Search PubMed⌕ Search

Biomedical subjects

J R Halpert

Publications and source records attributed to J R Halpert.

At least 73 records · Page 4Linked to original sources
PubMed1995

Selectivity and kinetics of inactivation of rabbit hepatic cytochromes P450 2B4 and 2B5 by N-aralkylated derivatives of 1-aminobenzotriazole.

The kinetics of mechanism-based inactivation of phenobarbital-inducible rabbit hepatic cytochromes P450 2B4 and 2B5 by N-benzyl-(BBT) and N-alpha-methylbenzyl (alpha MB) 1-aminobenzotriazole were investigated using reconstituted P450 2B4, a stable heterologous expression system, and hepatic microsomes. Low micromolar concentrations of the 1-aminobenzotriazole derivatives caused reversible inhibition as well as rapid inactivation of reconstituted P450 2B4 and recombinant P450 2B4 and 2B5. In contrast, even at a 1000-fold higher concentration, aminobenzotriazole inactivated the expressed P450 2B enzymes less rapidly. Preincubation of phenobarbital-induced hepatic microsomes with BBT and alpha MB resulted in concentration-dependent decreases in marker activities of P450 2B4 and 2B5, benzyloxyresorufin O-debenzylase and androstenedione 15 alpha-hydroxylase, respectively. BBT caused the inactivation of P450 2B4 and 2B5 in hepatic microsomes with apparent Kl values of 1.9 and 2.4 microM and maximal rate constants of 0.29 and 0.18 min-1, respectively. alpha MB inactivated both P450 2B enzymes with similar Kl values (approximately 7 microM) and maximal rate constants only slightly higher for 2B4 compared with 2B5 (0.68 vs. 0.55 min-1). Similar P450 2B selectivity of BBT and alpha MB in both hepatic microsomes and the stable expression system further validates this new expression system and the use of the selective markers identified for 2B4 and 2B5 in hepatic microsomes. The results also provide a mechanistic basis for the high potency of the N-aralkylated 1-aminobenzotriazole derivatives in vivo and suggest that treatments that inactivate 2B4 will also lead to 2B5 inactivation.

Alkylation↗
PubMed1995

Characterization of the progesterone 21-hydroxylase activity of canine cytochrome P450 PBD-2/P450 2B11 through reconstitution, heterologous expression, and site-directed mutagenesis.

Canine hepatic cytochrome P450 PBD-2 metabolizes 2,2',4,4',5,5'-hexachlorobiphenyl and catalyzes the 21-hydroxylation of progesterone, thereby distinguishing PBD-2 as unique among 2B P450s. Heterologous expression of the PBD-2 cDNA, P450 2B11, in COS and yeast systems produced a protein capable of androstenedione metabolism; however, this P450 did not metabolize progesterone in a manner consistent with PBD-2. Modification of PBD-2 reconstitution parameters resulted in significantly increased catalytic activities and further emphasized differences between PBD-2 and the heterologously expressed enzyme. Subsequent Escherichia coli expression of 2B11 generated a protein that possessed substrate specificities indistinguishable from those of PBD-2 and provided a system in which the determinants of 2B11 progesterone 21-hydroxylation could be examined via site-directed mutagenesis. Site-directed mutants of 2B11 expressed in E. coli revealed that substitution of Ile with Val at position 363 converted 2B11 into a highly active and specific progesterone 16 alpha-hydroxylase. Mutants Val-114 --> Ile, Asp-290 --> Ile, and Ile-365 --> Phe exhibited decreased progesterone 21- and 16 alpha-hydroxylase activities, in accordance with decreases in androstenedione hydroxylase activities. In contrast, replacement of Ile-365 with Val or Leu resulted in much greater changes in progesterone than androstenedione hydroxylation. Thus, the combination of P450 reconstitution techniques, heterologous expression, and site-directed mutagenesis has revealed PBD-2 to be an important progesterone 21-hydroxylase in canine liver and has identified several amino acid residues that alter progesterone metabolism by 2B11.

Animals↗
PubMed1994

Escherichia coli expression and characterization of cytochromes P450 2B11, 2B1, and 2B5.

Dog CYP2B11, rat CYP2B1, and rabbit CYP2B5 have been expressed in Escherichia coli from cDNAs modified at the N-terminus (Barnes et al., 1991, Proc. Natl. Acad. Sci. USA 88, 5597-5601). Using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps), solubilized membranes representing > 100 nmol of P450 2B11, > 35 nmol of P450 2B1, and > 7 nmol of P450 2B5 were efficiently extracted (40-70% yield) from a 1-liter culture. Chaps-solubilized preparations produced a reduced CO/reduced difference spectrum devoid of P420 and were used directly in a reconstituted system. The E. coli-expressed 2B enzymes retained the same functional characteristics as the purified hepatic enzymes or enzymes expressed in COS cells in terms of androstenedione metabolite profiles. Hydroxylation rates were determined under a variety of conditions, including two concentrations of NADPH-cytochrome P450 reductase (2 and 16 nmol/nmol P450) and the absence or presence of cytochrome b5 (2 nmol/nmol P450). The androstenedione hydroxylase activities of expressed 2B1 and 2B5 were stimulated by cytochrome b5, whereas P450 2B11 was inhibited slightly by cytochrome b5. Purified expressed 2B11 (specific content, 8 nmol/mg protein) had similar activities as the Chaps-solubilized membrane preparation. The solubilized membranes containing 2B11 were also tested with 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB). Three major metabolites, 2-hydroxy-4,5,2',4',5'-pentachlorobiphenyl, 3-hydroxy-2,4,5,2',4',5'-hexachlorobiphenyl, and 2-hydroxy-3,4,5,2',4',5'-hexachlorobiphenyl were produced from 245-HCB. These metabolites are identical to those produced by 2B11 purified from liver microsomes. The 245-HCB hydroxylation rates were similar for E. coli-expressed 2B11, dog liver microsomes, and purified liver 2B11. When only the second codon in the 2B1 was changed to GCT, > 25 nmol of P450 was extracted from a 1-liter culture, suggesting that the full Barnes et al. modification scheme may not be necessary for high-level expression. An efficient method of expressing, extracting, and analyzing different P450 2B enzymes has thus been achieved. In addition, rabbit P450 2B5, which has never been purified from liver, as well as different P450 2B mutants can now be expressed at much higher levels than previously reported. The ability to express different 2B wild-type and mutant P450s in E. coli provides an excellent opportunity to study the molecular basis of species differences in substrate metabolism.

Amino Acid Sequence↗
PubMed1994

Substrate-regulated, cAMP-dependent phosphorylation, denaturation, and degradation of glucocorticoid-inducible rat liver cytochrome P450 3A1.

The major rat glucocorticoid-inducible cytochrome P450 (CYP3A1) is known to be regulated at a transcriptional level by glucocorticoids and at a post-translational level by substrate-dependent stabilization. We have investigated mechanisms of substrate/ligand stabilization using primary hepatocytes, isolated liver microsomes from dexamethasone-treated rats, and purified enzymes. Treatment of hepatocytes with glucagon caused a 3-fold increase in CYP3A1 phosphorylation as well as an enhanced degradation rate of the enzyme. Specific CYP3A1 substrates or ligands, such as erythromycin, triacetyloleandomycin, and clotrimazole (CTZ) protected the enzyme from degradation in hepatocytes and inhibited in a concomitant manner (r = 0.99) glucagon-induced phosphorylation of the enzyme. In vitro experiments with purified CYP3A1 and isolated liver microsomes revealed one major site (Ser393) phosphorylated by the catalytic subunit of cAMP-dependent kinase, a reaction inhibited by ligands. Experiments in microsomes showed the presence of an endogenous cAMP-dependent kinase active on CYP3A1. Addition of exogenous cAMP-dependent kinase increased the rate of microsomal CYP3A1 phosphorylation, a reaction further stimulated by NADPH, but inhibited by CTZ. The microsomal phosphorylation caused a pronounced denaturation of cytochrome P450, as revealed spectrophotometrically, whereas CTZ protected from this reaction. Similar effects were noted when the CYP3A1-dependent 6 beta-hydroxylation of testosterone was monitored. It is suggested that the cellular CYP3A1 level is regulated to a significant extent posttranslationally by substrate-regulated cAMP-dependent phosphorylation on Ser393, followed by denaturation and degradation in the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1994

Epoxidation of arachidonic acid as an active-site probe of cytochrome P-450 2B isoforms.

In the present study we determined the regioselectivity of arachidonic acid epoxidation by several members of the cytochrome P-450 2B subfamily, including rat P-450 2B1, 2B1-WM (an allelic variant of 2B1 expressed in Wistar-Munich rats), 2B2, and rabbit 2B4 and 2B5. The major products formed with all isoforms were the four regioisomeric epoxides, but each isoform produced a distinct distribution of the four epoxides. P-450 2B1 produced predominantly 14,15-epoxyeicosatrienoic acid (EET), while P-450 2B1-WM produced the 11,12-EET as the major product. P-450 2B2, 2B4, and 2B5 catalyzed the formation of all four epoxides in nearly equal amounts. The single Gly-478-->Ala substitution in the variant P-450 2B1-WM was sufficient to cause a dramatic change in the ratio of epoxides when compared with P-450 2B1. The Gly-478-->Ala mutation also changed the regioselective epoxidation of gamma-linolenic acid at the three double bonds. Four site-directed mutants of P-450 2B1 were also evaluated. The mutations included two single mutants where Ile-114 was changed to either Val or Ala and two double mutants where the Ala-478 mutation was coupled with either Val or Ala at position 114. When Ile-114 was mutated to Val, the degree of epoxidation of arachidonic acid at all four double bonds was nearly equal. However, substitution of Ile-114 with Ala, resulted in a significant reduction in the degree of epoxidation at the 14,15- and 11,12-double bonds, and the 8,9- and 5,6-EETs were the major products. When Ala was introduced at position 478 in conjunction with Val at position 114 the regioselective epoxidation of the mutant enzyme more closely resembled P-450 2B1-WM in that 11,12-EET was the major metabolite. The double mutation with Ala at both positions 114 and 478 produced a unique distribution of epoxide products with 5,6-EET as the major metabolite. The results of these studies indicate that residues 114 and 478 in the P-450 2B subfamily are important for the orientation of fatty acids in the active site.

Animals↗
PubMed1994

Structural determinants of cytochrome P450 2B1 specificity: evidence for five substrate recognition sites.

Twelve site-directed mutants of rat cytochrome P450 2B1 distributed over seven positions and four putative substrate recognition sites (SRS) were constructed and expressed in COS cells. Function was examined using androstenedione and testosterone as substrates. Substitutions at positions 303, 360, and 473 did not markedly affect the regio- or stereoselectivity of androgen metabolism, whereas mutants in positions 206 (SRS-2), 302 (SRS-4), and 363 and 367 (SRS-5) exhibited markedly different steroid metabolite profiles compared with parental P450 2B1. In particular, the Phe-206-->Leu substitution conferred androgen 6 alpha- and testosterone 7 alpha-hydroxylase activities, and the Thr-302-->Ser substitution suppressed androgen 16 beta-hydroxylation in favor of androstenedione 16 alpha- and testosterone 15 alpha-hydroxylation. Replacement of Val-363 or Val-367 with Ala conferred androgen 15 alpha-hydroxylase and 6 beta-hydroxylase activities, respectively, and suppressed susceptibility to mechanism-based inactivation by the P450 2B1-selective chloramphenicol analog N-(2-p-nitrophenethyl)chlorofluoroacetamide. The Val-367-->Ala mutant was also resistant to chloramphenicol itself. The Leu mutant at position 363 exhibited increased specificity for androstenedione and testosterone 16 beta-hydroxylation, whereas the Leu mutant at position 367 exhibited decreased stereospecificity. Most interestingly, the size of key residues identified plays a critical role in governing steroid hydroxylation from the alpha-face or beta-face and hydroxylation on the D-ring or the B-ring.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Role of residues 363 and 206 in conversion of cytochrome P450 2B1 from a steroid 16-hydroxylase to a 15 alpha-hydroxylase.

Four double and four triple site-directed mutants of cytochrome P450 2B1 were constructed, expressed in COS cells, and assayed for androstenedione and testosterone hydroxylation. The mutants combined a Val-363-->Ala substitution with an Ile-114-->Val or Ala substitution and/or a Gly-478-->Ala or Ser substitution. Each of the individual mutations enhances androgen 15 alpha-hydroxylation, and the appropriate combination of Val or Ala at position 114 with Ala or Ser at position 478 has recently been shown to convert P450 2B1 from an androstenedione and testosterone 16 beta-hydroxylase to a 15 alpha-hydroxylase (Halpert, J. R., and He, Y.-A. (1993) J. Biol. Chem. 268, 4453-4457). All eight mutants containing the Val-363-->Ala substitution preferentially hydroxylated androstenedione and testosterone in the 15 alpha-position and thus functionally resemble mouse P450 2A4. However, unlike P450 2A4, various single and multiple 2B1 mutants at positions 114, 363, and 478 mainly hydroxylated progesterone in the 16 alpha- rather than 15 alpha-position. By combining the Ile-114-->Ala substitution with a Phe-206-->Leu mutation (corresponding to Ala-117 and Leu-209 in P450 2A4), P450 2B1 was converted to a progesterone 15 alpha-hydroxylase with retention of testosterone 15 alpha-hydroxylase activity. These studies document the importance of residues 363 and 206 in determining the substrate specificity of P450 2B1 and strongly support the hypothesis that the judicious combination of a small number of discrete mutations can be used to confer new specificities on P450 enzymes.

Androstenedione↗
PubMed1994

Selective inhibitors of cytochromes P450.

The balance between detoxification and bioactivation of a compound in a particular species or organ is highly dependent on the relative amounts and activities of the different forms of cytochrome P450 (P450) that are expressed. Therefore, knowledge of the catalytic specificities and regulation of individual P450 forms is of paramount importance in predicting and/or rationalizing species, strain, and individual differences in xenobiotic metabolism as well as metabolic interactions between compounds, both endogenous and exogenous. The emergence in recent years of a battery of isoform-selective chemical inhibitors that can be used in vitro and in vivo in experimental animals and humans has greatly facilitated the identification of individual cytochromes P450 responsible for specific bioactivation and detoxification reactions. Many of these inhibitors are mechanism-based and owe their selectivity to metabolism by the target enzyme. Such compounds have also proven valuable as probes of the catalytic mechanism of cytochromes P450, for identifying amino acid residues of importance for the various functions of the enzyme, for assessing the physiological roles of P450-derived oxidation products of endogenous compounds, in chemical-induced models of acute hepatic porphyria, and for studying protein turnover. The identification of isoform-selective, nontoxic inhibitors of individual human cytochromes P450 raises the real possibility of modulation of human drug metabolism for therapeutic purposes.

Animals↗
PubMed1994

Inactivation of cytochromes P450 2B protects against cocaine-mediated toxicity in rat liver slices.

Mechanism-based inactivators of rat liver cytochrome P450 2B1 and 2B2 were used to evaluate the role of these enzymes in the hepatotoxicity of cocaine. Loss of liver microsomal androstenedione 16 beta-hydroxylation was monitored to determine the extent of P450 2B1/2 inactivation by chloramphenicol (CAP) or its 2B-selective analogue, N-(2-p-nitrophenethyl)chlorofluoroacetamide (pNO2C1FA). The effect of P450 2B1/2 inactivation on cocaine-mediated hepatotoxicity was assessed in rat liver slices. Exposure of slices from phenobarbital-induced Lewis rats to CAP concentrations ranging from 100 to 500 microM resulted in a concentration-dependent decrease in P450 2B activity and a corresponding decrease in cytotoxicity as measured by K+ loss following exposure to 1 mM cocaine. Treating slices from PB-induced rats with 250 microM pNO2C1FA protected slices against cocaine-mediated cytotoxicity after exposure to 500 microM cocaine. In vivo administration of 300 mg/kg CAP or 200 mg/kg pNO2C1FA to phenobarbital-induced Lewis rats decreased androstenedione 16 beta-hydroxylation to 30 or 39% of control, respectively, and blocked cocaine-mediated K+ loss in rat liver slices. Rat liver microsomes from animals treated with either CAP or pNO2C1FA displayed approximately 40% of the control rate of cocaine N-demethylation. Experiments with phenobarbital-treated Munich Wistar (WM) rats, which lack 2B2, revealed similar rates of microsomal N-demethylation and comparable in vitro hepatotoxicity to Lewis rats. The capacity of a specific P450 2B1/2 inactivator to protect against cocaine-mediated hepatotoxicity both in vivo and in vitro and the results with the WM rats support the identification of P450 2B1 as a major cocaine bioactivating form.

Animals↗
PubMed1994

Catalytic selectivity and mechanism-based inactivation of stably expressed and hepatic cytochromes P450 2B4 and 2B5: implications of the cytochrome P450 2B5 polymorphism.

Cytochrome P450 (P450) 2B5 was recently found to be functionally distinct from three other rabbit P450 2B forms, based on androstenedione hydroxylase activities. In this investigation, we examined the frequency of the P450 2B5-null phenotype and the functional consequences of polymorphic P450 2B5 expression in hepatic microsomes from phenobarbital-treated rabbits. Four of the 10 animals examined did not have detectable levels of P450 2B5 mRNA and exhibited much lower microsomal androstenedione 15 alpha- and 16 alpha-hydroxylase activities. The 15 alpha-hydroxylase activity was found to correlate (r = 0.91) with liver P450 2B5 mRNA. P450 2B4 and 2B5 were stably expressed in human kidney 293 cells to further characterize substrate specificities and to investigate mechanism-based inactivation by phencyclidine. P450 2B4 was 4-16-fold more active than 2B5 towards benzphetamine, 7-ethoxycoumarin, methylenedioxybenzene, and pentoxyresorufin. Benzyloxyresorufin O-debenzylase activity was 160-fold higher for P450 2B4 than P450 2B5. Anti-P450 2B4 IgG inhibited benzyloxyresorufin O-debenzylation nearly completely in untreated and phenobarbital-induced liver microsomes. Phencyclidine selectively inactivated P450 2B4, compared with 2B5, in both human kidney 293 cell and liver microsomes. Poor inactivation of P450 2B5 by phencyclidine was found to be a result of its low maximal rate constant. Results of this study establish the idea that the metabolic consequences of phenobarbital induction depend on the potential of animals to express functionally variant P450 2B forms. Furthermore, we conclude that one or more of the 11 amino acid differences between these highly related P450 forms are critical to their substrate specificities and selective inactivation.

Androstenedione↗
PubMed1994

Application of 3-dimensional homology modeling of cytochrome P450 2B1 for interpretation of site-directed mutagenesis results.

Three-dimensional structures of cytochrome P450 2B1 were modeled based on the crystallographic structure of P450cam. The effect of the alignment, loop choice, and minimization with or without water was assessed. Although final models were similar in overall structure, the identity of active site residues depended upon the alignment. An example is Phe-206, which may or may not form part of the active site. The choice of the loop conformation had a lesser effect, while including water in the final minimization step was essential for preserving the shape and size of the active site. The best model (model 2) was in good agreement with the data from site-directed mutagenesis studies, and correctly predicted the effect of substitutions at 9 out of 10 amino acid positions. Thus, residues important for P450 2B1 activity, such as Ile-114, Phe-206, Ile-290, Thr-302, Val-363, and Gly-478, constitute part of the active site and are able to interact with the substrate androstenedione through hydrophobic interactions. On the other hand, Ser-303, Ser-360 and Lys-473 are far from the active site and/or cannot interact with the substrate, in agreement with experimental data. The model indicates other residues likely to be important for enzyme function, such as Tyr-111, Leu-209, Ile-477, and Ile-480, which can be tested experimentally. The substrate may assume numerous binding orientations consistent with observed patterns of hydroxylation at C15 and C16. The replacement in the model of certain amino acid residues to mimic residue substitutions from site-directed mutagenesis studies and docking of the substrate into the modified active site allowed a plausible explanation for alterations in regio- and stereospecificities of some mutants of P450 2B1, such as Gly-478-->Ala or Val-363-->Ala.

Amino Acid Sequence↗
PubMed1994

Selective suppression of rat hepatic cytochrome P450 2C11 by chloramphenicol.

Chloramphenicol produces mechanism-based inactivation of several rat hepatic microsomal P450 enzymes including 2C6, 2C11, 2B1/2 and 3A1/2. A preliminary study by this laboratory reported that 48 hr after in vivo treatment with chloramphenicol (CAP) 2C11 activity remained low, whereas activities catalyzed by 3A2 and 2C6 were almost fully restored (Halpert et al., Biochem. Pharmacol. 37: 3046-3048, 1988). Therefore, in experiments conducted to examine whether CAP affects P450 expression, Sprague-Dawley (SD) rats were treated with CAP (single i.p. injection, 300 mg/kg) and sacrificed at various times post-treatment. The loss of P450 2C6, 2C11 and 3A2 catalytic activities which is characteristic of inactivation was demonstrated 1 hr after CAP administration. However, at 4 and 6 days, 2C11-mediated progesterone 2 alpha-hydroxylase activity remained diminished by 52 and 45%, respectively. Similar decreases in anti-P450 2C11-reactive protein and 2C11 mRNA were observed at 6 days, suggesting that the compound acts at a pretranslational step. Evaluation of 2C11 regulators indicated that CAP causes a decrease in plasma thyroxine level in proportion to the loss of 2C11 activity, whereas testosterone appears to be unaffected. To minimize intragroup variability, the inbred Fischer 344 strain was then examined at 2 and 6 days after CAP treatment. Surprisingly, CAP caused no loss in 2C11 protein, although the compound does inactivate 2C11 in liver microsomes from Fischer 344 rats. These results suggest that CAP alters P450 expression in a manner distinct from previously described compounds.

Animals↗
PubMed1994

Site-directed mutagenesis of putative substrate recognition sites in cytochrome P450 2B11: importance of amino acid residues 114, 290, and 363 for substrate specificity.

Eleven amino acid residues unique to dog cytochrome P450 (P450) 2B11, compared with rat 2B1 and 2B2, rabbit 2B4 and 2B5, and mouse 2B10, in the putative substrate recognition sites [J. Biol. Chem. 267:83-90 (1992)] were mutated to the residues found in 2B1 or 2B5. The mutants were expressed initially in COS cells and screened for activity toward androstenedione and 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB). P450 2B11 mutants V107I, M199L-N200E-V204R, V234I, A292L, Q473R, and I475S showed no differences from wild-type P450 2B11 in metabolite profiles with either substrate. Mutants V114I, D290I, and L363V exhibited altered androstenedione metabolite profiles and were expressed in Escherichia coli for further study with androstenedione, testosterone, 7-ethoxycoumarin, (R)- and (S)-warfarin, and 245-HCB. With V114I, hydroxylation of steroids and warfarin and 2-hydroxylation of 245-HCB were decreased, whereas 7-ethoxycoumarin O-dealkylation and 3-hydroxylation of 245-HCB were unaltered. For D290I, activities toward all substrates were decreased, except for 16 beta-hydroxylation of testosterone. The activity of L363V was increased 5-6-fold for 16 alpha-hydroxylation of androstenedione and testosterone but was decreased to 40-50% of wild-type activity with 7-ethoxycoumarin and warfarin and to 6-8% of control for 2-hydroxylation of 245-HCB. Alignment of P450 2B11 with P450 101 and super-imposition of the 11 mutated 2B11 residues on a P450 101 three-dimensional model suggest that only residues 114, 290, and 363 represent substrate contact residues, in excellent agreement with the experimental results. The data indicate the importance of the three residues 114, 290, and 363 in substrate specificity and regio- and stereoselectivity of P450 2B11 and also demonstrate that the effects of the mutations vary considerably with different substrates.

Amino Acids↗
PubMed1993

Cloning, sequencing, and functional studies of phenobarbital-inducible forms of cytochrome P450 2B and 4B expressed in rabbit kidney.

Expression of several forms of cytochrome P450 2B and of P450 4B1 in rabbit kidney was investigated by cloning from cDNA libraries constructed with renal mRNA from animals treated with phenobarbital. Isolation and sequencing of several cDNAs demonstrated that: (i) cytochrome P450 2B-B0 can be found in rabbit kidney along with a newly discovered form of P450 2B termed "P450 2B-Bx." P450 2B-Bx differs from P450 2B-B0 at 25 nucleotide positions and at four positions in the derived sequence of 491 amino acids. Two previously identified forms of cytochrome P450 2B, 2B-B1 and 2B-B2, are not detected in rabbit kidney. cDNA encoding cytochrome P450 4B1 was also cloned from the kidney library and found to be identical in sequence to cDNAs cloned from rabbit hepatic and pulmonary libraries. Analysis of renal mRNA indicates that forms of cytochrome P450 2B and P450 4B1 are expressed in a number of species but induced by phenobarbital in rabbit only (4B1) or rabbit and hamster (2B). Relatively high levels of mRNA related to P450 4B1 were detected in samples from untreated and phenobarbital-treated mice. Analysis of protein by immunoblotting was less sensitive but produced results consistent with those obtained by analysis of mRNA; protein related to cytochrome P450 2B was detected in renal microsomal samples from rabbit and hamster (phenobarbital > untreated), and protein related to P450 4B1 in samples from rabbits (phenobarbital > untreated) and mice (phenobarbital = untreated). The four forms of cytochrome P450 2B were expressed in COS-7 cells, and their activities were evaluated with androstenedione, testosterone, and 7-ethoxycoumarin as substrates. Three of the P450 2B forms, B0, B1, and Bx, metabolize these substrates in a manner characterized by preference for 16 beta-hydroxylation of androstenedione, low testosterone 16-hydroxylation, and high ethoxycoumarin O-deethylation. The fourth form, P450 2B-B2, is catalytically distinct from the others, with activities characterized by high androstenedione 16 alpha- and 15 alpha-hydroxylation and high testosterone 16-hydroxylation. Since P450 2B-B2 is catalytically distinct from the other forms, the metabolic profiles of phenotypes that include P450 2B-B2 might differ significantly from those of phenotypes that lack P450 2B-B2.

Amino Acid Sequence↗
PubMed1993

Hybrid enzymes for structure-function analysis of cytochrome P-450 2B11.

Previous work has shown that P-450 2B11 is responsible for the unique ability of dogs to metabolize and eliminate certain highly-chlorinated biphenyls such as 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB), whereas the related P-450 2B forms in rat and rabbit are unable to metabolize the compound to any significant degree. To determine the structural basis for this functional diversity, hybrid enzymes were generated. Success with this approach required a careful choice of second enzyme and common substrate with which to assess the functional integrity of the hybrid proteins. The choices of P-450 2B5 from rabbit as the second enzyme and androstenedione as the substrate were based in part on the finding that P-450 2B11 and P-450 2B5 hydroxylate androstenedione with similar overall activities but distinct profiles. Enzymatic studies with eight hybrid enzymes provided evidence for two regions of P-450 2B11 and 2B5, between residues 95-239 and 240-370, that appear to be involved in defining substrate specificity for androstenedione, and three regions of P-450 2B11, between residues 95-239, 240-370, and 371-494, that contain amino acids necessary for metabolism of 245-HCB. This deliberate approach to the creation of hybrid cytochromes P-450 has generated a series of enzymes that will be central to further structure-function studies of the cytochromes P-450 2B.

Androstenedione↗
PubMed1993

Engineering of cytochrome P450 2B1 specificity. Conversion of an androgen 16 beta-hydroxylase to a 15 alpha-hydroxylase.

Six site-directed mutants of cytochrome P450 2B1 were constructed, and function was evaluated in COS cell microsomes by monitoring testosterone and androstenedione hydroxylation and inactivation by chloramphenicol. Mutants Ile-114-->Val and Ile-114-->Ala exhibited marked decreases in androgen 16 beta-OH:16 alpha-OH ratios and increases in 15 alpha-OH:16-OH ratios. Since substitution of Gly-478 with Ala or Ser reduces 16 beta-hydroxylation in favor of 15 alpha-hydroxylation, four double mutants containing Val or Ala at position 114 and Ala or Ser at position 478 were examined. For any given residue at position 114 (Ile, Val, or Ala), the 15 alpha-OH:16-OH ratio increased as residue 478 was changed from Gly to Ala to Ser, and for any residue at position 478, this ratio increased as residue 114 was changed from Ile to Val to Ala. As a consequence, the Ile-114-->Ala, Gly-478-->Ser mutant displayed an approximately 1000-fold higher androgen 15 alpha-OH:16-OH ratio compared with the parental enzyme and functionally resembles mouse P450 2A4 much more closely than P450 2B1. All three mutants with Val at position 114 retained susceptibility to inactivation by chloramphenicol, whereas inactivation was suppressed by Ala at this position. The results suggest the feasibility of an empirical approach to P450 engineering involving the appropriate combination of residues at a few critical sites to confer new regio- and stereoselectivity with retention of overall monooxygenase activity.

Androstenedione↗
PubMed1992

Role of residue 478 as a determinant of the substrate specificity of cytochrome P450 2B1.

Two allelic variants and eight site-directed mutants of cytochrome P450 2B1 differing at residue 478 have been expressed in COS cells and assayed for androstenedione hydroxylase activities. The 478Gly and 478Ala variants and five mutants (Ser, Thr, Val, Ile, and Leu) exhibited 16 beta-OH:16 alpha-OH ratios ranging from 0.7 to 9.3, whereas the Pro, Glu, and Arg mutants were expressed but inactive. The seven samples active toward androstenedione also exhibited testosterone 16 beta-OH:16 alpha-OH ratios ranging from 0.4 to 2.3. With both steroids, the Gly variant had the highest 16 beta-hydroxylase activity, and the 16 beta-OH:16 alpha-OH ratio increased with the size of aliphatic size chains (Ala, Val, and Ile/Leu). The highest ratio of androgen 15 alpha:16-hydroxylation was observed with the Ser mutant. On the basis of previous work indicating decreased susceptibility of the 478Ala variant in liver microsomal and reconstituted systems to inactivation by chloramphenicol analogs, methodology was refined for monitoring enzyme inactivation in COS cell microsomes. The Gly and Ala variants were inactivated by chloramphenicol with similar rate constants, whereas the Ser and Val mutants were inactivated more slowly, and the Leu mutant was refractory. Only the Gly variant was inactivated by the chloramphenicol analog N-(2-p-nitrophenethyl)chlorofluoroacetamide. Thus, the side chain of residue 478 appears to be a major determinant of enzyme inactivation as well as of androgen hydroxylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗