Biochemical Properties of Cytochrome P-450 in Relation to Steroid Oxygenation a

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73858/1/j.1749-6632.1985.tb14606.x.pd

Properties of the tryptophan residue in rabbit liver microsomal cytochrome P-450 isozyme 2 as determined by fluorescence

Cytochrome P-450 isozyme 2 from rabbit liver microsomes fluoresces upon excitation at 295 nm due to the single tryptophyl residue (Trp121) in the protein. The fluorescence spectrum, which is not altered by the presence of phospholipid or substrates, has a maximum at 335 nm, which suggests that the environment of the residue is hydrophobic. The fluorescence intensity decreases linearly with increase of specific content of the cytochrome preparations, and the holoenzyme was estimated to exhibit, at most, 6% as much fluorescence as the apoenzyme. This indicates that the fluorescence of the tryptophan is quenched by energy transfer to the heme. The distance between the tryptophyl residue and the heme was estimated to be less than 40 A. From enhancement of the fluorescence by methanol and ethanol, 30 to 50% of the Trp residue was found to be accessible to these solvents. On the other hand, the accessibility to iodide and cesium ions, as estimated by quenching effects, is less than 14%. From such evidence, the tryptophyl residue is believed to be partly buried. Since Trp121 is conserved at or near the same position in all mammalian P-450's so far sequenced, the results obtained may be applicable to these related cytochromes as well.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25697/1/0000251.pd

Simple, rapid, and highly efficient separation of amino acid phenylthiohydantoins by reversed-phase high-performance liquid chromatography

A rapid and efficient separation of amino acid phenylthiohydantoins by high-performance liquid chromatography has been accomplished by step-gradient elution with use of a mobile phase of acetate-buffered aqueous acetonitrile and an octadecylsilyl stationary phase. Typical analyses are completed in less than 12 min. Peak elution positions in this procedure are highly reproducible (with about 0.2% variance) and allow unambiguous identification of all residues. A procedure for the optimal positioning phenylthiohydantoin-Arg and -His is given. Molar extinction coefficients at 254 nm, which are particularly useful with common fixed wavelength detectors, are given for 25 amino acid phenylthiohydantoins.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24018/1/0000267.pd

Alcohol-inducible cytochrome P-450 (P-450 ALC )

Of the family of P-450 cytochromes occurring in rabbit liver microsomes, only isozyme 3 a (P-450 ALC ) is induced by alcohol administration and is effective in catalyzing the reaction: ethanol+0 2 +NADPH+H + → acetaldehyde +2H 2 O+NADP + . As judged by immuno-chemical quantitation, P-450 ALC is also induced in the animals by other diverse agents, including imidazole, trichlorethylene, acetone, pyrazole, and isoniazid. Evidence has been obtained for the occurrence of a protein immuno-chemically related to P-450 ALC in human liver microsomes and of a similar alcohol-inducible protein in the rat and in the normal and alcohol dehydrogenase-deficient deer-mouse. P-450 ALC catalyzes the activation of foreign compounds such as acetaminophen, various nitrosamines, and carbon tetrachloride and is therefore believed to play an important role in the enhanced toxicity of these substances accompanying alcohol administrationPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46156/1/204_2004_Article_BF00296940.pd

Role of alcohol P-450-oxygenase (APO) in microsomal ethanol oxidation

The form of liver microsomal cytochrome P-450 induced by chronic administration of ethanol to rabbits, designated as P-450 or P-450 isozyme 3a, has been purified to homogeneity as judged by several criteria, including NH2- and COOH-terminal amino acid sequence determination. The reconstituted alcohol-P-450 oxygenase (APO) system containing P-450 and NADPH-cytochrome P-450 reductase catalyzes the oxidation of a variety of primary and secondary alcohols to aldehydes and ketones, including methanol, ethanol, n-propanol, n-butanol, 2-butanol, n-pentanol, and cyclohexanol. Other purified P-450 cytochromes, including isozymes 2, 3b, 3c, 4, and 6, are much less active than P-450 in the oxidation of alcohols. That P-450 functions in ethanol oxidation in liver microsomal membranes as well as in the reconstituted system was shown by immunochemical experiments involving inhibition by sheep anti-P-450 antibodies. We conclude that P-450 is the predominant ethanol-oxidizing cytochrome present after induction by chronic alcohol administration and that the other P-450 cytochromes have low but significant activity in both control and ethanol-induced animals.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25797/1/0000359.pd

Drug and fatty acid hydroxylation by solubilized human liver microsomal cytochrome P-450--Phospholipid requirement

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/22148/1/0000577.pd

Isolation and characterization of a novel cytochrome P-450-like pseudogene

SummaryA rabbit liver P-450-like pseudogene has been isolated from a [lambda] phage genomic library. Sequence analysis revealed structural homology with respect to the rat P-450b and P-450e genes as well as a similar intron-exon organization. A 5'-proximal TATA box-like sequence and two 3'-distal putative polyadenylation signals were identified, and all putative intronexon boundaries except at the 3'-splice site of intron 2 were found to follow the GT/AG rule. With allowance for apparent deletions and insertions, the structural homology of the amino acid sequence deduced from the pseudogene with respect to rabbit P-450 isozyme 2 is lower for exons 1 through 4 (18-28%) than for exons 5 through 9 (42-65%). S1 nuclease mapping showed that mRNAs complementary to the DNA sequence of exon 9 are expressed. However, due to the alterations in the pseudogene, it appears that functional P-450 would not be produced from such mRNAs.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26288/1/0000373.pd

Comparison of six rabbit liver cytochrome P-450 isozymes in formation of a reactive metabolite of acetaminophen

This laboratory has recently reported the isolation of an ethanol-inducible form of rabbit liver microsomal cytochrome P-450, designated isozyme 3a. In view of the reports of others that the hepatotoxicity of acetaminophen is increased in ethanol-treated animals and the human alcoholic, we have determined the activity of the six available P-450 isozymes in the activation of the drug to give an intermediate which forms a conjugate with reduced glutathione. Isozymes 3a, 4, and 6, all of which are present in significant amounts in the liver microsomes from rabbits chronically admini-stered ethanol, exhibited the highest activities in the reconstituted enzyme system, whereas isozymes 3b and 3c were 10- to 20-fold less effective, and phenobarbital-inducible isozyme 2 was essentially inactive, even in the presence of cytochrome 5. The results obtained thus indicate that induction by ethanol of P-450 isozyme 3a (or a homologous enzyme in other species) may contribute to the toxicity of acetaminophen but that other cytochromes also play a significant role.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25240/1/0000682.pd

Studies on hydroperoxide-dependent substrate hydroxylation by purified liver microsomal cytochrome P-450

Highly purified liver microsomal cytochrome P-450 catalyzes the hydroperoxide-dependent hydroxylation of a variety of substrates in the absence of NADPH, NADPH-cytochrome P-450 reductase, and molecular oxygen. The addition of phosphatidylcholine is necessary for maximal activity. The absence of flavoproteins and cytochrome b5 from the cytochrome P-450 preparations rules out the involvement of other known microsomal electron carriers. The ferrous form of cytochrome P-450 is not involved in peroxide-dependent hydroxylation reactions, as indicated by the lack of inhibition by carbon monoxide. With cumene hydroperoxide present, a variety of substrates is attacked, including N-methylaniline, N,N-dimethylaniline, cyclohexane, benzphetamine, and aminopyrine. With benzphetamine as the substrate, cumene hydroperoxide may be replaced by other peroxides, including hydrogen peroxide, or by peracids or sodium chlorite. A study of the stoichiometry indicated that equimolar amounts of N-methylaniline, formaldehyde, and cumyl alcohol ([alpha],[alpha]-dimethylbenzyl alcohol) are formed in the reaction of N,N-dimethylaniline with cumene hydroperoxide. Since H218O is incorporated only slightly into cyclohexanol in the reaction of cyclohexane with cumene hydroperoxide, it appears that the oxygen atom in cyclohexanol is derived primarily from the peroxide. The data obtained are in accord with a peroxidase-like mechanism for the action of cytochrome P-450.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/21710/1/0000102.pd