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W Levin

Publications and source records attributed to W Levin.

At least 127 records · Page 7Linked to original sources
PubMed1985

Stereoselectivity of microsomal epoxide hydrolase toward diol epoxides and tetrahydroepoxides derived from benz[a]anthracene.

We have examined the selectivity of rat liver microsomal epoxide hydrolase (EC 3.3.2.3) toward all of the possible positional isomers of benzo-ring diol epoxides and tetrahydroepoxides of benz[a]anthracene, as well as the 1,2-diol 3,4-epoxides of triphenylene. This set includes compounds with no bay region in the vicinity of the benzo-ring, a bay-region diol group, a bay-region epoxide group, and (for the triphenylene derivatives) both a bay-region diol and a bay-region epoxide. In all cases where both the tetrahydroepoxides and the corresponding diol epoxides were examined, there is a large retarding effect of hydroxyl substitution on the rate of the enzyme-catalyzed hydration. When the tetrahydroepoxides are fair or poor substrates (epoxide group in the 1,2-, 8,9-, or 10,11-position), the additional retardation introduced by adjacent hydroxyl groups causes the enzyme-catalyzed hydrolysis of the corresponding diol epoxides to be insignificantly slow or nonexistent. In contrast, a benz[a]anthracene derivative with an epoxide group in the 3,4-position, (-)-tetrahydrobenz[a]anthracene (3R,4S)-epoxide, has been identified as the best substrate known for epoxide hydrolase, with a Vmax at 37 degrees C and pH 8.4 of 6800 nmol/min/mg of protein, and the two diastereomeric (+/-)-benz[a]anthracene 1,2-diol 3,4-epoxides, unlike all the other diol epoxides examined to date, are moderately good substrates for epoxide hydrolase. This novel observation is accounted for by the fact that the very high reactivity of the tetrahydrobenz[a]anthracene 3,4-epoxide system towards epoxide hydrolase is large enough to overcome a kinetically unfavorable effect of hydroxyl substitution. The enantioselectivity and positional selectivity of the enzyme have been determined for the tetrahydro-1,2- and -3,4-epoxides of benz[a]anthracene as well as the 1,2-diol 3,4-epoxides. When the epoxide is located in the 3,4-position, the benzylic carbon is the preferred site of attack, whereas for the enantiomers of the bay-region tetrahydro-1,2-epoxides, the chemically less reactive non-benzylic carbon is preferred. The regio- and enantioselectivity of epoxide hydrolase are discussed in terms of a possible model for the hydrophobic binding site of this enzyme.

Animals↗
PubMed1985

CT demonstration of gastrointestinal involvement in Henoch-Schonlein syndrome.

The CT appearance of gastrointestinal involvement in Henoch-Schonlein syndrome is described. The protean manifestations of this disorder are easily confused both clinically and radiographically with those of many other conditions. Mural thickening, thickened folds, ulceration, and spasm are seen radiographically. The CT appearance of segmental mural thickening and luminal narrowing correlates well with the abnormalities seen on the small-bowel series and upper endoscopy.

Duodenal Diseases↗
PubMed1985

Evidence for a family of four immunochemically related isozymes of cytochrome P-450 purified from untreated rats.

Hepatic microsomal cytochromes P-450f, P-450g, P-450h, and P-450i have recently been purified to electrophoretic homogeneity from untreated adult rats and identified as distinct isozymes [Ryan et al. (1984) J. Biol. Chem. 259, 1239-1250; Haniu et al. (1984) Arch. Biochem. Biophys. 235, 304-311]. In Ouchterlony double-diffusion plates, as well as enzyme-linked immunosorbent assays and Western blots, purified immunoglobulin G (IgG) raised in rabbits against cytochromes P-450f or P-450g show strong cross-reactions with the heterologous protein, indicating apparent partial identity. Anti-P-450f and anti-P-450g also show strong cross-reactivity with cytochromes P-450h and P-450i but not with five previously characterized rat hepatic cytochromes, P-450 (i.e., P-450a-P-450e), indicating a high degree of immunochemical and structural relatedness among cytochromes P-450f, P-450g, P-450h, and P-450i.

Animals↗
PubMed1985

PCBs: structure-function relationships and mechanism of action.

Numerous reports have illustrated the versatility of polychlorinated biphenyls (PCBs) and related halogenated aromatics as inducers of drug-metabolizing enzymes and the activity of individual compounds are remarkably dependent on structure. The most active PCB congeners, 3,4,4',5-tetra-, 3,3',4,4'-tetra-, 3,3',4,4',5-penta- and 3,3',4,4',5,5'-hexachlorobiphenyl, are substituted at both para and at two or more meta positions. The four coplanar PCBs resembled 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) in their mode of induction of the hepatic drug-metabolizing enzymes. These compounds induced rat hepatic microsomal benzo(a)pyrene hydroxylase (aryl hydrocarbon hydroxylase, AHH) and cytochromes P-450a, P-450c and P-450d. 3,4,4',5-Tetrachlorobiphenyl, the least active coplanar PCB, also induced dimethylaminoantipyrine N-demethylase and cytochromes P-450b+e and resembled Aroclor 1254 as an inducer of the mixed-function oxidase system. Like Aroclor 1254, all the mono-ortho- and at least eight di-ortho-chloro analogs of the coplanar PCBs exhibited a "mixed-type" induction pattern and induced microsomal AHH, dimethylaminoantipyrine NM-demethylase and cytochromes P-450a-P-450e. Quantitative structure-activity relationships (QSARs) within this series of PCBs were determined by comparing their AHH induction potencies (EC50) in rat hepatoma H-4-II-E cells and their binding affinities (ED50) for the 2,3,7,8-TCDD cytosolic receptor protein. The results showed that there was an excellent correlation between AHH induction potencies and receptor binding avidities of these compounds and the order of activity was coplanar PCBs (3,3',4,4' -tetra-, 3,3',4,4',5-penta- and 3,3',4,4',5,5'-hexachlorobiphenyls) greater than 3,4,4',5-tetrachlorobiphenyl approximately mono-ortho coplanar PCBs greater than di-ortho coplanar PCBs. It was also apparent that the relative toxicities of this group of PCBs paralleled their biological potencies. The coplanar and mono-ortho coplanar PCBs also exhibit differential effects in the inbred C57BL/6J and DBA/2J mice. These compounds induce AHH and cause thymic atrophy in the former "responsive" mice whereas at comparable or higher doses none of these effects are observed in the nonresponsive DBD/2J mice. Since the responsiveness of these two mice strains is due to the presence of the Ah receptor protein in the C57BL/6J mice and its relatively low concentration in the DBA/2J mice, the results for the PCB cogeners support the proposed receptor-mediated mechanism of action.

Animals↗
PubMed1985

Role of cul-de-sac aspiration cytology in the management and follow-up of patients with ovarian carcinoma. A preliminary report.

In a pilot study involving 44 patients on combined chemotherapy for epithelial ovarian carcinoma, cul-de-sac aspiration cytology was compared with second-look surgery in the evaluation of disease status. There was a 92% correlation. Our preliminary conclusion was that an aspirate from the cul-de-sac or peritoneal cavity can be obtained with ease and that if this finding is confirmed in a larger series now in progress, the procedure may in part replace second-look operations in the management of these patients.

Adenocarcinoma↗
PubMed1984

Use of monoclonal antibody probes against rat hepatic cytochromes P-450c and P-450d to detect immunochemically related isozymes in liver microsomes from different species.

Nine distinct monoclonal antibodies raised against purified rat liver cytochrome P-450c react with six different epitopes on the antigen, and one of these epitopes is shared by cytochrome P-450d. None of these monoclonal antibodies recognize seven other purified rat liver isozymes (cytochromes P-450a, b, and e-i) or other proteins in the cytochrome P-450 region of "Western blots" of liver microsomes. Each of the monoclonal antibodies was used to probe "Western blots" of liver microsomes from untreated, or 3-methylcholanthrene-, or isosafrole-treated animals to determine if laboratory animals other than rats possess isozymes immunochemically related to cytochromes P-450c and P-450d. Two protein-staining bands immunorelated to cytochromes P-450c and P-450d were observed in all animals treated with 3-methylcholanthrene (rabbit, hamster, guinea pig, and C57BL/6J mouse) except the DBA/2J mouse, where no polypeptide immunorelated to cytochrome P-450c was detected. The conservation of the number of rat cytochrome P-450c epitopes among these species varied from as few as two (guinea pig) to as many as five epitopes (C57BL/6J mouse and rabbit). The relative mobility in sodium dodecyl sulfate-gels of polypeptides immunorelated to cytochromes P-450c and P-450d was similar in all species examined except the guinea pig, where the polypeptide related to cytochrome P-450c had a smaller Mr than cytochrome P-450d. With the use of both monoclonal and polyclonal antibodies, we were able to establish that purified rabbit cytochromes P-450 LM4 and P-450 LM6 are immunorelated to rat cytochromes P-450d and P-450c, respectively.

Animals↗
PubMed1984

Effects of a 6-fluoro substituent on the metabolism of benzo(a)pyrene 7,8-dihydrodiol to bay-region diol epoxides by rat liver enzymes.

Metabolism of trans-7,8-dihydroxy-7,8-dihydro-6-fluorobenzo(a)pyrene by liver microsomes from 3-methylcholanthrene-treated rats and by a highly purified monooxygenase system, reconstituted with cytochrome P-450c, has been examined. Although both the fluorinated and unfluorinated 7,8-dihydrodiol formed from benzo(a)pyrene by liver microsomes share (R,R)-absolute configuration, the fluorinated dihydrodiol prefers the conformation in which the hydroxyl groups are pseudodiaxial due to the proximate fluorine. The fluorinated 4,5- and 9,10-dihydrodiols are also greater than 97% the (R,R)-enantiomers. For benzo(a)pyrene, metabolism of the (7R,8R)-dihydrodiol to a bay-region 7,8-diol-9,10-epoxide in which the benzylic hydroxyl group and epoxide oxygen are trans constitutes the only known pathway to an ultimate carcinogen. With the microsomal and the purified monooxygenase system, this pathway accounts for 76-82% of the total metabolites from the 7,8-dihydrodiol. In contrast, only 32-49% of the corresponding diol epoxide is obtained from the fluorinated dihydrodiol and this fluorinated diol epoxide has altered conformation in that its hydroxyl groups prefer to be pseudodiaxial. Much smaller amounts of the diastereomeric 7,8-diol-9,10-epoxides in which the benzylic hydroxyl groups and the epoxide oxygen are cis are formed from both dihydrodiols. As the fluorinated diol epoxides are weaker mutagens toward bacteria and mammalian cells relative to the unfluorinated diol epoxides, conformation appears to be an important determinant in modulating the biological activity of diol epoxides. One of the more interesting metabolites of 6-fluorinated 7,8-dihydrodiol was a relatively stable arene oxide, probably the 4,5-oxide, which is resistant to the action of epoxide hydrolase.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1984

Stereoselectivity of cytochrome P-450c in the formation of naphthalene and anthracene 1,2-oxides.

Absolute configurations of the arene 1,2-oxides formed from napththalene and anthracene by cytochrome P-450c, the predominant isozyme of cytochrome P-450 found in the livers of rats treated with 3-methylcholanthrene, were determined via two different approaches. The first consisted of trapping the arene oxides with N-acetyl-L-cysteine to form S-conjugates, methylation of the conjugates with diazomethane, and separation of the resulting diastereomeric esters by reversed phase high performance liquid chromatography. Analysis by this procedure of the arene oxides formed from radioactive naphthalene and anthracene by a highly purified and reconstituted monooxygenase system containing cytochrome P-450c indicated that 73 and greater than or equal to 95%, respectively, of the metabolically formed arene oxides consisted of the (+)-(1R,2S)-enantiomer. In the second approach, each hydrocarbon was incubated with a reconstituted system containing both cytochrome P-450c and epoxide hydrolase. Under these conditions, the predominant metabolites are trans-1,2-dihydrodiols formed by epoxide hydrolase catalyzed trans-addition of water to the arene oxide intermediates. In both cases, the (-)-(1R,2R)-dihydrodiols predominated; 92% for naphthalene and 99% for anthracene. Enzyme-catalyzed addition of water to (+)- and (-)-anthracene 1,2-oxide and (+)-napthalene 1,2-oxide occurred exclusively (greater than 99%) at the allylic 2-position. The (-)-(1S,2R)-naphthalene 1,2-oxide, however, is converted to a 40:60 mixture of the (-)-(1R,2R)- and (+)-(1S,2S)-dihydrodiols by benzylic and allylic attack, respectively, resulting in increased enantiomeric purity of the dihydrodiol relative to the oxide. Thus, qualitatively and quantitatively both approaches indicate that the (+)-arene (1R,2S)-oxides predominate. The results are discussed in terms of the steric constraints of a proposed model for the catalytic binding site of cytochrome P-450c.

Animals↗
PubMed1984

Structural analysis of the cysteine-containing peptides from the major 3-methylcholanthrene-induced isozyme of cytochrome P-450 (P-450c) in rat liver microsomes.

Cytochrome P-450c, the major 3-methylcholanthrene-inducible isozyme of cytochrome P-450 in rat liver microsomes, was subjected to proteolytic digestion after S-carboxymethylation of the protein, and the peptides were resolved by high-pressure liquid chromatography. Since it is now recognized that cytochromes P-450 contain a thiolate as the axial fifth ligand of the heme, seven peptides containing eight cysteines were subjected to microsequence analysis. One cysteine-containing peptide (Tsa-56) was shown to possess 46-69% homology with a common peptide found in five other cytochromes P-450 but is not anticipated to be the heme-binding segment on the basis of X-ray crystallographic results obtained with Pseudomonas putida cytochrome P-450cam. Analysis of the other cysteine-containing peptides in cytochrome P-450c revealed two peptides (Tsa-54 and T-46) of only limited homology with the highly conserved region of cytochromes P-450cam, P-450LM2, P-450b, and P-450e that are all presumed to contain the heme-binding cysteine. Another peptide that contained two cysteines and a stretch of hydrophobic residues (Tsa-47) had limited sequence homology with a similar peptide found in several other cytochromes P-450. This domain is located a short distance from the proposed heme-binding cysteine in other cytochromes P-450. Sequence analysis of a cysteine-containing peptide (T-30) from another 3-methylcholanthrene-inducible rat liver cytochrome P-450 (cytochrome P-450d) revealed 91% homology with peptide T-46 from cytochrome P-450c, but this peptide shows no significant homology with any of the cysteine-containing peptides from other cytochromes P-450.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1984

Characterization of nine monoclonal antibodies against rat hepatic cytochrome P-450c. Delineation of at least five spatially distinct epitopes.

Spleen cells from a BALB/cByJ mouse previously immunized with purified rat liver microsomal cytochrome P-450c were fused with myeloma cells (P3X63Ag8.653) and 10 hybridoma clones secreting antibody against cytochrome P-450c were selected for characterization. The monoclonal antibodies (C1-C10) were purified from mouse ascites fluid and nine were determined to be distinct immunoglobulins. C6 was an IgG2b, whereas the rest were of the IgG1 subclass. A competitive enzyme-linked immunoassay was used to show that the antibodies were directed against at least five spatially distinct epitopes on cytochrome P-450c. Additional evidence for the recognition of distinct epitopes was provided by Ouchterlony immunoprecipitation of cytochrome P-450c with mixtures of appropriate monoclonal antibodies. Differences in antibody reactivity provided evidence for a sixth overlapping epitope that was recognized by two antibodies (C4 and C6). Three monoclonal antibodies to the same epitope on cytochrome P-450c, (CD2, CD3, and CD5) cross-reacted strongly with cytochrome P-450d, another isozyme induced by 3-methylcholanthrene treatment of rats. The antibodies that did not cross-react with cytochrome P-450d contained kappa light chains, whereas the three cross-reacting antibodies contained lambda light chains. None of the monoclonal antibodies cross-reacted with purified cytochromes P-450a, P-450b, P-450e, P-450f, P-450g, or P-450h or any other cytochrome P-450 in "Western blots" of liver microsomes from untreated or 3-methylcholanthrene-treated rats. C8 was a potent inhibitor of metabolism catalyzed by cytochrome P-450c in a reconstituted system as well as microsomes from 3-methylcholanthrene-treated rats. This antibody effected maximal inhibition of catalytic activity at an approximately 0.5:1 molar ratio of IgG to cytochrome P-450c, i.e. one antibody-binding site per epitope on cytochrome P-450c.

Animals↗
PubMed1984

Stereoselective monooxygenation of carcinostatic 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea by purified cytochrome P-450 isozymes.

Three highly purified forms of liver microsomal cytochrome P-450 (P-450a, P-450b and P-450c) from Aroclor 1254-treated rats catalyzed 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea (CCNU) and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (MeCCNU) monooxygenation in the presence of purified NADPH-cytochrome P-450 reductase, NADPH, and lipid. Differences in the regioselectivity of CCNU and MeCCNU monohydroxylation reactions by the cytochrome P-450 isozymes were observed. Cytochrome P-450-dependent monooxygenation of CCNU gave only alicyclic hydroxylation products, but monooxygenation of MeCCNU gave alicyclic hydroxylation products, an alpha-hydroxylation product on the 2-chloroethyl moiety, and a trans-4-hydroxymethyl product. A high degree of stereoselectivity for hydroxylation of CCNU and MeCCNU at the cis-4 position of the cyclohexyl ring was demonstrated. All three cytochrome P-450 isozymes were stereoselective in primarily forming the metabolite cis-4-hydroxy-trans-4-Methyl-CCNU from MeCCNU. The principal metabolite of CCNU which resulted from cytochromes P-450a and P-450b catalysis was cis-4-hydroxy CCNU, whereas the principal metabolites from cytochrome P-450c catalysis were the trans-3-hydroxy and the cis-4-hydroxy isomers. Total amounts of CCNU and MeCCNU hydroxylation with cytochrome P-450b were twice that with hepatic microsomes from Aroclor 1254-treated rats. Catalysis with cytochromes P-450a and P-450c was substantially less effective than that observed with either cytochrome P-450b or hepatic microsomes from Aroclor 1254-treated rats.

Animals↗
PubMed1984

Characterization of three highly purified cytochromes P-450 from hepatic microsomes of adult male rats.

Three hepatic microsomal cytochromes P-450 (P-450f, P-450g, and P-450h) have been purified to electrophoretic homogeneity from both untreated and ethanol-treated adult male rats. By all criteria examined, the hemoproteins isolated from untreated rats are indistinguishable from the corresponding enzymes purified from rats administered ethanol. Highly purified cytochromes P-450f, P-450g and P-450h are characterized by minimum Mr of 51,000, 50,000, and 51,000, respectively, and unique coordinates in two-dimensional isoelectric focusing-sodium dodecyl sulfate-polyacrylamide gels. The CO-reduced spectral maxima of cytochromes P-450f and P-450g are at 447-448 nm, and the peak of cytochrome P-450h is at 451 nm. Cytochrome P-450h is a versatile catalyst exhibiting high activity toward benzphetamine, hexobarbital, and estradiol-17 beta and moderate activity toward benzo[alpha]pyrene and zoxazolamine. In contrast, cytochromes P-450f and P-450g have low metabolic activity for these substrates. The three hemoproteins catalyze the metabolism of testosterone with different regio- and stereospecificities and overall rates. Both cytochromes P-450f and P-450h catalyze the hydroxylation of testosterone at the 16 alpha-position; however, cytochrome P-450h also oxidizes the steroid at the 2 alpha- and 17 beta-position (androstenedione formation). Testosterone is oxidatively metabolized at the 6 beta-, 15 alpha- and an unknown position by cytochrome P-450g. Peptide maps, generated by proteolytic or chemical digestion of the hemoproteins, indicate that cytochromes P-450f, P-450g, and P-450h differ structurally from each other and five previously characterized rat hepatic microsomal cytochromes P-450 (P-450a, P-450b, P-450c, P-450d, and P-450e). Cytochromes P-450f, P-450g, and P-450h do not react with antibodies directed against these inducible hemoproteins by Ouchterlony immunodiffusion in the presence of detergent; however, in the absence of detergent, cytochrome P-450f cross-reacts weakly with anti-P-450b. Results of this study indicate that rat hepatic microsomal cytochromes P-450 are composed of at least four hemoproteins with CO-reduced absorbance maxima between 447-448 nm. Furthermore, a minimum of four microsomal cytochromes P-450 are now known to 16 alpha-hydroxylate testosterone.

Animals↗
PubMed1984

NH2-terminal sequence analyses of four rat hepatic microsomal cytochromes P-450.

Cytochromes P-450f, P-450g, P-450h, and P-450i are four hepatic microsomal hemoproteins that have been purified from adult rats. Whereas cytochromes P-450g and P-450h appear to be male-specific hemoproteins, cytochrome P-450i is apparently a female-specific enzyme purified from untreated adult female rats. Cytochrome P-450f has been purified from adult male and female rats with equivalent recoveries. Amino-terminal sequence analyses of the first 15-20 amino acid residues of each of these cytochromes P-450 has been accomplished in the current investigation. Each protein possesses a hydrophobic leader sequence consisting of 65-87% hydrophobic amino acids, and only one charged amino acid (Asp) in the amino-terminal region. Although differences in the amino-terminal sequences of cytochromes P-450f, P-450g, P-450h, and P-450i are identified, these hemoproteins all begin with Met-Asp, and marked structural homology is observed among certain of these enzymes. Cytochromes P-450g and P-450h, two male-specific proteins, have 11-12/15 identical residues with cytochrome P-450i, a female-specific isozyme. Cytochromes P-450f and P-450h have 16/20 identical amino-terminal residues. Only limited sequence homology is observed between the amino-terminal sequences of cytochromes P-450f-i compared to rat liver cytochromes P-450a-e. The results demonstrate that cytochromes P-450f, P-450g, P-450h, and P-450i are isozymic to each other and five additional rat hepatic microsomal cytochrome P-450 isozymes (P-450a-e).

Amino Acid Sequence↗
PubMed1984

Rat hepatic cytochrome P-450 isozyme specificity for the metabolism of the steroid sulfate, 5 alpha-androstane-3 alpha, 17 beta-diol-3,17-disulfate.

Nine rat hepatic microsomal cytochromes P-450 (P-450a-P-450i) have been purified to electrophoretic homogeneity and assayed as potential catalysts of 15 beta-hydroxylation of 5 alpha-androstane-3 alpha,17 beta-diol-3,17-disulfate in a reconstituted system containing NADPH-cytochrome c reductase and dilauroylphosphatidylcholine. Of the nine isozymes, only cytochrome P-450i, which is present in adult female but not adult male rats, catalyzes the hydroxylation of 5 alpha-androstane-3 alpha,17 beta-diol-3,17-disulfate. The reaction has an absolute requirement for cytochrome P-450i, NADPH-cytochrome c reductase, and NADPH, as well as a marked dependence on dilauroylphosphatidylcholine. Under optimal conditions, the Km,app was 69 microM, and the Vmax was 7.8 nmol min-1 nmol cytochrome P-450i-1. The affinity of cytochrome P-450i for the substrate, as expressed by the apparent spectral dissociation constant (Ks,app), was 20 microM. This female-specific isozyme had very low catalytic activity toward testosterone, and the metabolite profile from testosterone was distinct compared to the profiles obtained with the other eight isozymes. The results with androstane disulfate indicated that cytochrome P-450i is responsible for the sex-specific microsomal 15 beta-hydroxylase system in adult female rat liver, originally described by Gustafsson and Ingelman-Sundberg [(1974) J. Biol. Chem. 249: 1940-1945].

Aging↗
PubMed1984

Apparent anomalies in the resolution of cytochrome P-450 isoenzymes by gel electrophoresis.

Cytochromes P-450b and P-450e are two phenobarbital-inducible rat hepatic microsomal isoenzymes that possess approx. 97% sequence homology and show apparent anomalous behaviour in SDS-PAGE and isoelectric-focusing under denaturing conditions. Although the two enzymes differ in Mr by less than 0.1% and possess no apparent net charge difference, then can be resolved in both electrophoretic systems. These apparent discrepancies are discussed in terms of the predicted secondary structure of the proteins and the potential for certain specific amino acid substitutions to cause anomalous behaviour in SDS-PAGE. The results of electrophoresis of these cytochrome P-450 isoenzymes indicate that much smaller differences in primary structure can be detected by these methodologies than would be predicted from theoretical considerations.

Amino Acid Sequence↗
PubMed1984

Structures of cysteine-containing peptides in isosafrole-inducible rat hepatic microsomal cytochrome P-450d: sequence homology with 3-methylcholanthrene-induced cytochrome P-450c.

Six cysteine-containing tryptic peptides were isolated from rat liver cytochrome P-450d, the major isosafrole-induced isozyme, by reversed-phase high performance liquid chromatography. The six peptides contained a total of seven cysteine residues. Five of the peptides have significant sequence homology (20/22, 10/16, 8/13, 13/18, and 5/9 identical residues) to cysteine-containing peptides in cytochrome P-450c, the major isozyme induced by 3-methylcholanthrene. One of the peptides (partial sequence, Cys-Ile-Gly-Glu-Ile-Pro-Ala-Lys-Trp-Glu-Val-Phe-Leu-) can be included in the highly conserved COOH-terminal domain found in all cytochromes P-450 that have been sequenced. Although this domain has been postulated as the heme-binding domain by some investigators, it is not homologous to the heme-binding region in cytochrome P-450cam. A second peptide (partial sequence, Asp-Pro-Thr-Ser-Val-Ser-Ser-Cys-Tyr-Leu-Glu-Glu-His-Val-Ser-Lys) is, however, a possible candidate for the heme attachment site to cysteine because of its weak homology to the heme-binding site in cytochrome P-450cam. These results indicate that either the location of the heme-binding site is different in various forms of cytochromes P-450 or the amino acid sequence surrounding the heme-binding cysteine is not highly conserved among these proteins.

Amino Acid Sequence↗