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PubMed1984

Role of supernatant protein factor and anionic phospholipid in squalene uptake and conversion by microsomes.

Supernatant protein factor (SPF), a cytosolic protein (Mr = 47,000) stimulates microsomal squalene epoxidase activity 4- to 10-fold in the presence of anionic phospholipid such as phosphatidylglycerol (PG) (Saat, Y., and Bloch, K. (1976) J. Biol. Chem. 251, 5155-5160). This effect has been ascribed to substrate translocation from inactive to active pools within the membrane of the endoplasmic reticulum (Friedlander, E. J., Caras, I. W., Lin, L. F. H., and Bloch, K. (1980) J. Biol. Chem. 255, 8042-8045). Here we show that SPF and PG also stimulate squalene uptake per se by microsomes as well as stimulate squalene epoxidase. Microsomes preloaded with substrate in the presence of SPF and PG show full epoxidase activity. They do not require further addition of these factors during enzyme assay. Addition of SPF and PG to assay mixtures containing microsomes preloaded with substrate in the presence of SPF and PG did not further increase epoxidase activity. We also show that PG tightly binds to microsomes. This binding of PG is essential for the response of microsomal epoxidase to SPF. Solubilized microsomal enzymes have been reconstituted and show high epoxidase activity. In this system, SPF and PG do not stimulate the conversion of squalene into products.

Enzyme Activation↗
PubMed1978

Characterization of the microsomal steroid-8-ene isomerase of cholesterol biosynthesis.

Rat liver microsomes contain an enzyme that catalyzes the isomerization of the nuclear double bond of steroids from the 8(9) position to the 7(8) position. The enzyme is most active with zymosterol, 5alpha-cholesta-8,24-dien-3beta-ol, which is a precursor of cholesterol. Properties of the microsomal isomerase have now been studied, and preliminary data are reported on both regulation of enzymic activity and first steps in the solubilization of the enzyme from membranes. After a brief lag period, the velocity of isomerase is relatively constant for about 5 min of incubation, and then isomerization subsides. The apparent Michaelis constant (52-70 micro M) is difficult to determine accurately, due to these complex kinetic changes. V(max) is 4.0-4.7 nmol/min per mg of microsomal protein. The apparent specific activity is more than ten times that of liver microsomal methyl sterol oxidase. The maximal specific activity of microsomal isomerase is approximately doubled when rats are fed an intestinal bile acid sequestrant, cholestyramine. Changes in specific activity of isomerase parallel changes in activities of other microsomal enzymes of cholesterol biosynthesis, such as 3-hydroxy-3-methylglutaryl-CoA reductase and 4-methyl sterol oxidase. Isomerase activity is destroyed by phospholipase A digestion, high concentration of bile salts, and solvent extraction, all of which are known either to remove phospholipid or to alter microsomal membrane integrity. On the other hand, isomerase remains active in the presence of a mild, nonionic detergent, Triton WR-1339; thus, solubilization with nonionic detergents is under study.

Animals↗
PubMed1983

Testosterone metabolism by cytochrome P-450 isozymes RLM3 and RLM5 and by microsomes. Metabolite identification.

Testosterone metabolism by cytochrome P-450 isozymes RLM3 and RLM5 in a reconstituted system and by rat liver microsomes was examined. Eleven metabolites were detected. Two of these, found in spots 2 and 4 of a thin layer plate, were only formed by the rat liver microsomes and may represent reductive metabolites of testosterone. A number of monohydroxy metabolites were conclusively identified by gas chromatography-mass spectrometry. These include the 2-, 6 beta-, 7 alpha-, and 16 alpha-hydroxy isomers. Liver microsomes formed the 2 alpha- and 2 beta-epimers in a 1:2 ratio and both co-chromatographed with a third reduced metabolite in thin layer plate spot 4. In contrast with RLM5 about 90% of the 2-hydroxy isomer was the 2 alpha-epimer. RLM3 did not perform the 2-hydroxylation in detectable amounts. The 6 beta-isomer was a major metabolite of RLM3 and microsomes, but a minor product of metabolism by RLM5. In contrast, the 7 alpha-isomer was a minor metabolite of RLM3, was not formed by RLM5, and was a major microsomal metabolite. Hydroxylation at position 16 alpha was a major activity of RLM5 and the heterogeneous microsomal cytochromes, but with RLM3 it was a minor reaction. One new metabolite was found which appeared to be hydroxylated in the D-ring, had a mass spectrum different from both 16 alpha- and 16 beta-hydroxytestosterone, and was tentatively identified as a 15-hydroxy isomer. In agreement with the literature, androstene-3,17-dione was found to be an oxidative metabolite of testosterone by both microsomes and purified cytochrome P-450. It was a major metabolite of RLM5 but was not produced by RLM3. Studies with 18O2 and H218O conclusively show that oxidation of testosterone at C-17 does not involve transient incorporation of an oxygen atom in this position. A mechanism is suggested whereby cytochrome P-450 acts as a peroxidase in the formation of androstenedione.

Animals↗
PubMed1983

The hepatic microsomal formation of bilirubin diglucuronide.

Although the formation of bilirubin monoglucuronide by hepatic microsomes has been easily demonstrable, that of bilirubin diglucuronide, the principal conjugate of bile, has been more difficult. Therefore, an examination of the uridine diphosphate glucuronate-dependent microsomal formation of these two conjugates has been made utilizing a high performance liquid chromatographic method which quantitates the isomeric forms of the products. Initial studies indicated that at high starting bilirubin concentrations, only bilirubin monoglucuronide was formed; whereas at lower concentrations (approximately 15 microM), bilirubin diglucuronide could be formed efficiently, but only under very specific conditions. Untreated microsomes and microsomes treated with Triton X-100 did not differ; each formed monoglucuronide efficiently, yet formed diglucuronide poorly. Digitonin or UDP-N-acetylglucosamine pretreatment, in contrast, was found to facilitate bilirubin diglucuronide formation, the former much more than the latter. The activity of mannose 6-phosphatase, an enzyme located on the inner surface of the microsomal vesicles, did not correlate well with the bilirubin diglucuronide formation. Time course studies with digitonin and UDP-N-acetylglucosamine indicated a precursor-product relation between bilirubin monoglucuronide and bilirubin diglucuronide, and product isomer composition studies indicated that the bilirubin tetrapyrroles were stable (no random dipyrrolic exchange had occurred). Temperature studies with the digitonin-treated preparation demonstrated an increase in monoglucuronide-forming activity over the 0-25 degrees C range, whereas diglucuronide formation increased dramatically over the range from 25 to 35 degrees C. The results indicate that microsomal diglucuronide-forming activity differs characteristically from monoglucuronide-forming activity, and that it is intensely sensitive to the manipulation of its microsomal membrane environment.

Animals↗
PubMed1983

Identification and characterization of a new epoxide hydrolase from mouse liver microsomes.

A new microsomal epoxide hydrolase (mEH2) has been identified and characterized. This enzyme has properties which distinguish it from previously described cytosolic (cEH) or membrane-bound (mEH1) epoxide hydrolases. The enzyme is an integral microsomal protein which is not dissociated from the membrane by repeated washing, high ionic strength salt, or chaotropic agent solutions, or by sonication. It is very different from the normally described microsomal epoxide hydrolase (mEH1) as shown by its different substrate specificity and kinetic properties and by immunological criteria. In contrast to the hitherto described microsomal epoxide hydrolase, mEH1, the new enzyme effectively catalyzes the hydration of transdisubstituted oxiranes such as trans-stilbene oxide and trans-beta-ethyl styrene oxide and has no appreciable activity toward benzo(a)pyrene 4,5-oxide. It is also structurally distinct, in that it does not cross-react with antibodies raised against the normally described microsomal epoxide hydrolase mEH1. This newly described microsomal epoxide hydrolase probably represents an important factor in the control of reactive epoxides; its location in the membrane ensures access to lipophilic epoxides generated by membrane-bound monooxygenases, and its substrate specificity is such that it can hydrolyze epoxides poorly metabolized by the previously described microsomal epoxide hydrolase.

Animals↗
PubMed1981

The participation of sterol carrier protein2 in the conversion of cholesterol to cholesterol ester by rat liver microsomes.

The purification of sterol carrier protein2 (SCP2), purified 1500-fold to homogeneity from the 303,000 x g supernatant (S303) of rat liver, has recently been described (Noland, B. J., Arebalo, R. E., Hansbury, E., and Scallen T. J. (1980) J. Biol. Chem, 255, 4282-4289). Since SCP2 is required for the synthesis of cholesterol by microsomal membranes, it was decided to test the hypothesis that SCP2 might also participate in enzymatic reactions which utilize cholesterol as a substrate. The reaction studied in the present investigation was the conversion of cholesterol to cholesterol ester (acyl-CoA cholesterol acyltransferase) by rat liver microsomes. The results show that when exogenously added [4-14C]cholesterol is the substrate, SCP2 produces a striking increase in cholesterol ester biosynthesis by rat liver microsomes. Although the effect of SCP2 was most clearly seen with exogenously added cholesterol, it was also demonstrated when [1-14C]oleoyl-CoA was the labeled substrate and the incorporation of labeled oleate into cholesterol ester was determined. Although it was demonstrated that microsomes could bind large amounts of cholesterol in the absence of SCP2, the bound cholesterol was ineffective as a substrate for microsomal acyl-CoA cholesterol acyltransferase. However, the microsomally bound cholesterol became an effective substrate for the enzyme upon the addition of SCP2. The results demonstrate that SCP2 participates in the utilization of cholesterol via the microsomal conversion of cholesterol to cholesterol ester. We also conclude that SCP2 may participate in the intracellular transport of cholesterol, in particular, the delivery of either exogenous (dietary) cholesterol or endogenous cholesterol to acyl-CoA cholesterol acyltransferase in the endoplasmic reticulum.

Animals↗
PubMed1982

Age-related changes in hepatic microsomal drug metabolism are substrate selective.

Microsomes were isolated from livers of male Fischer 344 rats at 3 to 5, 14 to 15 and 24 to 25 months of age for the determination of monooxygenase components and drug metabolism activities. Microsomal cytochrome P-450, cytochrome b5, NADPH-cytochrome c reductase activity and phospholipid were decreased in middle-aged and old rats compared with young-adult rats, but the enzymatic reduction of microsomal cytochrome P-450 was unchanged. Drug metabolism activities both decreased and increased as a consequence of aging, depending upon the substrate used. Differences were observed between young-adult and old rats in the CO maximum of reduced microsomal cytochrome P-450, in microsomal fatty acid composition and in the amounts of microsomal polypeptides having molecular weights of 52,500 and 53,000. The substrate selectivity of the age-related alterations in hepatic microsomal drug metabolism may be due to qualitative changes in the cytochrome P-450 and phospholipid components of the monooxygenase system.

Aging↗
PubMed1982

Studies on the O-demethylation of misonidazole by rat liver microsomes.

The role of rat liver microsomes in the O-demethylation of misonidazole to desmethylmisonidazole was studied. The rate of the microsomal-dependent formation of desmethylmisonidazole was linear up to a protein concentration of 2 mg/ml and over a 10-minute interval. The metabolism was optimal in a system comprised of microsomes, O2, and NADPH. Metabolism in incubation mixtures continuously flushed with N2 was inhibited by 78%. The O-demethylase activity was competitively inhibited by the addition of SKF 525-A, with a Ki of approximately 1 x 10(-5) M. The Km and Vmax values of normal microsomes were 1.87 +/- 0.30 mM and 413 +/- 14 pmols/minute/mg of microsomal protein, respectively. Pretreatment of rats with phenobarbital for 7 days prior to preparation of the microsomes resulted in no significant change in the Km, but the Vmax was considerably increased to 1033 +/- 203 pmols/minute/mg of microsomal protein. The results demonstrate that the O-demethylation of misonidazole is mediated by cytochrome P-450.

Animals↗
PubMed1982

Thyroid-stimulating hormone subunit processing and combination in microsomal subfractions of mouse pituitary tumor.

Mouse pituitary thyrotropic tumor minces were labeled with [35S]methionine and fractionated into rough microsomes, intermediate, and low density smooth microsomes. Thyroid-stimulating hormone subunits were mainly in rough microsomes after a 10-min pulse, but with increasing chase times the proportion in smooth microsomes increased. In rough microsomes, small amounts of an alpha subunit precursor of Mr = 11,000 and larger amounts of an alpha form of Mr = 18,000 were rapidly processed to a form of Mr = 21,000, while small amounts of a beta-subunit precursor of M r = 11,000 were processed to a form of Mr = 18,000. Most of the Mr = 18,000 and Mr = 21,000 subunit forms were converted by endoglycosidase H to forms of Mr = 11,000 to 12,000. Small amounts of endoglycosidase H-resistant forms appeared in low density smooth microsomes after a 30-min chase. Subunit combination was not detected at 10 min; combination was first detected at 20 min and increased progressively to a maximum of 61% of beta in the low density smooth microsomes at 60 min of chase. Although alpha of Mr = 11,000 and 18,000, and beta of Mr - 11,000, were not detected in thyroid-stimulating hormone, both endoglycosidase H-sensitive and -resistant alpha subunit of Mr = 21,000 and beta subunit of Mr = 18,000 were found combined. Thus, the rough endoplasmic reticulum (ER) contains only small amounts of nonglycosylated subunits (Mr = 11,000). The major subunit precursors contain one high mannose oligosaccharide (Mr = 18,000), with a second unit being added onto alpha in the rough ER. Combination of alpha (Mr = 21,000) with beta (Mr = 18,000) begins in the rough ER but occurs predominantly in the smooth ER/Golgi. Oligosaccharides of both combined and uncombined subunits are processed from high mannose to complex forms predominantly in the smooth ER/Golgi.

Animals↗
PubMed1982

Comparative study on oxidation of aromatic compounds by rat liver and rabbit lung microsomes.

A systematic comparative investigation of the kinetics of naphthalene hydroxylation by rabbit lung and rat liver microsomes has been carried out over a temperature range of 4-40 degrees C. A higher catalytic activity of cytochrome P-450 from rabbit lung to naphthalene as compared to rat liver microsomes has been stated. The temperature dependence of naphthalene hydroxylation is characterized by a break in the Arrhenius plot for the enzyme systems of liver microsomes from rats untreated and treated with phenobarbital (PB) and 3-methylcholanthrene (MC), respectively, whereas no such break is observed when aniline is oxidized by rat liver microsomes in combination with NADPH and O2, and also in the cumene hydroperoxide supported (CHP) aniline hydroxylation involving highly purified cytochrome P-450 LM2. A break in the Arrhenius plot is also observed for maximal rates of the naphthalene hydroxylation by lung microsomes from untreated and PB-treated rabbits, whereas for MC-induced lung microsomes it is absent. Glycerol eliminates this break in the Arrhenius plot while propranolol shifts the break point by 3-4 degrees C. The data obtained are discussed in terms of the physical state of microsomal phospholipids and their effect on the components of the hydroxylating system.

Animals↗
PubMed1981

[Stoichiometric protein-lipid interactions in liver microsomal membranes measured by the induction-resonance energy transfer method].

The stoichiometry of proteins and lipids in the microsomal membranes of the liver were studied by the induction-resonance energy transfer method. The energy was transferred between the fluorescent tryptophane residues of membrane proteins and fluorescent probes (pyrene and 4-dimethylaminochalcone) located in the microsomal lipid bilayer. It was assumed that the rate of protein fluorescence is correlated with protein mass. All microsomal proteins were arbitrarily divided into two groups, i.e. proteins located at a distance of more ("peripheral" proteins) and less than 30 A from the lipid bilayer surface. The former proteins made up to 14-24% of total microsomal protein, while microsomal ghosts--less than 10%. In the microsomes the bulk of other proteins is located at an average distance of about 19-26 A from the lipid bilayer surface, while the grosts--at about 4-9 A. Treatment of the ghosts with pronase results in approximation of the protein mass locus with the lipid by about 5 A. In this way the generation of ghosts and their treatment with pronase cause a loss of proteins predominantly from the peripheral parts of the microsomal membrane.

Animals↗
PubMed1981

Structural gene products of the murine Ah complex. Differences in ontogenesis and glucosamine incorporation between liver microsomal cytochromes P1-450 and P-448 induced by polycyclic aromatic compounds.

Antibodies against mouse-liver microsomal cytochromes P1-450 and P-448, two polycyclic aromatic inducible cytochromes, were previously developed [Negishi, M. and Nebert, D.W. (1979) J. Biol. Chem. 254, 11015-11023]. Liver microsomes from 3-methylcholanthrene-treated and phenobarbital-treated and control adult mice and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated adult and fetal mice were examined. Immunoprecipitable radioactivity was measured, following labeling with pyridoxal phosphate/NaB[3H]4 or with 125I-labeled p-aminosulfobenzoic acid/NaNO2 in vitro or with [3H]leucine, [14C]glucosamine, or [32P]O4 in vivo. (a) Induction of cytochrome P1-450 occurs developmentally earlier in gestation than induction of cytochrome P-448 when the mother is treated with polycyclic aromatic compounds. (b) There appears to be a basal form of cytochrome P-448 but no cytochrome P1-450 in control liver microsomes; inducibility of cytochrome P-448 thus ranges between 5--12-fold, whereas that of P1-450 is infinite. (c) Phenobarbital pretreatment induces no detectable P1-450 or P-448. (d) P-448 appears to be either greater in concentration than P1-450 in the membrane or more exposed than P1-450 on the microsomal membrane surface. (e) By the radioimmunoassay methods used, 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced P1-450 and P-448 in Ah-nonresponsive mice are indistinguishable from those in Ah-responsive mice; this is true in both the fetus and the adult. (f) Compared with P-448 expression, the expression of P1-450 is more closely associated with 3-methylcholanthrene-induced aryl hydrocarbon hydroxylase activity, and these two structural gene products are apparently regulated independently. (g) P-448 but not P1-450 appears to be a glycoprotein. These data illustrate further differences between two forms of polycyclic aromatic-inducible P-450 in mouse liver. Neither P1-450 nor P-448 appears to be a phosphoprotein. Neither anti-(P1-450) nor anti-(P-448) precipitates any forms of liver microsomal P-450 from beta-naphthoflavone-treated adult rabbits and, conversely, anti-LM4 (the antibody to rabbit liver microsomal P-450 form 4) does not precipitate any forms of liver microsomal P-450 from 3-methylcholanthrene-treated C57BL/6N mice.

Animals↗
PubMed1981

Metabolism of alpha-naphthoflavone by rat liver microsomes.

alpha-Naphthoflavone (ANF) or 7,8-benzoflavone, a synthetic flavonoid, has been widely used in biochemical and biological studies concerning the mechanisms of action of chemical carcinogens. It has been shown previously that ANF inhibits benzo(a)pyrene metabolism by beta-naphthoflavone (BNF)-induced rat liver microsomes but has no inhibitory effects on benzo(a)pyrene metabolism in phenobarbital (PB)-induced rat liver microsomes. This study shows that ANF gives type 1 binding spectra with and is metabolized by both BNF- and PB-induced rat liver microsomes. Specific metabolites identified by ultraviolet and mass spectra and in some cases by cochromatography with authentic standards were: 6-hydroxy-alpha-naphthoflavone, 9-hydroxy-alpha-naphthoflavone, alpha-naphthoflavone-5,6-oxide, and 5,6-dihydro-5,6-dihydroxy-alpha-naphthoflavone. Metabolism at the 5,6 bond of ANF accounted for 73 and 86% of the total organic soluble metabolites produced by PB- and BNF-induced microsomes, respectively. This result is in concert with previous observations on the role of 6 substitution and the loss of inhibitory activity of ANF in BNF-induced rat liver microsomes. Metabolism of ANF is mediated by the cytochrome P-450 mixed-function oxidases, because it is dependent on NADPH and inhibited by carbon monoxide and other cytochrome P-450 inhibitors. BNF-induced microsomes metabolize ANF to 5,6-dihydro-5,6-dihydroxy-alpha-naphthoflavone to a much greater extent than do PB-induced microsomes.

Animals↗
PubMed1995

Activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in human lung microsomes by cytochromes P450, lipoxygenase, and hydroperoxides.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent tobacco-specific carcinogen, has been demonstrated to induce lung tumors in animals and is suspected to be a human carcinogen. Cytochromes P450 are the major enzymes responsible for the activation of NNK in microsomes from the lung and liver of rat and mouse, as well as human liver. The present study investigated the enzymes responsible for the metabolic activation of NNK in human lung microsomes. In the presence of a NADPH-generating system, the formation of keto aldehyde and keto alcohol (alpha-hydroxylation products, measured together), keto acid, hydroxy acid, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol was observed in human lung microsomes. Carbon monoxide (90%) decreased the rate of NNK oxidation by 5-49%, depending on the human lung microsomal samples analyzed. Coumarin decreased the oxidation of NNK by 9-34%, and an antibody against human P450 2A6 decreased the metabolism of NNK by 8-37%, suggesting the involvement of P450 2A6 in NNK oxidation. alpha-Napthoflavone inhibited NNK oxidation by 6-26%, possibly due to the inhibition of P450 1A1. P450 1A1-expressed microsomes catalyzed the formation of keto aldehyde and keto alcohol, exhibiting Km values of 1400 microM and 371 microM, respectively. In the absence of NADPH, NNK metabolism resulted in the formation of keto acid, keto aldehyde, and keto alcohol, and the activities in different lung samples were decreased by indomethacin (100 microM; cyclooxygenase inhibitor) or nordihydroguaiaretic acid (100 microM; lipoxygenase inhibitor) by 0-27% or 30-66%, respectively. The addition of arachidonic acid (10-100 microM) increased the rate of the formation of keto aldehyde and keto alcohol approximately 2-fold but inhibited the formation of keto acid. Soybean lipoxygenase increased the rate of formation of keto aldehyde and keto alcohol in a concentration-dependent manner. The increased rate in NNK oxidation by arachidonic acid or lipoxygenase was inhibited completely by nordihydroguaiaretic acid. Catalase, thiourea, and conjugated linoleic acid decreased the rate of NNK oxidation by 47, 20, and 45%, respectively. tert-Butyl-hydroperoxide, cumene hydroperoxide, and hydrogen peroxide increased the rate of formation of keto aldehyde and keto alcohol by 210, 40, and 50%, respectively. The results suggest that P450 enzymes are only partially responsible for the activation of NNK in human lung microsomes, and P450 2A6 or a P450 2A6-related enzyme seems to be involved in the activation. Furthermore, lipoxygenase and lipid hydroxperoxides may play important roles in the oxidation of NNK in human lung microsomes.

Aldehydes↗
PubMed1995

In vitro metabolism of terfenadine by a purified recombinant fusion protein containing cytochrome P4503A4 and NADPH-P450 reductase. Comparison to human liver microsomes and precision-cut liver tissue slices.

The metabolism of terfenadine was studied with a cDNA-expressed/purified recombinant fusion protein containing human liver microsomal cytochrome P4503A4 (CYP3A4) linked to rat NADPH-P450 reductase (rF450[mHum3A4/mRatOR]L1) and was compared with that observed in the presence of human liver microsomes and precision-cut human liver tissue slices. In all three cases, [3H]terfenadine was metabolized to at least three major metabolites. LC/MS (electrospray) analysis confirmed that these metabolites were alpha, alpha-diphenyl-4-piperidinomethanol (M5), t-butyl hydroxy terfenadine (M4), and t-butyl carboxy terfenadine (M3), although the level of M5 detected in the presence of fusion protein was greater than that found with microsomes or tissue slices. Two additional metabolites, M1 (microsomes and tissue slices) and M2 (fusion protein), were also detected, but remain uncharacterized. Consumption of parent drug (microsomes: KM = 9.58 +/- 2.79 microM, Vmax = 801 +/- 78.3 pmol/min/nmol CYP; fusion protein: KM = 14.1 +/- 1.13 microM, Vmax = 1670 +/- 170 pmol/min/nmol CYP) and t-butyl hydroxylation to M4 (microsomes: KM = 12.9 +/-3.74 microM, Vmax = 643 +/- 62.5 pmol/min/nmol CYP, ; fusion protein: KM = 30.0 +/- 2.55 microM, Vmax = 1050 +/- 141 pmol/min/nmol CYP) obeyed Michaelis-Menten kinetics over the terfenadine concentration range of 1-200 microM. Ketoconazole, a well-documented CYP3A inhibitor, effectively inhibited terfenadine metabolism in all three models. The conversion of M4 to M3, studied with human liver microsomes and fusion protein, was NADPH-dependent and inhibited by ketoconazole. It is concluded that cDNA-expressed CYP3A4, in the form of a NADPH-P450 reductase-linked fusion protein, may also serve as a model for studying the metabolism of terfenadine in vitro and many other drugs.

Adult↗
PubMed1979

Prostaglandin and thromboxane synthesis by microsomes of rabbit ocular tissues.

Microsomes of albino rabbit ocular tissues were incubated with (1-14C)-arachidonic acid for 15 min at 37 degrees C. Thin-layer chromatography revealed that ciliary body-iris microsomes were capable of synthesizing prostaglandin F2alpha (PGF2alpha), PGE2, PGD2, thromboxane B2(TXB2), and 6-keto-PGF1alpha. Indomethacin 14 micrometer in the incubation medium essentially abolished all prostaglandin synthesis detectable by this method. Imidazole 10 mM in the incubation medium inhibited only TXB2 synthesis. Ciliary body-iris microsomes were incubated for 2 min at 0 degrees C with PGH2. The products of this reaction were superfused over spiral strips of rabbit aorta and produced the strong contractions typical of TXA2. Addition to imidazole to the incubation medium blocked the formation of the contracting substance. Incubation of ciliary body-iris microsomes with (1-14C)--8,11,14-eicosatrienoic acid produced PGF1alpha, PGD1, and PGE1 but no evidence of any thromboxane product or 6-keto-PGF1alpha. Conjunctival and corneal microsomes synthesized prostaglandins, although less effectively than ciliary body-iris microsomes, when incubated with (1-14C)-arachidonic acid. Microsomes of sclera, retina-choroid, and lens synthesized little, if any, prostaglandins.

8,11,14-Eicosatrienoic Acid↗
PubMed1994

Induction of hepatic microsomal cytochrome P450 by dexamethasone in rhesus monkey (Macaca mulatta).

Intraperitoneal administration of 150 mg dexamethasone (DEX) Kg-1 body wt for four days to rhesus monkeys resulted in statistically significant increases in the activities of hepatic tyrosine aminotransferase (3 fold), microsomal cytochrome P450 (2 fold) and erythromycin N-demethylase (4 fold), but no change in the activities of aminopyrine N-demethylase and NADPH cytochrome c reductase. Three peaks were obtained from control or DEX-treated monkey livers on fractionation of detergent solubilized microsomes by anion exchange chromatography on DE-52. Peak II obtained from DEX-treated monkey microsomes on DE-52 demonstrated the highest specific activity of cytochrome P450 (5.84 nmol mg-1 protein) as compared to other peaks from the same microsomes or any of the peaks obtained from the control microsomes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the microsomes from control and experimental animals and peak II obtained after anion exchange chromatography of DEX-treated microsomes demonstrated the intensification of two polypeptides of 52.5 and 50 kDa. The results indicate that DEX is an inducer of cytochrome P450 and dependent erythromycin N-demethylase in non-human primate, Macaca mulatta.

Aminopyrine N-Demethylase↗
PubMedDate not supplied

Characterization of verlukast metabolites arising from an epoxide intermediate produced with hepatic microsomes from beta-naphthoflavone-treated rodents (P-4501A1).

Verlukast, (R)3-((((3-(2-(7-chloroquinolin-2-yl)-(E)-ethenyl)phenyl)-3- dimethylamino-3-oxopropylthio)methyl)thio)-propionic acid (also known as MK-0679 and L-668,019), is a potent leukotriene D4 antagonist. Verlukast was incubated with hepatic microsomes from beta-naphthoflavone (beta NF) or isosafrole-treated rodents to evaluate whether P-4501A1 or 1A2 mediated biotransformations could occur. With beta NF-induced mouse or rat microsomes, in which the induction of P-4501A1 had been proven by Western blot analysis, incubations produced new metabolites that were separated by reversed-phase HPLC and were initially characterized by UV (photodiode array). Metabolites were subsequently isolated and characterized by NMR and MS, and were assigned as the 5",6"-dihydrodiol and 6"-phenol (on the quinoline ring). The presumed 5",6"-epoxide intermediate was also detected and was characterized by UV (photodiode array) and MS. Microsomes from isosafrole-treated rodents produced the dihydrodiol to a much lesser extent and did not yield any other new metabolites. alpha-Naphthoflavone inhibited the dihydrodiol formation in incubations with microsomes from isosafrole- and beta NF-treated rats. In incubations with microsomes from beta NF-treated rats, to which the epoxide hydrolase inhibitor 3,3,3-trichloropropene 1,2-oxide had been added, the formation of dihydrodiol was inhibited, consistent with a microsomal epoxide hydrolase hydrolysis of the epoxide intermediate. When glutathione was added to incubations with microsomes from beta NF-treated rats, the dihydrodiol, phenol, and epoxide peaks were reduced in size and a new material, the glutathione adduct, was formed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗