Biomedical subjects

M A Lang

Publications and source records attributed to M A Lang.

At least 37 records · Page 2Linked to original sources

PubMed1994

Cocaine N-demethylation and the metabolism-related hepatotoxicity can be prevented by cytochrome P450 3A inhibitors.

Cocaine is eliminated and detoxified principally through the metabolism of nonspecific plasma and tissue esterases. Microsomal oxidative metabolism is of importance in cocaine N-demethylation, this being a principal pathway of cocaine bioactivation and hepatotoxicity. The contribution of different cytochrome P450 (CYP) enzymes to cocaine N-demethylase activity was studied in vitro with DBA/2 mouse and human liver microsomes, and cocaine hepatotoxicity was examined in vivo in DBA/2 male mice. Species dependent enzyme kinetics was observed. Cocaine N-demethylase displayed two Km values in murine liver (40-60 microM and 2-3 mM), whereas only one Km value was observed in human liver microsomes (2.3-2.7 mM). We suggest that CYP3A plays a prominent role in the N-demethylation of cocaine for the following reasons: (i) pregnenolone-16 alpha-carbonitrile, an inducer of CYP3As increases cocaine N-demethylase in parallel with testosterone 6 beta-hydroxylase activity and immunoreactive 3A protein in mouse liver; (ii) human and mouse cocaine N-demethylase and testosterone 6 beta-hydroxylase activities can be inhibited by triacetyloleandomycin, cannabidiol, or gestodene, all selective inhibitors of CYP3A P450s; (iii) antibodies directed against P450s within subfamilies 1A, 2A, 2B, 2C, or 2E inhibited cocaine N-demethylase activity only marginally, and finally, (iv) treatment of mice with triacetyloleandomycin or cannabidiol in vivo significantly attenuated the cocaine-elicited hepatotoxicity as assessed by the serum alanine aminotransferase activity and liver histology in parallel with decreased cocaine N-demethylase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1994

Induction of specific cytochrome P450s involved in aflatoxin B1 metabolism in hepatitis B virus transgenic mice.

The relative roles of hepatitis B virus (HBV) and aflatoxin and their possible mechanism of interaction in the etiopathogenesis of hepatocellular carcinoma (HCC) are not understood. One hypothesis is that viral infection and associated liver injury alter expression of carcinogen-metabolizing enzymes. We tested this hypothesis in an HBV-transgenic mouse model in which a synergistic interaction occurs between aflatoxin B1 (AFB1) and HBV in the induction of HCC (Sell et al., Cancer Res 51:1278-1285, 1991). In this transgenic mouse lineage, overproduction of the HBV large envelope protein results in progressive liver cell injury, inflammation, and regenerative hyperplasia. Initially, two cytochrome P450s of importance in AFB1 metabolism in the mice were identified, namely Cyp2a-5 and Cyp3a, using specific antibodies and chemical inhibitors. The expression of these P450 isoenzymes and an alpha-class glutathione S-transferase (GST) isoenzyme, YaYa, were examined. Increased expression and altered distribution of Cyp2a-5 were demonstrated, by immunohistochemical analysis, to be associated with the development of liver injury in mice and to increase with age between 1 and 12 months. Cyp3a expression was also increased in HBV-transgenic mice, but the increase was not as clearly related to age. GST YaYa levels were the same in HBV-transgenic mice and their nontransgenic littermates of all ages. These results show that expression of specific cytochrome P450s is altered in association with overexpression of HBV large envelope protein and liver injury in this model. This may have general relevance to human HCC, the etiology of which is associated with a diverse range of liver-damaging agents.

Aflatoxin B1↗

PubMed1994

Association of liver fluke (Opisthorchis viverrini) infestation with increased expression of cytochrome P450 and carcinogen metabolism in male hamster liver.

Synergy between exposure to chemical carcinogens (nitrosamines) and infestation with the liver fluke Opisthorchis viverrini has been demonstrated in a hamster model of hepatocarcinogenesis (Flavell et al., Carcinogenesis 4:927-930, 1983; Thamavit et al., Carcinogenesis 8:1351-1353, 1987). To elucidate the mechanisms of this interaction we tested the hypothesis that liver parasitism might influence the expression and activity of carcinogen metabolizing enzymes. We found that one, and perhaps more, hamster liver cytochrome P450 (CYP) isozymes immunorelated to mouse CYP2A5 contributed up to 50 or 60% of the hepatic aflatoxin B1 (AFB) and N-nitrosodiethylamine (NDEA) metabolism, respectively. As inferred from average enzyme activities and from western blot, immunoinhibition, and substrate (coumarin) inhibition analyses, O. viverrini infestation increased the expression of enzymes detectable by anti-CYP2A5 antibody as well as NDEA metabolism in male but not in female hamsters. Immunohistochemical analysis of CYP2A expression by anti-mouse CYP2A5 antibody demonstrated that the O. viverrini-associated increase was not uniformly distributed throughout the liver but occurred in hepatocytes immediately adjacent to areas of inflammation. Immunohistochemical analysis of AFB-DNA adducts in the livers of O. viverrini-infested hamsters treated with AFB showed that the highest levels of adducts were found in the regions of liver where hepatocellular expression of enzymes detectable by anti-CYP2A5 antibody is induced. These results suggest that a high local expression of CYP isozymes in O. viverrini-infested livers could be a contributing risk factor in the development of liver cancers associated with parasitic hepatitis.

Animals↗

PubMed1994

Metabolism of nitrosamines and aflatoxin B1 by hamster liver CYP2A enzymes.

Using a specific antibody against mouse CYP2A5 and coumarin as inhibitors, and pretreatments known to affect the activities of CYP2A-related enzymes, we studied the contribution of hamster liver CYP2As in the dealkylation of N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA) and hydroxylation of aflatoxin B1 (AFB1). CYP2A5 antibody inhibited AFB1 and NDEA metabolism by 40-60% in untreated hamsters and after treatment with phenobarbital (PB) or 3-methylcholanthrene (MC). After pyrazole (PYR) treatment, there was no inhibition, although PYR increased the metabolism above the controls. NDMA metabolism was inhibited only weakly in all groups. This suggests that CYP2As may contribute significantly to NDEA and AFB1 metabolism and less to NDMA metabolism in untreated hamster liver and after MC or PB treatments, but that PYR treatment may change the expression pattern of CYPs so that the metabolism is mainly catalysed by CYPs not recognized by the antibody. Coumarin inhibited NDEA and NDMA metabolism in all groups of hamsters, including pyrazole-pretreated animals, suggesting that it interacts not only with CYP2As but also with other CYPs that are important in nitrosamine metabolism. On the other hand, coumarin inhibited AFB1 metabolism significantly only in control and PB-treated animals but not at all after PYR or MC treatment. This suggests that enzymes involved in AFB1 metabolism may be different depending on the pretreatment. It is concluded that CYP2As may contribute significantly to nitrosamine and aflatoxin metabolism in hamster liver. However, in view of previous results showing essential differences in their regulation between mouse and hamster, interspecies comparisons may be difficult.

Aflatoxin B1↗

PubMed1993

Comparison of hamster and mouse reveals interspecies differences in the regulation of hepatic CYP2A isozymes.

Three CYP2A-related activities [coumarin 7-hydroxylase (COH), testosterone 7 alpha- (test7 alpha) and 15 alpha-hydroxylases (test15 alpha)], identified in hamster liver and analysed by immunoinhibition, and western and northern blotting, were found to be similar to mouse and human CYP2As. In the microsomal fractions, anti-mouse CYP2A5 antibody recognised three bands of about 48, 49 and 52 kDa, suggesting the presence of at least three proteins immunologically similar to mouse CYP2A5. The 49 kDa band migrated close to mouse CYP2A5 and changes in its expression followed COH and test15 alpha activities. Test7 alpha activity did not associate with any of the individual bands detected on western blots despite its strong inhibition by the antibody. Despite the immunological and catalytic similarities between mouse and hamster CYP2A enzymes, their regulation is different. In mice, the enzyme activities are higher in females than males, are induced by pyrazole (PY) and phenobarbital (Pb), and are not affected by 3-methylcholanthrene (MC). In hamsters, activities are not higher in females, induced by MC and reduced by PY. MC and PY appear to regulate expression at the mRNA level, while Pb seems to act post-transcriptionally by increasing either the synthesis or the stability of the protein. Our data indicate that the modes of expression and regulation of CYP2A-related enzymes make the hamster different from mice and humans with respect to the mechanism of metabolism of certain drugs and carcinogens.

Animals↗

PubMed1993

High variability of nitrosamine metabolism among individuals: role of cytochromes P450 2A6 and 2E1 in the dealkylation of N-nitrosodimethylamine and N-nitrosodiethylamine in mice and humans.

We undertook this study to answer several questions regarding nitrosamine metabolism. Kinetics of nitrosamine metabolism showed the involvement of at least two enzymes in the dealkylation of N-nitrosodiethylamine (NDEA) and N-nitrosodimethylamine (NDMA) in mouse liver microsomes. Coumarin inhibited both reactions competitively. On the other hand, microsomal coumarin 7-hydroxylase was inhibited by NDMA (Ki 2.7 mM) and NDEA (Ki 0.013 mM). The big difference in the Ki values suggests a higher affinity of NDEA than NDMA to Cyp2a-5 (mouse cytochrome P450coh). A specific antibody against Cyp2a-5 inhibited more of the microsomal NDEA (up to 90%) than NDMA (up to 40%) dealkylation. The converse was true with anti-Cyp2e-1 antibody. These results suggest that the primary substrate for Cyp2a-5 is NDEA and for Cyp2e-1, NDMA. Western blot analysis of human liver microsomes showed a great interindividual variation in the amounts of CYP2A6 (human cytochrome P450coh) and CYP2E1. Also, coumarin 7-hydroxylation and nitrosamine dealkylation varied greatly among individuals. A high correlation (r = 0.93, P < 0.001) was found between NDEA and coumarin metabolism. Both activities were associated with CYP2A6. On the other hand, little or no correlation was found between microsomal CYP2A6 and CYP2E1 or between CYP2E1 and NDEA dealkylation. Immunoinhibition of human microsomal NDEA metabolism by CYP2a-5 antibody varied greatly among individuals (10-90%), suggesting, as in the case of mice, that NDEA is metabolized primarily by CYP2A6, at least in some individuals. Taken together the data suggest that (1) the metabolic activation of nitrosamines in humans varies greatly among individuals; (2) different nitrosamines may partially be metabolized by different cytochrome P450 isozymes; and (3) because of similarities between nitrosamine metabolism in mice and humans, inbred strains of mice would be relevant experimental models for studying nitrosamine activation.

Animals↗

PubMed1993

High expression of cytochrome P450 2a-5 (coumarin 7-hydroxylase) in mouse hepatomas.

A high level of Cyp2a-5 was found in spontaneous and transplanted mouse hepatomas compared with normal liver. Increased expression of Cyp2a-5 was associated with an increase in coumarin 7-hydroxylation, a marker activity of Cyp2a-5, and the corresponding mRNA, suggesting that regulation of Cyp2a-5 in hepatomas is pretranslational. In contrast, the total P450 content and arylhydrocarbon hydroxylase and amidopyrene demethylase activities decreased. Pyrazole, a strong inducer of Cyp2a-5 in normal mouse livers, also increases this isozyme in hepatomas. A parallel increase in the corresponding mRNA suggests that pyrazole, like the formation of hepatomas, affects the regulation of Cyp2a-5 pretranslationally.

Aminopyrine N-Demethylase↗

PubMed1993

Cytochrome P4502A-mediated coumarin 7-hydroxylation and testosterone hydroxylation in mouse and rat lung.

Pulmonary coumarin 7-hydroxylase, testosterone hydroxylase and other P450-mediated activities were compared in the mouse and rat. Coumarin 7-hydroxylase activity was 20 pmol/mg/min. in mouse and 4 pmol/mg/min. in rat lung microsomes. Liver values were 180 (mouse) and 1 (rat) pmol/mg/min. Km values of rat and mouse lung coumarin 7-hydroxylase were about 1 microM whereas the rat liver Km value was > 100 microM. Phenobarbital and 3-methylcholanthrene did not affect rat lung (or liver) coumarin 7-hydroxylase activity. Anti-Cyp2a-5 antibody effectively inhibited mouse and rat lung coumarin 7-hydroxylase and testosterone 15 alpha-hydroxylations but failed to block these activities in the rat liver. In immunoblot analysis anti-Cyp2a-5 antibody recognized the 50-kDa Cyp2a-4/5 protein in mouse lung microsomes. A P450 protein co-migrating with Cyp2a-5 was also detected in rat lung microsomes. Cyp2a-5 cDNA probe hybridized with a 1.8-kb mRNA species in rat lung RNA fraction. The hybridization signal was not increased by 3-methylcholanthrene or phenobarbital. These data suggest that the mouse lung expresses Cyp2a-5 which differs from the liver enzyme only in its regulation and that the rat lung contains a P450 isoform(s) belonging to the 2A subfamily which may be orthologous with the mouse Cyp2a-4/5 catalyzing coumarin 7-hydroxylase and testosterone 15 alpha-hydroxylase activities. The recently reported rat lung CYP2A3 (Kimura et al.) gene product is a candidate for the observed coumarin 7-hydroxylase activity in the rat lung.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1992

Effect of pyrazole, cobalt and phenobarbital on mouse liver cytochrome P-450 2a-4/5 (Cyp2a-4/5) expression.

Pyrazole, cobalt and phenobarbital increase the activity of coumarin 7-hydroxylase (COH) in mouse liver. To study the mechanism of this increase, we measured the expression of the cytochrome P-450 2a-4/5 (Cyp2a-4/5) complex, which mediates testosterone 15 alpha-hydroxylase and COH activities, as a function of dose and time after the treatment of C57BL/6 (B6) and DBA/2 (D2) male mice with the inducers. COH activity and Cyp2a-4/5 steady-state mRNA levels were increased in both strains in response to the inducers. No marked effect occurred with testosterone 15 alpha-hydroxylase or activities associated with Cyp1a-1 or Cyp2e-1. A 2-7-fold increase in response to the inducers was seen in the amount of P-450Coh (cytochrome P-450 isoenzyme catalysing coumarin 7-hydroxylation) protein in Western immunoblots. PCR amplification of a 1 kb region in Cyp2a-4/5-mRNA-derived cDNA, followed by cutting at the diagnostic PstI site, showed that most of the steady-state mRNA consisted of Cyp2a-5, which is also the form most affected by pyrazole. Nuclear run-off analysis revealed no increase in the transcription rate of Cyp2a-4/5 after pyrazole or cobalt treatment, whereas a 2-3-fold increase occurred after phenobarbital pretreatment in B6 mice. Together with previous reports [Aida & Negishi (1991) Biochemistry 30, 8041-8045], the current data suggest that both pyrazole and cobalt increase COH catalytic activity by affecting Cyp2a-5 by post-transcriptional mechanisms in mice.

Animals↗

PubMed1992

Regulation of the mouse liver cytochrome P450 2B subfamily by sex hormones and phenobarbital.

The sex-dependent expression and inducibility of the cytochrome P450 2B subfamily was studied in DBA/2 and Balb/c mice, and their F1 recombinants, at the mRNA, protein and activity levels. Analysis of poly(A)+ RNA with specific oligonucleotide probes directed to known mRNAs within the mouse 2B subfamily revealed that the levels of P450 2b-10 and 2b-9 mRNAs were co-regulated with two proteins (56 and 53 kDa) detected by a 2B-specific polyclonal antibody. Other mRNAs related to the 2B subfamily were barely or not at all detectable, and did not coincide with protein expression, suggesting that P450s 2b-9 and 2b-10 are the major 2B isoenzymes present in mouse liver. Specifically, castration of males increased the expression of 2b-9 and 2b-10 mRNAs and protein up to female levels, and testosterone administration to castrated mice reversed these changes. Ovariectomy of females appears to increase the expression of these P450s slightly. 2b-10, but not 2b-9, mRNA and protein were induced by phenobarbital. Based on immunoinhibition studies and the levels of these isoenzymes, P4502b-10 appears to be the major catalyst of 7-pentoxyresorufin O-dealkylation. Both P4502b-9 and P4502b-10 contribute up to 30% of the testosterone 16 alpha-hydroxylation, the balance being catalysed by P450s within the 2D subfamily. These experiments show that the female-predominant expression of the two mouse liver isoenzymes P4502b-9 and P4502b-10 is dependent on sex hormones. The fact that P4502b-9 does not respond to phenobarbital, while P4502b-10 is regulated by both phenobarbital and sex hormones, demonstrates the complexity of P450 expression even within one subfamily.

Amino Acid Sequence↗

PubMed1992

Highly homologous cytochromes P-450 and b5: a model to study protein-protein interactions in a reconstituted monooxygenase system.

Cytochrome b5 from mouse and rat liver formed a type I spectral complex with two murine cytochrome P-450 isozymes, the P450Coh and P450PBI. Mouse b5 stimulated the reactions catalyzed by reconstituted P450Coh and an equimolar amount of b5 to P450Coh was needed for maximal effect. In contrast, rat b5 inhibited P450Coh-mediated reactions progressively starting from 1:1 ratio of b5 to P-450. Neither b5 had any effect on reactions catalyzed by P45015 alpha, an isozyme highly homologous to P450Coh, but with a point mutation (Arg-129----Ser) at site considered important for P-450-b5 interactions. In case of P450PBI, neither b5 protein had any effect on the associated activities at b5: P-450 ratios below 1, and a progressive inhibition occurred when b5: P-450 ratio was above 1. The results were similar with either rat or mouse liver NADPH-cytochrome P-450 reductase used in reconstitution demonstrating that the critical differences take place in P-450-b5 interactions. Kinetic and spectral experiments revealed that the stimulatory and inhibitory effects of b5 on the enzymatic reactions were due to corresponding changes in the reaction velocity, and that b5 does not compete with the flavoprotein nor with the substrate for binding to P-450. These results indicate that the high spin shift of P-450 does not necessarily correlate with enhanced reaction rates. Also, the increase in the coupling efficiency of P450PBI may result from the increased affinity for substrate in the presence of b5. Sequenation of mouse b5 peptides generated with proteinases revealed three amino acid changes between the mouse and rat b5, two of which appeared at the hydrophobic domain necessary for the P-450-b5 interaction. This could explain the species specificity of b5 proteins in supporting the P-450-mediated reactions. This is the first time functionally important differences in the interaction of highly homologous cytochromes P-450 and b5 have been demonstrated. Isozymes P45015 alpha and P450Coh, and mouse and rat b5 could serve as an excellent model for further studies on the nature and significance of P-450-b5 interactions.

Amino Acid Sequence↗

PubMed1992

Distinct induction profiles of three phenobarbital-responsive mouse liver cytochrome P450 isozymes.

The dose- and time-responses of three liver cytochrome P450 (P450) isozymes to 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and phenobarbital (PB) were studied in DBA/2 mice at activity, protein and mRNA levels. We found that the maximal induction ranged from about 3-fold (P4502a-4/5) and 5-fold (P4502c-x) to more than 50-fold (P4502b-10). Only P4502a-4/5 and associated mRNA displayed a biphasic time-response after TCPOBOP induction: a transient increase occurring 3-8 hr after administration with a subsequent decline at 24 hr before the maximal induction at 72 hr. The changes in P450 isozyme content reflected those in mRNA levels suggesting that the induction by TCPOBOP and PB is controlled largely at pretranslational stages. The isozyme P4502c-x and associated immunoinhibited benzphetamine N-demethylase and testosterone 16 beta-hydroxylase activities were induced half-maximally by 6-30 times smaller doses of TCPOBOP and by three to four times smaller doses of PB than isozymes P4502a-4/5, P4502b-10 or related activities. Furthermore, larger doses of TCPOBOP decreased the expression of P4502c-x to sub maximal levels. Our data show that the three isozymes, although all inducible by TCPOBOP and PB, have distinct dose dependencies and different time-responses to induction. This indicates that the induction by TCPOBOP and PB of P450s belonging even to the same subfamily may proceed by different mechanisms.

Animals↗

PubMed1992

Human and mouse liver coumarin 7-hydroxylases do not metabolize warfarin in vitro.

1. The contribution of human P450 2A6 and mouse P450 2a-5 isoenzymes, both highly active in coumarin 7-hydroxylation, to the metabolism of warfarin was studied in several in vitro systems with human and mouse liver preparations. 2. The reconstituted P450 2a-5 purified from DBA/2 mouse liver did not metabolize warfarin. 3. An anti-P450 2a-5 antibody did not consistently inhibit any of the warfarin biotransformation reactions catalyzed by human or mouse liver microsomes, although coumarin 7-hydroxylation was inhibited by over 90%. 4. In some human microsomal samples, 4- and 8-hydroxylations of warfarin were inhibited to some extent by the anti-P450 2a-5 antibody. 5. Warfarin (less than 1 mM) did not inhibit coumarin 7-hydroxylation by human or mouse liver microsomes in vitro. 6. We conclude that mouse and human coumarin 7-hydroxylases do not oxidise warfarin.

Adult↗

PubMed1991

Comparative studies on coumarin and testosterone metabolism in mouse and human livers. Differential inhibitions by the anti-P450Coh antibody and metyrapone.

We have studied coumarin 7-hydroxylase (COH) and testosterone 15 alpha-hydroxylase (15 alpha OH) activities in human liver microsomes and compared them with corresponding activities catalysed by members of the P450IIA sub-family in DBA/2N mouse liver microsomes. Human liver contained low levels of 15 alpha OH (about 5-30 pmol/min/mg protein) when compared with control mouse liver microsomes (about 200 pmol/min/mg protein). The anti-P450Coh antibody efficiently inhibited mouse liver 15 alpha OH, also 7 alpha OH (which is a member of the P450IIA sub-family), but it did not inhibit human 15 alpha OH or other testosterone hydroxylases. In mouse liver microsomes, metyrapone preferentially inhibited 15 alpha OH, but in human liver microsomes it inhibited all testosterone hydroxylations measured, including 15 alpha OH (IC50 = 2.0-5.0 microM). Metyrapone clearly inhibited COH in mouse liver microsomes, but interestingly it had no effect on COH activity in human liver microsomes, although these two isozymes have earlier been shown to be immunologically similar. On the basis of available evidence human and mouse P450Coh isozymes seem to be orthologous enzymes whereas the present results indicate that the human 15 alpha OH is different from the mouse P45015 alpha.

Animals↗

PubMed1991

Comparison between cobalt and pyrazole in the increased expression of coumarin 7-hydroxylase in mouse liver.

The data in this report show that administration of both cobalt and pyrazole results in an elevation in the amount of hepatic mRNA encoding for microsomal P45015 alpha/P450Coh, an increase in the amount of P450Coh protein, and an activation of COH and to a lesser extent testosterone 15 alpha-hydroxylase in two inbred strains of mice. Considerable quantitative differences between the two compounds and the two mouse strains in the response suggest that the effects of cobalt and pyrazole are mediated, at least partly, through different mechanisms. It is of interest that human hepatic COH resembles very closely that in the mouse liver.

Animals↗

PubMed1990

Immunochemical detection of human liver cytochrome P450 forms related to phenobarbital-inducible forms in the mouse.

Polyclonal antibodies generated to four distinct mouse liver phenobarbital-inducible cytochrome P450 isoforms were used to analyse related forms in human liver. N-terminal sequence analysis and biochemical properties of the P450s used as antigens suggest that they belong to P450 subfamilies IIB (P450PBI), IA (P450PBII), IIC (P450PBIII) and IIA (P450Coh). In immunoblot analysis, anti-P450PBII detected a single protein presumed to be P450IA2 in all the human livers tested. No proteins corresponding with P450IA1 could be detected. Anti-PBIII and anti-P450Coh antibodies each detected one band (54 and 48 kDa, respectively) in the liver samples. No bands were revealed by anti-P450PBI antibody. Protein dot-immunobinding analysis showed that P450s immunodetectable by anti-P450PBII, anti-P450PBIII and anti-P450Coh antibodies are expressed in human liver (range 9 to 69 pmol P450/mg protein). In immunoinhibition experiments the activity of 7-ethoxyresorutin O-deethylase (EROD) was blocked up to 90% by the anti-P450PBII antibody. Aryl hydrocarbon hydroxylase (AHH) was inhibited only by anti-P450PBIII, and coumarin 7-hydroxylase (COH) only by anti-P450Coh antibody. Testosterone hydroxylations in positions 6 beta, 7 alpha, 15 alpha and 16 alpha were not affected significantly by any of the antibodies. These data suggest that the human liver P450IA2 is responsible for most of the elevated EROD activity, P450s in the IIC subfamily for constitutive AHH and P450s in the IIA subfamily for all of COH activity.

Adult↗

PubMed1990

Identification of the human liver cytochrome P-450 responsible for coumarin 7-hydroxylase activity.

1. We have constructed a full-length human liver cytochrome P450IIA cDNA from a partial-length clone by oligonucleotide-directed mutagenesis, and subcloned it into the monkey kidney (COS-7) cell expression vector, pSVL. 2. The cDNA encodes a 49 kDa protein with coumarin 7-hydroxylase (COH) activity which cross-reacts with antisera to the mouse cytochrome P-450 isoenzyme responsible for COH activity and comigrates with a human liver microsomal protein. 3. Western blot analysis of a panel of human livers indicates that the level of the 49 kDa protein, detected using antisera to either the mouse COH P-450 or rat P450IIA1 protein, correlates very highly with COH activity. 4. Antisera to the rat P450IIA1 protein can inhibit COH activity in human liver microsomes. Taken together, these data indicate that a member of the P450IIA subfamily is responsible for most, if not all, of the COH activity in human liver.

Amino Acid Sequence↗

PubMed1989

Comparison of the immunochemical properties of human placental and bovine adrenal cholesterol side-chain cleavage enzyme complex.

The immunochemical relatedness between human and bovine proteins catalyzing the cholesterol side-chain cleavage reaction was investigated. In dot-immunobinding analysis, antibodies against bovine adrenocortical cytochrome P-450SCC, adrenodoxin, and adrenodoxin reductase recognized the corresponding proteins in a dose-dependent manner in mitochondrial preparations from human placenta. Limited proteolysis with trypsin cleaved bovine P-450SCC into fragments F1 and F2, which represent the NH2- and C-terminal parts of P-450SCC, respectively. Identical trypsin treatment yielded similar-size fragments from human placental P-450SCC. In Western immunoblots, anti-F1 and anti-F2 antibodies recognized the corresponding fragments in both trypsin-digested bovine and human P-450SCC. Antibodies against bovine P-450SCC, fragments F1 and F2, adrenodoxin and adrenodoxin reductase inhibited cholesterol side-chain cleavage activity in bovine adrenocortical mitochondria by 24-51%, but failed to affect the activity in human placental mitochondria. These data indicate that human and bovine P-450SCC share common antigenic determinants located outside the enzyme active site. The immunological similarity between bovine adrenodoxin and human ferredoxin allowed for a simple purification protocol of human placental P-450SCC by adrenodoxin affinity chromatography. The P-450SCC obtained by this method was electrophoretically homogeneous and showed characteristics typical to P-450SCC.

Adrenal Glands↗