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Biomedical subjects

D E Ryan

Publications and source records attributed to D E Ryan.

At least 55 records · Page 3Linked to original sources
PubMed1986

Chemical modification and inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4'-nitroacetophenone.

The alkylating agent 2-bromo-4'-nitroacetophenone (BrNAP) binds covalently to each of 10 isozymes of purified rat liver microsomal cytochrome P-450 (P-450a-P-450j) but substantially inhibits the catalytic activity of only cytochrome P-450c. Regardless of pH, incubation time, presence of detergents, or concentration of BrNAP, treatment of cytochrome P-450c with BrNAP resulted in no more than 90% inhibition of catalytic activity. Alkylation with BrNAP did not cause the release of heme from the holoenzyme or alter the spectral properties of cytochrome P-450c, data that exclude the putative heme-binding cysteine, Cys-460, as the major site of alkylation. Two residues in cytochrome P-450c reacted rapidly with BrNAP, for which reason maximal loss of catalytic activity was invariably associated with the incorporation of approximately 1.5 mol of BrNAP/mol of cytochrome P-450c. Two major radio-labeled peptides were isolated from a tryptic digest of [14C]BrNAP-treated cytochrome P-450c by reverse-phase high performance liquid chromatography. The amino acid sequence of each peptide was determined by microsequence analysis, but the identification of the residues alkylated by BrNAP was complicated by the tendency of the adducts to decompose when subjected to automated Edman degradation. However, results of competitive binding experiments with the sulfhydryl reagent 4,4'-dithiodipyridine identified Cys-292 as the major site of alkylation and Cys-160 as the minor site of alkylation by BrNAP in cytochrome P-450c.

Acetophenones↗
PubMed1986

Age- and sex-related expression of cytochromes p450f and P450g in rat liver.

We have previously shown that rat hepatic cytochromes P450f, P450g, P450h, and P450i possess a high degree of immunochemical and, presumably, structural relatedness. Polyclonal antibodies directed against cytochromes P450f and P450g were made monospecific by immunoabsorption against the cross-reactive proteins. The specificity of the immunoabsorbed antibodies was established by using Ouchterlony double diffusion analyses, enzyme-linked immunosorbent assays (ELISA), and immunoblots. Since factors regulating the expression of cytochromes P450f and P450g are unknown, a competitive ELISA employing the monospecific antibodies was developed to quantitate each of these isozymes in hepatic microsomes from control and treated rats. The results obtained showed that expression of cytochrome P450f is developmentally regulated in both male and female rat liver. Cytochrome P450f levels rise from less than 1% in young animals to approximately 7 and 14% of total cytochrome P450 in adult male and female rats, respectively. Cytochrome P450g is sex-specific since it is expressed only in male rat liver where it also is developmentally regulated. Levels of cytochrome P450g rise from less than 1% in 3-week-old male rats to an average value of 17% of total cytochrome P450 in 6-week-old adult animals. However, there appear to be at least two subpopulations of adult male Long Evans rats, one of which expresses low levels (less than 1%) of cytochrome P450g and the other high levels (greater than or equal to 10%). This expression appears to be independent of serum testosterone levels. Treatment of immature and adult male rats with 20 xenobiotics that are known inducers of certain cytochrome P450 isozymes revealed that cytochromes P450f and P450g are relatively refractory to induction, although Kepone appears to be a weak inducer of cytochrome P450f.

Age Factors↗
PubMed1986

Evidence that isoniazid and ethanol induce the same microsomal cytochrome P-450 in rat liver, an isozyme homologous to rabbit liver cytochrome P-450 isozyme 3a.

Cytochrome P-450j has been purified to electrophoretic homogeneity from hepatic microsomes of adult male rats administered ethanol and compared to the corresponding enzyme from isoniazid-treated rats. The enzymes isolated from ethanol- and isoniazid-treated rats have identical chromatographic properties, minimum molecular weights, spectral properties, peptide maps, NH2-terminal sequences, immunochemical reactivities, and substrate selectivities. Both preparations of cytochrome P-450j have high catalytic activity in aniline hydroxylation, butanol oxidation, and N-nitrosodimethylamine demethylation with turnover numbers of 17-18, 37-46, and 15 nmol product/min/nmol of P-450, respectively. A single immunoprecipitin band exhibiting complete identity was observed when the two preparations were tested by double diffusion analysis with antibody to isoniazid-inducible cytochrome P-450j. Ethanol- and isoniazid-inducible rat liver cytochrome P-450j preparations have also been compared and contrasted with cytochrome P-450 isozyme 3a, the major ethanol-inducible isozyme from rabbit liver. The rat and rabbit liver enzymes have slightly different minimum molecular weights and somewhat different peptide maps but similar spectral, catalytic, and immunological properties, as well as significant homology in their NH2-terminal sequences. Antibody to either the rat or rabbit isozyme cross-reacts with the heterologous enzyme, showing a strong reaction of partial identity. Antibody against isozyme 3a specifically recognizes cytochrome P-450j in immunoblots of induced rat liver microsomes. Aniline hydroxylation catalyzed by the reconstituted system containing cytochrome P-450j is markedly inhibited (greater than 90%) by antibody to the rabbit protein. Furthermore, greater than 85% of butanol or aniline metabolism catalyzed by hepatic microsomes from ethanol- or isoniazid-treated rats is inhibited by antibody against isozyme 3a. Results of antibody inhibition studies suggest that cytochrome P-450j is induced four- to sixfold by ethanol or isoniazid treatment of rats. All of the evidence presented in this study indicates that the identical cytochrome P-450, P-450j, is induced in rat liver by either isoniazid or ethanol, and that this isozyme is closely related to rabbit cytochrome P-450 isozyme 3a.

1-Butanol↗
PubMed1986

Stereoselective metabolism of the (+)-(S,S)- and (-)-(R,R)-enantiomers of trans-3,4-dihydroxy-3,4-dihydrobenzo[c]-phenanthrene by rat and mouse liver microsomes and by a purified and reconstituted cytochrome P-450 system.

Metabolism of (+)-, (-)-, and (+/-)-trans-3,4-dihydroxy-3, 4-dihydrobenzo[c]phenanthrenes by liver microsomes from rats and mice and by a purified monooxygenase system reconstituted with cytochrome P-450c has been examined. Bay-region 3,4-diol 1,2-epoxides are minor metabolites of both enantiomers of the 3,4-dihydrodiol with liver microsomes from 3-methylcholanthrene-treated rats or with the reconstituted system (less than 10% of total metabolites). Microsomes from control and phenobarbital-treated rats and from control mice form higher percentages of these diol epoxides (13-36% of total metabolites). Microsomes from 3-methylcholanthrene-treated rats and cytochrome P-450c in the reconstituted system form exclusively the diol expoxide-1 diastereomer, in which the benzylic hydroxyl group and oxirane oxygen are cis to each other, from the (+)-(3S,4S)-dihydrodiol. The same enzymes selectively form the diol expoxide-2 diastereomer, with its oxirane oxygen and benzylic hydroxyl groups trans to each other, from the (-)-(3R,4R)-dihydrodiol (77% of the total diol epoxides). Liver microsomes from control rats show similar stereoselectivity whereas liver microsomes from phenobarbital-treated rats and from control mice are less stereoselective. Three bis-dihydrodiols and three phenolic dihydrodiols are also formed from the enantiomeric 3,4-dihydrodiols of benzo[c]phenanthrene. A single diastereomer of one of these bis-dihydrodiols with the newly introduced dihydrodiol group at the 7,8-position accounts for 79-88% of the total metabolites of the (-)-(3R,4R)-dihydrodiol formed by liver microsomes from 3-methylcholanthrene-treated rats or by the reconstituted system containing epoxide hydrolase. In contrast, the (+)-(3S,4S)-dihydrodiol is metabolized to two diastereomers of this bis-dihydrodiol, a third bis-dihydrodiol, and two phenolic dihydrodiols.

Animals↗
PubMed1986

The primary structure of cytochrome P-450d purified from rat liver microsomes: prediction of helical regions and domain analysis.

The complete amino acid sequence of rat liver microsomal cytochrome P-450d (induced by isosafrole) was deduced by microsequence analysis of the tryptic peptides after separation by reverse-phase HPLC and alignment by comparison to the cDNA sequence reported by K. Kawajiri, O. Gotoh, K. Sogawa, Y. Tagashira, M. Muramatsu, and Y. Fujii-Kuriyama (1984, Proc. Natl. Acad. Sci. USA 81, 1649-1653). Results from the two approaches are in complete agreement with the exception of two residues, Lys-30 (Arg in cDNA) and Phe-261 (Ser in cDNA). As previously reported by us (L. H. Botelho, D. E. Ryan, P.-M. Yuan, R. Kutney, J. E. Shively, and W. Levin (1982, Biochemistry 21, 1152-1155) the NH2-terminal sequence of the mature protein lacks the NH2-terminal Met residue. Comparison of the rat cytochrome P-450d sequence with the mouse cytochrome P3-450 cDNA sequence reported by S. Kimura, F.J. Gonzalez, and D.W. Nebert (1984, Nucl. Acids Res. 12, 2917-2928) reveals a high sequence homology with a total of 32 amino acid differences including six conferring charge changes. Prediction of the secondary structure of cytochrome P-450d yields a maximum of 17 helices, two of which may be poly(Pro)-like helices adjacent to potential membrane-spanning alpha-helices. Four of the alpha-helices are sufficiently hydrophobic to traverse the endoplasmic reticulum. The remaining helices are largely amphiphilic. Analysis of the helices in reference to predicted membrane topology suggests that cytochrome P-450d either has one large and one small globular domain separated by a transmembrane domain and anchored by NH2-terminal and COOH-terminal transmembrane domains, or has one large globular domain anchored at both ends by transmembrane domains.

Amino Acid Sequence↗
PubMed1986

N-demethylation of N-nitrosodimethylamine catalyzed by purified rat hepatic microsomal cytochrome P-450: isozyme specificity and role of cytochrome b5.

Metabolism of the potent hepatocarcinogen N-nitrosodimethylamine (NDMA) was evaluated in reconstituted monooxygenase systems containing each of 11 purified rat hepatic cytochrome P-450 isozymes. The reaction has an absolute requirement for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH, as well as a partial dependence on dilauroylphosphatidylcholine. Of the cytochrome P-450 isozymes evaluated, only cytochrome P-450j, purified from livers of ethanol- or isoniazid-treated rats, had high catalytic activity for the N-demethylation of NDMA. At substrate concentrations of 0.5 and 5 mM, rates of NDMA metabolism to formaldehyde catalyzed by cytochrome P-450j were at least 15-fold greater than the rates obtained with any of the other purified isozymes. At the pH optimum (approximately 6.7) for the reaction, the Km,app and Vmax were 3.5 mM and 23.9 nmol/min/nmol cytochrome P-450j, respectively. With hepatic microsomes from ethanol-treated rats, which contain induced levels of cytochrome P-450j, the Km,app and Vmax were 0.35 mM and 3.9 nmol/min/nmol cytochrome P-450, respectively. Inclusion of purified cytochrome b5 in the reconstituted system containing cytochrome P-450j caused a six-fold decrease in Km,app (0.56 mM) of NDMA demethylation with little or no change in Vmax (29.9 nmol/min/nmol cytochrome P-450j). Trypsin-solubilized cytochrome b5, bovine serum albumin, or hemoglobin had no effect on the kinetic parameters of the reconstituted system, indicating a specific effect of intact cytochrome b5 on the Km,app of the reaction. These results demonstrate high isozyme specificity in the metabolism of NDMA to an ultimate carcinogen and further suggest an important role for cytochrome b5 in this biotransformation process.

Animals↗
PubMed1986

A simplified technique of inferior border wiring in the sagittal split osteotomy.

The inferior border wire, as originally described by Booth, is a well-accepted means of fixation of the proximal and distal segments in the sagittal split osteotomy. However, it appears that there is some criticism of the technique because of the difficulty in placing drill holes at the appropriate position and angulation and subsequent difficulty with passage of a fixation wire. The technique presented here eliminates the technical difficulties and provides for a rapid, safe, and stable fixation.

Bone Wires↗
PubMed1985

Microheterogeneity of a male-specific rat hepatic cytochrome P-450: existence of three allozymic forms.

Preparations of hepatic cytochrome P-450 h [D. E. Ryan, et al. (1984) J. Biol. Chem. 259, 1239] and cytochrome P-450 2c [D. J. Waxman (1984) J. Biol. Chem. 259, 15481] from outbred Sprague-Dawley rats were analyzed using two-dimensional electrophoresis and in situ peptide mapping. Both preparations consisted of the same isozyme which was previously characterized as a developmentally regulated, male-specific cytochrome P-450 active in the 16 alpha-hydroxylation of steroids. Each preparation evidenced microheterogeneity which was shown, in part, to result from the existence of two genetically determined variant forms of cytochrome P-450 h/2c. Analyses of hepatic microsomes from several inbred strains of rat revealed that each was characterized by a single variant form of this isozyme, with some strains expressing a variant that was not present in Sprague-Dawley rats. Genetic crosses indicated that these electrophoretic variants represent allozymic forms of cytochrome P-450 h/2c which are codominantly expressed at a single autosomal locus. Additional microheterogeneity of each allozymic form of cytochrome P-450 h/2c was shown to result from a specific in vitro modification that may involve limited proteolysis near its C terminus by a microsome-bound protease.

Alleles↗
PubMed1985

Monoclonal antibodies distinguish among isozymes of the cytochrome P-450b subfamily.

Polyclonal antibody has been shown previously to react identically with cytochromes P-450b and P-450e purified from Long Evans rats and a strain variant of cytochrome P-450b purified from Holtzman rats (P-450bH). In the present study, an array of 12 different monoclonal antibodies produced against cytochrome P-450b has been used to distinguish among these closely related phenobarbital-inducible rat hepatic cytochromes P-450. In immunoblots and enzyme-linked immunosorbent assays, 10 monoclonal antibodies bind to cytochromes P-450b, P-450e, and P-450bH; one monoclonal antibody (B50) recognizes cytochromes P-450b and P-450bH but not cytochrome P-450e; and one monoclonal antibody (B51) is specific for cytochrome P-450b. In addition, one monoclonal antibody (BEF29) reacts strongly with cytochrome P-450f, and another antibody (BEA33) reacts weakly with cytochrome P-450a. No cross-reactions with cytochromes P-450c, P-450d, and P-450g-P-450j were detected with any of the monoclonal antibodies in these assays. Six spatially distinct epitopes on cytochrome P-450b were identified, and differences in antibody reactivity provided evidence for three additional overlapping epitopes. Several monoclonal antibodies are potent inhibitors of testosterone and benzphetamine metabolism supported by cytochrome P-450b in a reconstituted system. B50 and BE52 do not inhibit metabolism of the two substrates by microsomes from untreated rats, but inhibit benzphetamine N-demethylation and testosterone metabolism to 16 alpha- and 16 beta-hydroxytestosterone as well as androstenedione formation 67-94% by microsomes from phenobarbital-treated rats. No other pathways of testosterone metabolism are inhibited by these monoclonal antibodies. The differential inhibition of microsomal metabolism of benzphetamine and testosterone by these monoclonal antibodies is a reflection of the content and inducibility of cytochromes P-450b and P-450e as well as other cytochrome P-450 isozymes.

Animals↗
PubMed1985

Differential stereoselectivity of cytochromes P-450b and P-450c in the formation of naphthalene and anthracene 1,2-oxides. The role of epoxide hydrolase in determining the enantiomer composition of the 1,2-dihydrodiols formed.

As is the case for cytochrome P-450c, arene 1,2-oxides have been identified as initial metabolites when naphthalene and anthracene are oxidized by cytochrome P-450b in a highly purified, reconstituted system. Overall rates of metabolism by cytochrome P-450b are greater than 3-fold and greater than 50-fold lower than the respective rates of metabolism by cytochrome P-450c. For both hydrocarbons, the (-)-(1S,2R)-oxide predominates (74%) with cytochrome P-450b as the terminal oxidant, based on trapping the labile arene oxides as N-acetyl-L-cysteine S-conjugates of known absolute configuration. This result is in marked contrast to data obtained with cytochrome P-450c where the (+)-(1R,2S)-oxides predominate (73-greater than 95%). In the absence of added epoxide hydrolase, the metabolically formed arene oxides rapidly isomerize to phenols. Addition of increasing amounts of epoxide hydrolase to the incubation medium results in the formation of trans-1,2-dihydrodiols at the expense of phenols from the common arene oxide intermediates. Evaluation of the kinetic parameters (Km and kcat) for the hydration of the (+)- and (-)-enantiomers of both arene oxides by epoxide hydrolase has indicated that the (+)-(1R,2S)-enantiomers exhibit lower values of Km (approximately 1 microM) whereas the values of kcat are similar for both enantiomers of a given arene oxide. These parameters have allowed construction of a mathematical model which predicts the enantiomer composition of the dihydrodiols formed from naphthalene in reconstituted systems containing specific epoxide hydrolase concentrations. The data reported argue against a selective functional coupling mechanism between cytochrome P-450c and epoxide hydrolase in the metabolism of naphthalene and anthracene to the 1,2-dihydrodiols.

Animals↗
PubMed1985

Absolute configuration of the 5,6-oxide formed from 7,12-dimethylbenz[a]anthracene by cytochrome P450c.

The absolute configurations of the enantiomeric 5,6-arene oxides of 7,12-dimethylbenz[a]anthracene (DMBA) were recently assigned such that the late eluting enantiomer from a chiral HPLC column has 5R,6S absolute configuration. [Mushtaq et al. (1984) BBRC 125, 539]. The authors further concluded that the 5R,6S-enantiomer predominates on metabolism of DMBA by cytochrome P450c in liver microsomes from 3-methylcholanthrene-treated rats. Their chemical assignment of absolute configuration is incorrect. Thus, metabolism of DMBA by these microsomes as well as by homogeneous cytochrome P450c produces 5,6-oxide highly enriched (95%) in the 5S,6R-enantiomer in accord with theoretical predictions.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1985

Characterization of a major form of rat hepatic microsomal cytochrome P-450 induced by isoniazid.

Cytochrome P-450j has been purified to electrophoretic homogeneity from isoniazid-treated adult male rats; and this enzyme appears to be a major protein induced in hepatic microsomes after administration of isoniazid, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hemoprotein has a minimum molecular weight of approximately 51,500, and the ferrous-carbonyl complex of cytochrome P-450j has a Soret maximum at 451-452 nm. The oxidized heme iron appears to be predominately in the high spin state as deduced from the Soret maximum at 395 nm. Ethylisocyanide binds to ferrous cytochrome P-450j to yield spectral maxima at approximately 458 and 430 nm with a resultant 458/430 ratio of 0.7 at pH 7.4. Cytochrome P-450j has no measurable catalytic activity for the metabolism of benzo[a]pyrene (3- and 9-hydroxylation), hexobarbital, testosterone, and 5 alpha-androstane-3 alpha,17 beta-diol-3,17-disulfate. Low, but detectable, catalytic activity is obtained for the metabolism of 7-ethoxycoumarin, benzphetamine, p-nitroanisole, zoxazolamine, and 2-hydroxylation of 17 beta-estradiol. In contrast, cytochrome P-450j effectively catalyzes p-hydroxylation of aniline with a turnover of 12.7 nmol/min/nmol cytochrome P-450j. Hydroxyl radical scavengers, Fe-EDTA, superoxide dismutase, and catalase have no effect on aniline p-hydroxylation catalyzed by cytochrome P-450j. Cytochrome P-450j is distinct from nine other rat hepatic microsomal cytochromes P-450 (P-450a-P-450i) previously purified in this laboratory, as well as different isozymes described by other investigators, based on several parameters including minimum molecular weight, spectral properties, and catalytic activity. In Ouchterlony double diffusion plates, antibodies against cytochromes P-450a-P-450f show no cross-reaction with cytochrome P-450j. Structural differences among cytochromes P-450a-P-450j are apparent from the NH2-terminal sequence of cytochrome P-450j, as well as the electrophoretic profiles of proteolytic digests of the hemoproteins.

Amino Acids↗
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

Neutron activation determination of mercury in waters after preconcentration by flotation of dithizone-mercury complexes.

Mercury in water samples, at levels below parts per billion, was collected by dithizone flotation. The statistical detection limit of Hg in seawater was 4 times better than that with a hydrous iron oxide--APDC flotation system in neutron activation determination. The same floated dithizone precipitate can be used repeatedly for collection from several volumes of the water sample. The Hg content in seawater was 0.017 micrograms/L. Average recovery from waters containing 0.025 micrograms/L, 0.05 micrograms/L and 0.1 micrograms/L was 98%. The method was applied successfully to the determination of Hg in NBS 1641b. Methyl mercury is also collected by the procedure described.

Azo Compounds↗
PubMed1985

Inhibition and stimulation of enflurane metabolism in the rat following a single dose or chronic administration of ethanol.

The effects of a single oral dose or chronic ingestion of ethanol on in vivo and in vitro metabolism of enflurane were studied in Fischer 344 rats. At various intervals after ethanol treatment, enflurane was administered ip and 1 h after enflurane administration fluoride and ethanol levels were measured in plasma and hepatic microsomes were prepared. The concentration of ethanol in plasma (+/- SE) was 0.138 +/- 0.035% at 9 h after the single dose of ethanol and decreased almost to control levels by 17 h. The hepatic microsomal defluorination of enflurane was enhanced 3.5-fold and 6.3-fold at 9 and 25 h after the ethanol dose and returned to the control level by 33 h. In vivo defluorination was inhibited almost completely at 8 h after the ethanol dose, increased to 3.4 times the control level at 24 h, and decreased to the control level by 32 h. At 1 h after the end of chronic ethanol treatment, the concentration of ethanol in plasma was 0.254 +/- 0.018%, and it decreased to the control level by 9 h. Hepatic microsomal enflurane defluorinating activity was increased 10.5-fold at 1 h after the end of chronic treatment and decreased to the control level by 13 h. Immediately following chronic treatment, enflurane defluorination in vivo was almost totally inhibited. It increased to 9.3 times the control level at 4 h after chronic treatment was stopped and then decreased to nearly the control level at 12 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcoholism↗