levels, whereas continuous administration of hGH to mimic the female secretory pattern caused a greater increase in P-450f mRNA levels in male and female hypophysectomized rats (25-fold and 9-fold respectively) to levels nearer female control levels. The responses of the other GH-stimulated P-450s, P-450-male and P-450-female, to the different modes of hGH treatment were different from that of P-450f. Since sex hormones are known to affect the regulation of other P-450s, the effect of sex hormones on P-450f mRNA was studied. Ovariectomy caused a 2.4-fold reduction in P-450f mRNA which was partially reversed by estradiol treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Polymorphism in hydroxylation of mephenytoin and hexobarbital stereoisomers in relation to hepatic P-450 human-2.
Stereoselective 4'-hydroxylations of R-(-)-mephenytoin and S-(+)-mephenytoin and 3'-hydroxylation of R-(-)-hexobarbital and S-(+)-hexobarbital were determined in liver microsomes of 14 Japanese subjects who were extensive metabolizers of mephenytoin and in five Japanese subjects who were poor metabolizers of mephenytoin. Content of P-450 human-2 assessed by Western blots was correlated to microsomal S-(+)-mephenytoin 4'-hydroxylation, R-(-)-hexobarbital 3' alpha-hydroxylation, and S-(+)-hexobarbital 3' beta-hydroxylation, and was less correlated to R-(-)mephenytoin 4'-hydroxylation, R-(-)-hexobarbital 3' beta-hydroxylation, and S-(+)-hexobarbital 3' alpha-hydroxylation. Antibodies raised against P-450 human-2 inhibited microsomal S-(+)-mephenytoin 4'-hydroxylation efficiently but was less efficient on R-(-)-mephenytoin 4'-hydroxylation in extensive metabolizers and on 4'-hydroxylation of mephenytoin enantiomers in poor metabolizers. The antibodies also inhibited R-(-)-hexobarbital 3' alpha-hydroxylation and S-(+)-hexobarbital 3' beta-hydroxylation but did not effectively inhibit the hydroxylation of the two other optical isomers of hexobarbital in extensive metabolizers and of four stereoisomers in poor metabolizers. These findings indicate the close relationship between polymorphic mephenytoin 4'-hydroxylation and two stereospecific hexobarbital hydroxylations, and they suggest that P-450 human-2 is a typical S-(+)-mephenytoin 4'-hydroxylase and a major hexobarbital 3'-hydroxylase in the livers of extensive metabolizers. The findings were further supported by the experiments that used P-450 human-2 complementary dexoyribonucleic acid-derived protein in yeast microsomes.
Cytochrome P-450 Enzyme System↗
Cytochrome b5 potentiation of cytochrome P-450 catalytic activity demonstrated by a vaccinia virus-mediated in situ reconstitution system.
A cDNA containing the full coding region of human cytochrome b5 was inserted into a vaccinia virus cDNA expression vector. Infection of human thymidine kinase-minus (TK-) 143 cells in culture with this recombinant virus resulted in production of 0.3 nmol of cytochrome b5 per mg of cell lysate protein. The expressed cytochrome had a reduced difference spectrum with a Soret peak at 424 nm, typical of pure cytochrome b5. TK- 143 cells have little detectable endogenous cytochrome b5, cytochrome P-450 (P450), and NADPH-P450 oxidoreductase. To test whether cytochrome b5 potentiated mixed-function monooxygenation in situ, these cells were coinfected with three recombinant vaccinia viruses individually carrying cDNAs encoding cytochrome b5, NADPH-P450 oxidoreductase, and P450 form IIB1. These triple-virus-infected cells were compared to cells infected with the P450IIB1 and NADPH-P450 oxidoreductase recombinant viruses with respect to P450IIB1-catalyzed monooxygenase activities. Cytochrome b5 specifically augmented the deethylation of p-nitrophenetole in microsomal membrane fractions of infected cells or when substrate was incubated directly with cells in situ. No significant increases were seen with P450IIB1-catalyzed testosterone, 7-ethoxycoumarin, or 7-pentoxyresorufin oxidations. These data demonstrate that cytochrome b5 is capable of specifically augmenting monooxygenase activities in intact cells.
Cell Line↗
Monomorphic and polymorphic isozymes of arylamine N-acetyltransferases in hamster liver: purification of the isozymes and genetic basis of N-acetylation polymorphism.
Two forms of cytosolic acetyltransferases, AT-I and AT-II, have been purified from hamster livers, and a comparison made of their chemical and catalytic properties and genetically expressed difference. Homogeneous AT-I and AT-II were 31 and 30 kd respectively on SDS-PAGE and catalyzed efficiently various N- and O-acetylations in their reconstitution systems. AT-I used both acetyl CoA and arylhydroxamic acids as acetyl donors, while AT-II did not utilize arylhydroxamic acids as acetyl donors. In the reconstitution system, purified AT-I, but not AT-II, catalyzed acetyl CoA-dependent O-acetylation of 2-N-hydroxyamino-6-methyldipyrido[1,2-alpha:3', 2'-d]imidazole (N-OH-Glu-P-1) and arylhydroxamic acid-dependent N-acetylation of 4-aminoazobenzene (AAB). On the other hand purified AT-II showed high activities of acetyl CoA-dependent N-acetylation of 2-aminofluorene (AF) and p-aminobenzoic acid (PABA). Polyclonal antibodies raised against AT-I inhibited cytosolic acetylations of N-OH-Glu-P-1 and AAB, and to a lesser extent of AF, while PABA N-acetylation was only marginally inhibited. Using Western blots, both AT-I and AT-II were recognized by the antibodies. AT-I was detectable in all the livers examined, and the content did not differ among the individuals (monomorphic distribution). In contrast, AT-II was distributed polymorphically, and the trimodal distribution of AT-II (high, intermediate and low) was correlated with the phenotype identified by cytosolic N-acetylations of AF and PABA (rapid, intermediate and slow). In addition, cross-mating experiments with intra- and inter-phenotype animals confirmed that hepatic AT-II isozyme is inherited by a Mendelian co-dominant trait. These results indicate that the polymorphic appearance of an acetyltransferase, AT-II, is responsible for the N-acetylation polymorphism in individual hamsters.
4-Aminobenzoic Acid↗
Purification and characterization of four catalytically active testosterone 6 beta-hydroxylase P-450s from rat liver microsomes: comparison of a novel form with three structurally and functionally related forms.
Four microsomal cytochrome P-450s (P-450), all of which are active testosterone 6 beta-hydroxylases, were purified to electrophoretic homogeneity from livers of phenobarbital-treated (P-4506 beta-1 and P-4506 beta-3) or dexamethasone-treated adult male rats (P-4506 beta-2 and P-4506 beta-4). Purified P-4506 beta-1, P-4506 beta-2, P-4506 beta-3, and P-4506 beta-4 had apparent molecular weights of 52,000, 51,000, 52,000, and 52,500 as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Absolute spectra revealed that all four P-450 forms had characteristic low-spin spectral patterns in their fully oxidized states. P-4506 beta-1 and P-4506 beta-3 displayed spectra of the reduced carbonyl complex with lambda max at 447 nm. P-4506 beta-2 and P-4506 beta-4 showed lambda max at 446 and 448 nm, respectively. Antibodies raised against each P-450 recognized all forms, although differences were observed with respect to the extents of cross-reactivities on Western blots. Form-specific peptide fragments were also detected among the four P-450 proteins after partial protease-digestion. P-4506 beta-1 was identical to P-4506 beta-3 in the first 26 residues of the NH2-terminal amino acid sequence, but differed by 13 residues from P-4506 beta-2. The amino-terminal sequence of P-4506 beta-2 was unique and was not identical with those of any rat P-450 previously reported. This P-450 form was detected in the livers of untreated male rats and was induced by treatment with dexamethasone, but not with phenobarbital.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Modulation of hepatic level of microsomal testosterone 7 alpha-hydroxylase, P-450a (P450IIA), by thyroid hormone and growth hormone in rat liver.
The effects of thyroid hormone and growth hormone on microsomal testosterone 7 alpha-hydroxylase, P-450a, were studied to understand the interaction of these hormone-mediated regulations in rats. In Western blots using anti-P-450a IgG, 1.7-fold higher content of P-450a was observed in livers of female than male adult rats, while no appreciable sex-related difference was detected in prepubertal rats and rats of 24 months of age. Treatment with n-propyl-2-thiouracil or thyroidectomy of male rats increased by 2-fold the hepatic content of P-450a, but neither regimen had a significant effect on the content in female rats. Levels of P-450a in both sexes of thyroidectomized rats were decreased by the supplementation of triiodothyronine (T3, 50 micrograms per kg, i.p. for 7 days) to levels similar to that observed in normal male rats. Hypophysectomy also caused an increase in microsomal P-450a content in male rats. Continuous infusion of human growth hormone, which mimicked the female secretion, further significantly increased the content in hypophysectomized rats to a level similar to that observed in normal female rats. In contrast, hepatic level of P-450a in hypophysectomized male and female rats was reduced by intermittent injection, which mimicked the male secretion. Clear suppression on the level of hepatic P-450a was also observed by the treatment of hypophysectomized rats with 5 or 50 micrograms/kg of T3 and of hGH-infused hypophysectomized rat with 50 micrograms/kg of T3.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Decrease in the metabolic activating capacities of arylamines in livers bearing hyperplastic nodules: association with the selective changes in hepatic P-450 isozymes.
The mechanism of the alteration in carcinogenic arylamine-activating capacities in livers bearing pre-neoplastic (or hyperplastic) nodules induced by the Solt-Farber protocol was investigated in relation to the changes in hepatic cytochrome P-450 isozymes. In the Salmonella mutagenesis test, the numbers of revertants induced with 2-amino-3-methylimidazo[4,5-f]quinoline and 2-aminofluorene were significantly lower in the presence of microsomes of nodule-bearing livers than of control livers. A similar tendency was also observed with another heterocyclic arylamine, 2-amino-6-methyldipyrido-[1,2-a:3',2'-d]imidazole. In Western blots using specific antibodies against 5 different forms of cytochrome P-450, hepatic contents of P-450-male (a main constitutive form) and P-450b (a main phenobarbital-inducible form) were decreased in the livers with hyperplastic nodules to 63% and 35% of the corresponding controls, while no significant decrease was observed in the contents of P-448-H (a main 3-methylcholanthrene-inducible form), P-450(6 beta-1) (testosterone 6 beta-hydroxylase) and P-450e (a phenobarbital-inducible form). In accordance with the reduction in P-450-male, capacities for microsomal 16 alpha- and 2 alpha-hydroxylations, but not 6 beta-hydroxylation, of testosterone were decreased in the livers with hyperplastic nodules. Although P-448-H has higher capacities for the activation of arylamines than does P-450-male, the hepatic content of P-450-male is more than ten-fold higher than that of P-448-H in both normal and nodule-bearing livers. These results indicate that the selective decrease in hepatic content of P-450-male is likely to be a main cause of the decrease in arylamine metabolic activating capacities in livers with hyperplastic nodules.
Animals↗
High capacity of human skin for N-acetylation of arylamines.
Human skin showed high activity of cytosolic N-acetylation for two typical arylamine substrates, p-aminobenzoic acid (PABA) and 2-aminofluorene (AF). Their specific activities (per milligram protein basis) were comparable with those of hamster skin, which is known to have high acetylating capacities for several arylamines. In hamster skin, two other types of acetylation, N-hydroxy-4-acetylaminobiphenyl-dependent N-acetylation of AF (N,N'-acetyltransfer) and acetyl CoA-dependent O-acetylation of 2-hydroxyamino-6-methyl-pyrido[1,2-a:3',2'-d]imidazole (O-acetylation) also occurred, but no appreciable activity was observed in human skin for these two reactions. These results suggest that arylamines including prohaptens and carcinogens may be modified metabolically through N-acetylation at the first contact site, human skin. In addition, a good correlation was observed for N-acetylations of PABA and AF in human skin, implying that these N-acetylations may be catalyzed by the same or a closely related enzyme in human skin.
4-Aminobenzoic Acid↗