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J R Halpert

Publications and source records attributed to J R Halpert.

At least 91 records · Page 5Linked to original sources
PubMed1992

Topological analysis of the active sites of cytochromes P450IIB4 (rabbit), P450IIB10 (mouse), and P450IIB11 (dog) by in situ rearrangement of phenyl-iron complexes.

The reaction of phenyldiazene with purified, phenobarbital-inducible rabbit cytochrome P450IIB4, mouse cytochrome P450IIB10, and dog cytochrome P450IIB11 yields complexes with absorbance maxima at 480 nm. Treatment of the cytochrome P450 complexes with K3Fe(CN)6 results in disappearance of the 480-nm absorption. Extraction of the prosthetic group from the proteins after these reactions yields the two isomers of N-phenylprotoporphyrin IX with the N-phenyl group on pyrrole rings A and D as the major products and the regioisomer with the N-phenyl on pyrrole ring C as a minor product. The A:C:D arylated pyrrole ring ratio is 3:2:3 for rabbit P450IIB4, 3:1:3 for mouse P450IIB10, and 4:1:2 for dog P450IIB11. Formation of the A and D regioisomers is consistent with the results obtained previously for rat isozymes IA1, IIB1, IIB2, and IIE1, but the rabbit, mouse, and dog P450IIB enzymes differ from the four rat enzymes in that a substantial amount of the isomer with the N-phenyl on pyrrole ring C is also formed. The results indicate that the region over pyrrole ring B is masked by protein residues in all the active sites and suggest that the region over pyrrole ring C is more hindered by protein residues in the rat than in the rabbit, mouse, or dog enzymes so far examined.

Animals↗
PubMed1991

Molecular basis for a functionally unique cytochrome P450IIB1 variant.

Liver microsomes from phenobarbital-treated rats of four inbred strains expressing distinct allelic variants of cytochrome P450IIB1 were analyzed. The Wistar Munich (WM) strain exhibited 5- to 10-fold lower androstenedione 16 beta-hydroxylase activity (a specific P450IIB1 marker) than the Lewis, Wistar Kyoto, and Wistar Furth strains. The androstenedione 16 beta-hydroxylase in the WM liver microsomes was refractory to inactivation by N-(2-p-nitrophenethyl)chlorofluoroacetamide, a selective P450IIB1 inactivator in the other three strains. Purified P450IIB1-WM was insensitive to the inactivator and exhibited 5-fold lower androstenedione 16 beta-hydroxylase, testosterone 16-hydroxylase, and 7-ethoxycoumarin deethylase activities but the same benzphetamine demethylase activity and slightly higher androstenedione 16 alpha-hydroxylase activity than a P450IIB1 purified from outbred Sprague-Dawley rats, which appears to correspond to the form in Lewis rats. The stereoselectivity of androstenedione 16-hydroxylation catalyzed by P450IIB1-WM (16 beta-OH:16 alpha-OH = 1.4) is thus distinct from that (16 beta-OH:16 alpha-OH = 12-15) of other P450IIB1 preparations described. A cDNA encoding P450IIB1-WM was cloned and sequenced, revealing a single amino acid substitution (Gly-478----Ala) compared with the published sequence (Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., and Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2793-2797). Heterologous expression of P450IIB1 and P450IIB1-WM confirmed the striking difference in androstenedione metabolite profiles, strongly implicating the involvement of Ala-478 in defining the distinctive catalytic properties of P450IIB1-WM.

Alleles↗
PubMed1991

Functional expression of mammalian cytochromes P450IIB in the yeast Saccharomyces cerevisiae.

Three mammalian cytochromes P450 from the IIB subfamily, P450IIB11 from canine and P450IIB4 and P450IIB5 from rabbit, have been expressed in the yeast Saccharomyces cerevisiae by use of an autonomously replicating vector containing the galactose-inducible gal10 promoter. Cytochromes P450IIB4 and P450IIB5 are closely related proteins, with only 11 amino acid substitutions between them. P450IIB11 is a homologous protein, likely orthologous with IIB4 or IIB5, with 102 amino acid substitutions compared with the P450IIB4 protein and 106 compared with the P450IIB5 protein. The expressed proteins are functional in yeast microsomes, exhibiting activity toward androstenedione, 7-ethoxycoumarin, and, in some cases, progesterone. Expressed cytochromes P450IIB4 and P450IIB11 hydroxylate androstenedione with regio- and stereoselectivity characteristic of the purified, reconstituted proteins. A striking difference in the androstenedione metabolite profiles of IIB4 and IIB5 was observed, with IIB4 producing almost exclusively the 16 beta-hydroxy metabolite and IIB5 producing the 16 alpha-hydroxy and 15 alpha-hydroxy products. This is the first time that 15 alpha-hydroxylase activity has been associated with IIB4/IIB5. This activity has also been detected in liver microsomes from some, but not all, individual phenobarbital-induced rabbits tested and is largely inhibited by anti-rabbit P450IIB immunoglobulin G. These studies illustrate the utility of the yeast expression system for defining catalytic activities of individual mammalian cytochromes P450 and identifying new marker activities that can be utilized in liver microsomes.

Androstenedione↗
PubMed1991

cDNA and deduced amino acid sequences of a dog liver cytochrome P-450 of the IIIA gene subfamily.

A 1.96 kbp cDNA encoding a male Beagle dog liver cytochrome P-450 of 503 amino acid residues (Mr 57,636) has been isolated and sequenced. The deduced amino acid sequence is 79.8%, 69.3% and 74.1% identical to the P450IIIA forms human NF25, rat PCN1 and rabbit LM3c, respectively. The amino terminal sequence is identical to the first 28 residues of the dog P450IIIA form PBD-1. Southern blot analysis yields restriction patterns consistent with IIIA gene subfamily multiplicity.

Amino Acid Sequence↗
PubMed1991

Porphyrinogenic effects in chick embryo liver cell culture of chloramphenicol analogues that are mechanism-based inactivators of cytochrome P-450.

Structural analogues of chloramphenicol (CAP) cause mechanism-based inactivation of rat liver cytochrome P-450 (P450) either via protein acylation or destruction of the heme prosthetic group. The goal of the present work was to determine whether CAP analogues that cause loss of the P450 heme moiety also cause porphyrin accumulation in chick embryo liver cell culture. The porphyrin profiles produced by exposure of cells to CAP analogues (160 microM) were determined by high-performance liquid chromatography with fluorescence detection. Of three CAP analogues that do not cause loss of the heme moiety of rat liver P450IIB1, two dichloroacetamides were not porphyrinogenic. The third compound, a chlorofluoroacetamide, caused porphyrin accumulation. This result may be due to the presence of P450 isozymes in chick embryo hepatocytes, distinct from rat liver P450IIB1, that are susceptible to destruction by this analogue. Of four CAP analogues that inactivate rat liver P450IIB1 with concomitant heme loss, a dichloroacetamide and two chlorofluoroacetamides caused porphyrin accumulation. The remaining compound, a monochloroacetamide, was not porphyrinogenic, perhaps because the P450 apoprotein cannot be reconstituted with fresh heme drawn from the regulatory "free heme pool" following inactivation by this analogue. Alternatively, there may be no P450 isozyme in chick embryo liver cell culture that is susceptible to inactivation by this compound.

Animals↗
PubMed1990

cDNA and deduced amino acid sequences of a dog hepatic cytochrome P450IIB responsible for the metabolism of 2,2',4,4',5,5'-hexachlorobiphenyl.

The nucleotide sequence of a cDNA that codes for the major phenobarbital (PB)-inducible male beagle dog hepatic cytochrome P450 has been determined. Using a rabbit P450IIB cDNA probe (R. Gasser, M. Negishi, and R. M. Philpot, 1988, Mol. Pharmacol, 32, 22-30), a cDNA clone with a 2.6-kilobase pair insert was isolated from a lambda gt11 library prepared from hepatic mRNA from a PB-treated dog. The cloned insert was sequenced and found to contain an open reading frame coding for a polypeptide of 494 amino acids (Mr 56,183). The encoded protein can be assigned to the P450IIB subfamily on the basis of homology to cytochromes P450 from other species. The deduced amino acid sequence is 79% identical to that reported for rabbit P450 BO (P450IIB4) and 75% identical to that for rat P450b (P450IIB1). The sequence identity decreases to less than 52% when the dog sequence is compared with other P450II subfamilies. The deduced NH2-terminal 30 amino acids encoded by the dog cDNA are identical to those determined by sequence analysis of purified dog cytochrome P450 PBD-2, and the amino acid composition concurs with that determined for the PBD-2 protein (D. B. Duignan, I. G. Sipes, T. B. Leonard, and J. R. Halpert, 1987, Arch. Biochem. Biophys. 255, 290-303). Northern blots revealed two mRNA species of approximately 1.9 and 2.9 kilobases in length, which hybridized to the coding region of the dog P450IIB cDNA. The level of total hybridizable mRNA was increased approximately sixfold in livers from PB-treated dogs compared with that in untreated animals. This increase correlates well with the reported nearly sixfold increase in the level of PBD-2 protein and the fivefold increase in the rate of hepatic metabolism of 2,2',4,4',5,5'-hexachlorobiphenyl following PB treatment. The two mRNA species may result from the use of different polyadenylation signals located in the 3'-noncoding region or from transcription of more than one gene for PBD-2. Southern blot analysis indicated that the dog P450IIB subfamily contains at least two closely related genes.

Amino Acid Sequence↗
PubMed1990

The biochemical basis for the species difference in hepatic microsomal 4-vinylcyclohexene epoxidation between female mice and rats.

Mice but not rats are susceptible to 4-vinylcyclohexene (VCH)-induced ovarian toxicity and carcinogenicity. This is due in part to a 4- to 6-fold greater rate of hepatic microsomal bioactivation of VCH to the ovotoxicant VCH-1,2-epoxide. The biochemical basis for this difference was investigated in microsomes using enzyme induction, enzyme inhibition with chloramphenicol or specific inhibitory antibodies, and correlation with marker steroid hydroxylase activities to associate VCH epoxidation with particular cytochrome P450 forms. Testosterone 6 beta- and 15 alpha-hydroxylase activities and VCH epoxidation were decreased in microsomes from chloramphenicol-treated mice, initially suggesting the possible involvement of P450IIIA and P450IIA forms in VCH metabolism. Although both testosterone 6 beta-hydroxylase and VCH epoxidase activities were increased by dexamethasone treatment (P450IIIA inducer), anti-rat P450IIIA IgG inhibited testosterone 6 beta-hydroxylase (68%) but not VCH epoxidase activity. These latter results do not support the involvement of mouse P450IIIA forms in VCH epoxidation. However, results were obtained which indicated that mouse P450IIA forms are involved in VCH epoxidation. In microsomes from untreated female mice VCH epoxidase activity was inhibited 48% by antibodies to mouse P45015 alpha (P450IIA3) at a concentration that inhibited testosterone 15 alpha-hydroxylase activity by 86%. No protein immunochemically related to mouse P45015 alpha was detected in female rat hepatic microsomes. VCH epoxidation by hepatic microsomes was increased in female mice and rats by phenobarbital treatment and was inhibited by approximately one-third by anti-rat-P450IIB1 IgG in microsomes from untreated animals of both species. Furthermore, microsomal VCH epoxidase and testosterone 16 alpha-hydroxylase activities were lower (34%) in female 129/J mice (deficient in constitutive expression of P450IIB forms) than in B6C3F1 mice. These results suggested partial involvement of P450IIB forms in the microsomal epoxidation of VCH. Therefore, P450 forms IIA and IIB account for the majority of VCH bioactivation in female mouse liver, which explains in part the susceptibility of mice to VCH-induced ovarian toxicity and carcinogenicity.

Animals↗
PubMed1989

Characterization of a phenobarbital-inducible dog liver cytochrome P450 structurally related to rat and human enzymes of the P450IIIA (steroid-inducible) gene subfamily.

A cytochrome P450 called PBD-1 isolated from liver microsomes of an adult male Beagle dog treated with phenobarbital (PB) is structurally and functionally similar to members of the P450IIIA gene subfamily in rat and human liver microsomes. The sequence of the first 28 amino-terminal residues of PBD-1 is identical in 15 and 20 positions, respectively, to the P450IIIA forms P450p from rat and P450NF (and HLp) from human. Upon immunoblot analysis, anti-PBD-1 IgG recognizes PCNa (P450p) and PCNb (PB/PCN-E) from rat, P450NF from human, and two proteins in liver microsomes from both untreated and PB-treated dogs. Similarly, anti-PCNb IgG cross-reacts with PBD-1 and with at least one protein in microsomes from untreated dogs and two proteins in microsomes from PB-treated dogs. P450IIIA-form marker steroid 6 beta-hydroxylase activities increase 2.5-fold upon PB-treatment of dogs and are selectively inhibited by anti-PBD-1 IgG. NADPH-dependent triacetyloleandomycin (TAO) complex formation and erythromycin demethylase, also marker activities for P450IIIA forms from rats and humans, increase 4- and 5-fold in dog liver microsomes upon PB treatment, whereas immunochemically reactive PBD-1 is induced 3-fold. In microsomes from PB-treated dogs, 5 mg anti-PBD-1 IgG/nmol P450 inhibits greater than 75 and 50% of TAO complex formation and erythromycin demethylase activity, respectively. TAO complex formation is not inhibited by chloramphenicol, a selective inhibitor of the major PB-inducible dog liver cytochrome P450, PBD-2. These data suggest that PBD-1 or another immunochemically related form is responsible for a major portion of macrolide antibiotic metabolism by microsomes from PB-treated dogs and for steroid 6 beta-hydroxylation by microsomes from both untreated and PB-treated dogs. Major species differences were noted, however, in the apparent Km for 6 beta-hydroxylation of androstenedione by liver microsomes from untreated rats (24 microM), humans (380 microM), and untreated dogs (4700 microM).

Amino Acid Sequence↗
PubMed1988

The metabolism of xenobiotics and endogenous compounds by the constitutive dog liver cytochrome P450 PBD-2.

We have investigated the metabolism of polychlorinated biphenyls and endogenous steroids by the major phenobarbital (PB)-inducible hepatic cytochromes P450 in dogs and rats, PBD-2 and PB-B, respectively. Previous results from our laboratory indicate that dog PBD-2 purified from microsomes of PB-treated animals is similar to rat PB-B with respect to structure and the regioselective metabolism of warfarin and androstenedione. The results also strongly suggest that PBD-2 is the P450 form responsible for metabolizing 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB) in liver microsomes from untreated dogs. In the present study, a cytochrome P450 with similar chromatographic behavior to that of PBD-2 has been purified from liver microsomes of untreated dogs. This protein is identical to PBD-2 based on (i) mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (ii) reactivity with anti-PBD-2 IgG, (iii) amino-terminal sequence, and (iv) 245-HCB metabolite profile. Induction and antibody-inhibition data suggest that PBD-2 is responsible for the metabolism of 2,2',3,3',6,6'-hexachlorobiphenyl (236-HCB) in microsomes obtained from both untreated and PB-treated dogs. In contrast, metabolism of 4,4'-dichlorobiphenyl (4-DCB) by dog microsomes is poor, and does not appear to be catalyzed to a significant extent by PBD-2. Antibody-inhibition studies with intact microsomes corroborate previous results that androstenedione is metabolized by purified PBD-2 to the same major metabolite (16 beta-OH androstenedione) produced by rat PB-B. Dog PBD-2 metabolizes progesterone primarily to the 21-OH metabolite, while metabolism by rat PB-B leads to the formation of the 16 alpha-OH product. On the other hand, upon Ouchterlony double-immunodiffusion analysis, anti-PBD-2 IgG reacts strongly with PB-B but not PB-C, the major rat liver progesterone 21-hydroxylase. The data suggest that dog PBD-2 is a constitutive P450 important in the metabolism of various PCBs and endogenous steroids. Dog PBD-2 and rat PB-B appear to be similar enzymes, yet they differ in their regioselective metabolism of progesterone.

Amino Acid Sequence↗
PubMed1988

Multiplicity of steroid-inducible cytochromes P-450 in rat liver microsomes.

Highly purified preparations of steroid-inducible cytochromes P-450 have been isolated from liver microsomes of adult male Sprague-Dawley rats treated with pregnenolone-16 alpha-carbonitrile (PCN), phenobarbital (PB), or triacetyloleandomycin (TAO). The interrelationships among these preparations as well as their relationship to the major forms of cytochrome P-450 of this gene family identified in other laboratories have been evaluated by amino-terminal sequence analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, chromatography on DEAE-Sephacel, and Ouchterlony double-immunodiffusion. The results indicate that a cytochrome P-450 previously isolated in this laboratory and referred to as PCNa (P.E. Graves, L.S. Kaminsky, and J. Halpert (1987) Biochemistry 26, 3887-3894) is the major cytochrome P-450 induced in rats by TAO, and corresponds to P-450p (K.A. Hostetler, S.A. Wrighton, P. Kremers, and P.S. Guzelian (1987) Biochem. J. 245, 27-33) and to P450PCN1 (F.J. Gonzalez, B.J. Song, and J.P. Hardwick (1986) Mol. Cell. Biol. 6, 2969-2976). A second previously isolated cytochrome P-450 termed PCNb in this laboratory appears to be identical to PB/PCN-E (F.P. Guengerich, G.A. Dannan, S.T. Wright, M.V. Martin, and L.S. Kaminsky (1982) Biochemistry 21, 6019-6030). PCNb as well as a third cytochrome P-450 termed PCNc isolated from PB-treated rats both correspond in amino-terminal sequence to the putative protein product of the pP450PCN2/cDNA clone of Gonzalez et al. These results document at the protein level the multiplicity of steroid-inducible rat liver cytochromes P-450.

Amino Acid Sequence↗
PubMed1987

Purification and characterization of the dog hepatic cytochrome P-450 isozyme responsible for the metabolism of 2,2',4,4',5,5'-hexachlorobiphenyl.

The biochemical basis for the marked difference in the rate of the hepatic metabolism of 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB) by Beagle dogs and Sprague-Dawley rats has been investigated. Control dog liver microsomes metabolize this substrate 15 times faster than control rat liver microsomes. Upon treatment with phenobarbital (PB), at least two cytochrome P-450 isozymes are induced in the dog, and the hepatic microsomal metabolism of 245-HCB is increased on both a per nanomole P-450 basis (twofold) and a per milligram protein basis (fivefold). One of the PB-induced isozymes, PBD-2, has been purified to a specific content of 17-19 nmol/mg protein and to less than 95% homogeneity, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In a reconstituted system containing cytochrome b5, this isozyme shows an activity toward 245-HCB which is greater than threefold that seen in intact liver microsomes from PB-induced dogs. A reconstituted system containing the major isozyme induced by PB in the rat (PB-B) metabolizes 245-HCB at 1/10 the rate observed with purified PBD-2. Antibody inhibition studies have shown that PBD-2 accounts for greater than 90% of the hepatic microsomal metabolism of 245-HCB in control and PB-induced dogs, while PB-B only accounts for about half of the metabolism of this compound by microsomes obtained from PB-treated rats. Immunoblot analysis has revealed that the level of PBD-2 in dog liver microsomes increases nearly sixfold with PB treatment, and this increase correlates well with the fivefold increase in the rate of hepatic microsomal metabolism of 245-HCB by dogs. Together these data support a primary role for isozyme PBD-2 in the hepatic metabolism of 245-HCB in control and PB-induced dogs. In addition, these results suggest that, in contrast to rats, dogs can readily metabolize 245-HCB as a result of the presence of a cytochrome P-450 isozyme with efficient 245-HCB metabolizing activity.

Amino Acid Sequence↗
PubMed1986

Dichloromethyl compounds as mechanism-based inactivators of rat liver cytochromes P-450 in vitro.

Twenty dichloromethyl compounds have been tested as potential mechanism-based inactivators of the major phenobarbital-inducible isozyme of rat liver cytochrome P-450 (PB-B) in a reconstituted system. With the exception of dichloromethane and dichloroacetamide, all the compounds decreased the ethoxycoumarin deethylase activity of the enzyme in a time- and NADPH-dependent manner. The inhibitory compounds could be divided into two classes according to whether the loss of monooxygenase activity was accompanied by a decrease in spectrally detectable cytochrome P-450. N-Monosubstituted dichloroacetamides in which the side-chain consisted of a phenyl or n-octyl group were able to mimic the action of chloramphenicol and inactivate the PB-B without destroying the heme moiety. In contrast, dichloroacetamides containing an n-hexyl, n-butyl, or methyl substituent caused a significant loss of heme, as did the five non-amides tested: 1,1,2,2-tetrachloroethane, 1,1-dichloroacetone, methyl dichloroacetate, alpha,alpha-dichlorotoluene, and alpha,alpha-dichloroacetophenone. Representative compounds were also examined as inactivators of the major beta-naphthoflavone-inducible isozyme of rat liver cytochrome P-450 (BNF-B), using a reconstituted system, as well as of constitutive cytochromes P-450, using intact liver microsomes from untreated rats. These studies suggested a marked difference in isozyme selectivity between certain of the compounds. For example, of the isozymes monitored, only the PB-B was affected by alpha,alpha-dichlorotoluene in an NADPH-dependent manner, whereas N-octyl dichloroacetamide inactivated not only the PB-B and BNF-B, but also certain constitutive cytochromes, as evidenced by decreases in microsomal S-warfarin hydroxylase activities. These studies help delineate the structural requirements for the use of dichloromethyl compounds as probes of cytochrome P-450 function and as potential isozyme-selective inhibitors.

Acetamides↗
PubMed1985

On the mechanism of the inactivation of the major phenobarbital-inducible isozyme of rat liver cytochrome P-450 by chloramphenicol.

The mechanism of the inactivation of the major phenobarbital-inducible isozyme of rat liver cytochrome P-450 (P-450 PB-B2) by chloramphenicol has been investigated. Preparations of the enzyme from animals treated in vivo with chloramphenicol (CAP PB-B2) have been isolated, and their catalytic, spectral, and physical properties have been compared with those of the native PB-B2. The CAP PB-B2 exhibited: 1) a 60-70% loss in the rate of NADPH-supported monooxygenase activity with the substrates benzphetamine, 7-ethoxycoumarin, and p-nitroanisole; 2) a 60% decrease in the extent of enzymatic P-450 reduction catalyzed by NADPH-cytochrome P-450 reductase under both aerobic and anaerobic conditions; 3) a 60% decrease in the steady-state level of the ferrous dioxygen complex in the presence of substrates; 4) a 60% decrease in the magnitude of the type I spectral change induced by benzphetamine; and 5) a shift in the wavelength maximum for the chemically reduced ferrous-carbonyl complex from 450 to 451.5 nm. On the other hand, the ability of the CAP PB-B2 to catalyze the iodosobenzene-supported metabolism of 7-ethoxycoumarin and p-nitroanisole was unaltered. The results are consistent with a scheme whereby the binding of metabolites of chloramphenicol to amino acid residues in the PB-B2 close to the heme moiety blocks electron transport from NADPH-cytochrome P-450 reductase, thereby leading to a loss of monooxygenase activity.

7-Alkoxycoumarin O-Dealkylase↗
PubMedDate not supplied

Effect of congestive heart failure on the intrinsic metabolic capacity of the liver in the dog.

The intrinsic metabolic capacity of the liver at end-stage heart failure in the pacing overdrive dog model of congestive heart failure was evaluated ex vivo. Congestive heart failure was induced in seven adult mongrel dogs (20-30 kg) by cardiac electrical pacing at a frequency of 240 stimuli/min until the development of overt heart failure; seven other dogs served as controls. The animals were then anesthetized and the right ventricular papillary muscles and samples from the left lateral hepatic lobes were collected. The degree of myocardial dysfunction as well as the total amount and the activities of cytochromes P-450 were evaluated. Tension, maximum rate of tension rise, and Vmax were significantly lower (40-60%) in the paced than in the control dogs, indicating a marked myocardial dysfunction. Moreover, significant decreases in total cytochrome P-450 (0.31 +/- 0.04 vs. 0.53 +/- 0.03 nmol/mg of microsomal protein, p less than 0.01) and in the intensity of four different electrophoretic protein bands (molecular masses of 46, 48, 50, and 59 kDa) occurred in the dogs with congestive heart failure. The decrease in total cytochrome P-450 was accompanied by a significant reduction in aminopyrine N-demethylase activity (1.74 +/- 0.25 vs. 2.91 +/- 0.40 nmol/min/mg of microsomal protein, p less than 0.05). Immunoblot analysis using antibodies to two different dog liver phenobarbital-inducible cytochromes P-450 demonstrated that PBD-1 (a P-450IIIA) was not affected by congestive heart failure, whereas PBD-2 (a P-450IIB) was markedly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Mechanism-based inactivation of the major beta-naphthoflavone-inducible isozyme of rat liver cytochrome P-450 by the chloramphenicol analog N-(2-p-nitrophenethyl)dichloroacetamide.

The effectiveness, selectivity, and mechanism of the inactivation of the major beta-naphthoflavone-inducible isozyme of rat liver cytochrome P-450 (BNF-B) by the chloramphenicol analog N-(2-p-nitrophenethyl)dichloroacetamide (pNO2DCA) have been investigated. Intraperitoneal administration of pNO2DCA to beta-naphthoflavone-treated rats at doses of 10 and 100 mg/kg resulted in 72 and 95% decreases, respectively, in the ethoxyresorufin deethylase activity of subsequently prepared liver microsomes. Similar decreases were observed in the warfarin R-6 and R-8 hydroxylase activities of the microsomes. At the lower dose of pNO2DCA, only those R-warfarin hydroxylase activities attributable to BNF-B were decreased, whereas, at the higher dose, inhibition of additional cytochromes P-450 was evident. In vitro, pNO2DCA was found to be a highly efficient inactivator of purified BNF-B in a reconstituted system. The maximal rate constant for inactivation and the apparent Km for the inhibitor were 0.52 min-1 and 2.7 microM, respectively. Inactivation of BNF-B by pNO2DCA appears to involve an impairment in electron transfer from NADPH-cytochrome P-450 reductase, as evidenced by a decrease in the NADPH- but not the iodosobenzene-supported metabolism of ethoxycoumarin by the modified enzyme. However, in the absence of substrate, there was no decrease in the NADPH oxidase activity or in the steady state level of ferrous carbonyl complex formed enzymatically. Likewise, the maximal level of isosafrole metabolite-P-450 complex formed by BNF-B was not decreased by modification with pNO2DCA, although the rate of complex formation was inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetamides↗