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PubMed1975

Influence of ouabain and dihydroouabain on the circular dichroism of cardiac plasmalemmal microsomes.

1. The influence of ouabain on the tertiary structure of cardiac plasmalemmal proteins was investigated by means of circular dichroism measurement. Purified plasmalemmal microsomes were obtained by sucrose gradient centrifugation. The CD-spectra of the membranal proteins were shifted to the red and the amplitudes were smaller than those of the same proteins after solubilization. 2. Ouabain induced an increase of the ellipticity bands at 210 and 222 nm of about 50% above the level yielded with microsomes after sonication. At 222 nm ouabain exhibited the half maximum effect at a concentration of 5 X 10(-9) M. The effect could, however, only be exerted if the inside of the microsomes was exposed to ouabain by sonication, thus reflecting the inside-out nature of the plasmalemmal microsomes. 3. The high specificity of the ouabain effect was underlined by the following experiments: a) Dihydroouabain, a much less cardioactive derivative of ouabain proved to be ineffective in corresponding concentrations, b) ouabain had no influence upon the CD spectrum of microsomes derived from cardiac sarcoplasmic reticulum, c) a detergent-like action of ouabain underlying the observed effect can be excluded since highly active tensides, i.e. desoxycholate and dodecylsulfate, only influence the CD spectra at concentrations exceeding 10(-3) M, d) electronmicrographs of microsomes exposed to ouabain demonstrated no alteration of either the appearance or size of the microsomes. 4. The magnitude of the observed ouabain effect indicates that a large portion of the membrane-bound proteins is involved. The number of binding sites and their isolated structural alteration induced by ouabain are not sufficient to account quantitatively for the enhanced amplitudes of the CD spctra. This suggests that ouabain evokes structural changes of membrane proteins different from actual binding sites. It seems, therefore highly improbable that changes of the Na-K-ATPase present in the plasmalemmal microsomes are responsible for the observed effect.

Animals↗
PubMed1992

Fluorospectroscopic analysis of the fluorescent substances in peroxidized microsomes of rat liver.

The fluorescent substances formed in rat liver microsomes in the course of lipid peroxidation were investigated by fluorescence techniques. The fluorescence emitted from peroxidizing microsomes continuously increased as lipid peroxidation progressed, while the steady-state fluorescence anisotropy increased and then reached a plateau. A similar increase was observed in the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene in peroxidizing microsomes. The fluorescence from peroxidized microsomes consisted of at least three species having short, middle or long fluorescence lifetimes. The lifetimes and relative amplitudes of fluorescence were unaffected by the extent of lipid peroxidation. Both fluorescence of the chromolipids extracted and the proteins isolated from peroxidized microsomes had the same characteristics in fluorescence lifetimes as the fluorescence from whole peroxidized microsomes. Thus, these lipids and proteins appear to be the major biological substances responsible for the fluorescence emanating from whole peroxidized microsomes. Furthermore, fluorescent substances formed in microsomes seem to increase in quantity rather than change in quality as lipid peroxidation progresses.

Animals↗
PubMed1986

Immobilization of pig liver microsomes. Stability of cytochrome P-450-dependent monooxygenase activities.

Microsomes from pig liver were covalently coupled to Sepharose activated by CNBr and to Sephadex activated by 1,1'-carbonyldiimidazole. Microsomes were also entrapped inside Ca-alginate and kappa-carrageenan gels. The concentration of immobilized cytochrome P-450 was determined by CO-difference spectra. The activity of the monooxygenase system was demonstrated by the N-demethylation of aminopyrine, the O-demethylation of p-nitroanisole, and the hydroxylation of perhexiline maleate. Upon immobilization, a 30-40% and a 60-70% decrease in Vappmax for the O- and N-demethylations were respectively observed. The Vappmax values for the hydroxylation of perhexiline maleate were essentially the same for the different immobilized forms and for the freely suspended microsomal cytochrome P-450. Under storage at 4 degrees C, microsomes entrapped inside kappa-carrageenan and Ca-alginate were less stable than the free microsomes, whereas immobilization on CNBr-activated Sepharose improved the stability of the hepatic microsomal monooxygenase system at the same temperature. These types of immobilized microsomes have the advantage of being easily recovered and reused for other assays. Finally, microsomes entrapped inside kappa-carrageenan or Ca-alginate can be used to follow up the continuous metabolization of p-nitroanisole for several hours in a stirred-batch reactor.

Animals↗
PubMedDate not supplied

The in vitro metabolism of desglymidodrine, an active metabolite of prodrug midodrine by human liver microsomes.

The human cytochrome P450 (CYP) isoforms catalyzing the oxidation metabolism of desglymidodrine (DMAE), an active metabolite of midodrine, were studied. Recombinant human CYP2D6, 1A2 and 2C19 exhibited appreciable catalytic activity with respect to the 5'-O-demethylation of DMAE. The O-demethylase activity by the recombinant CYP2D6 was much higher than that of other CYP isoforms. Quinidine (a selective inhibitor of CYP2D6) inhibited the O-demethylation of DMAE in pooled human microsomes by 86%, while selective inhibitors for other forms of CYP did not show any appreciable effect. Although the activity of CYP2D6 was almost negligible in the PM microsomes, the O-demethylase activity of DMAE was found to be maintained by about 25% of the pooled microsomes. Furafylline (a selective inhibitor of CYP1A2) inhibited the M-2 formation in the PM microsomes by 57%. The treatment of pooled microsomes with an antibody against CYP2D6 inhibited the formation of M-2 by about 75%, whereas that of the PM microsomes did not show drastic inhibition. In contrast, the antibody against CYP1A2 suppressed the activity by 40 to 50% in the PM microsomes. These findings suggest that CYP2D6 have the highest catalytic activity of DMAE 5'-O-demethylation in human liver microsomes, followed by CYP1A2 to a small extent.

Animals↗
PubMed2005

Role of MMP-2 in inhibiting Na+ dependent Ca2+ uptake by H2O2 in microsomes isolated from pulmonary smooth muscle.

Treatment of microsomes (preferentially enriched with endoplasmic reticulum) isolated from bovine pulmonary artery smooth muscle tissue with H2O2 (1 mM) markedly stimulated matrix metalloproteinase activity and also inhibited Na+ dependent Ca2+ uptake. Electron micrograph revealed that H2O2 (1 mM) does not cause any damage to the microsomes. MMP-2 and TIMP-2 were determined to be the ambient protease and corresponding antiprotease of the microsomes. Pretreatment with vitamin E (1 mM) and TIMP-2 (50 microg/ml) reversed the effect produced by H2O2 (1 mM) on Na+ dependent Ca2+ uptake in the microsomes. However, H2O2 (1 mM) caused changes in MMP-2 activity and Na+ dependent Ca2+ uptake were not reversed upon pretreatment of the microsomes with a low concentration of 5 microg/ml of TIMP-2 which otherwise reversed MMP-2 (1 microg/ml) mediated increase in 14C-gelatin degradation and inhibition of Na+ dependent Ca2+ uptake. Combined treatment of the microsomes with a low dose of MMP-2 (0.5 microg/ml) and H2O2 (0.5 mM) inhibited Na+ dependent Ca2+ uptake in the microsomes compared to the respective low dose of either of them. Direct treatment of TIMP-2 (5 microg/ml) with H2O2 (1 mM) abolished the inhibitory effect of the inhibitor on 14C-gelatinolytic activity elicited by 1 microg/ml of MMP-2. Thus, one of the mechanisms by which H2O2 activates MMP-2 could be due to inactivation of TIMP-2 by the oxidant. The resulting activation of MMP-2 subsequently inhibits Na+ dependent Ca2+ uptake in the microsomes.

Animals↗
PubMed2006

Determination of a human hepatic microsomal scaling factor for predicting in vivo drug clearance.

PURPOSE: To determine a microsomal scaling factor for human liver suitable for prediction of in vivo drug clearance from in vitro data and to explore the role of inter-liver variability in this factor on the reported underprediction from microsomal parameters. METHODS: Cytochrome P450 (henceforth P450) content in whole homogenates and microsomes from 38 donor livers was used to determine a microsomal scaling factor. In a subset (n = 20) of these preparations, individual P450 enzymes were examined by Western blotting and selective probe activities were determined. RESULTS: The scaling factor from 38 livers averaged 40 mg microsomal protein per gram liver with a coefficient of variation of 31%. Western blotting experiments indicated that there was no P450 enzyme-specific trend in the distribution of individual P450 enzymes in liver microsomes relative to whole homogenate. Predictions based on an average scaling factor resulted in a satisfactory prediction of intrinsic clearance of three benzodiazepines similar to that obtained using individual factors for the same livers. CONCLUSION: A value for human liver microsomal scaling of 40 mg microsomal protein per gram liver has been established. The reason for underprediction previously reported for 52 different drug substrates was not the use of an incorrect value for the scaling factor.

Benzodiazepines↗
PubMed1983

Distinct rat hepatic microsomal epoxide hydrolases catalyze the hydration of cholesterol 5,6 alpha-oxide and certain xenobiotic alkene and arene oxides.

Metabolism of cholesterol 5,6 alpha-oxide to the 5,6-glycol is catalyzed by a rat liver microsomal epoxide hydrolase that is distinct from the microsomal epoxide hydrolase that metabolizes a wide range of xenobiotic alkene and arene oxides. The two enzymes are antigenically distinct, and the purified microsomal epoxide hydrolase that metabolizes xenobiotic oxides does not catalyze the hydration of cholesterol 5,6 alpha-oxide. In vivo treatment of rats with inducers of microsomal epoxide hydrolase does not enhance the activity of cholesterol 5,6 alpha-oxide hydrolase and, in some cases, actually depresses enzyme activity in the resultant microsomal preparations. Octene 1,2-oxide and benz[a]anthracene 5,6-oxide, both good substrates for xenobiotic epoxide hydrolase, are not competitive inhibitors of cholesterol oxide hydration by rat liver microsomes. The above results establish the existence of a liver microsomal epoxide hydrolase that is under different regulatory control and that appears to have a different substrate specificity than the well-characterized microsomal epoxide hydrolase involved in the metabolism of a widely diverse group of alkene and arene oxides.

Alkenes↗
PubMed1984

Immunochemical evidence for a role of cytochrome P-450 in liver microsomal ethanol oxidation.

Antibodies to cytochrome P-450 isozyme 3a, the ethanol-inducible isozyme in rabbit liver, were used to determine the role of this enzyme in the microsomal oxidation of alcohols and the p-hydroxylation of aniline. P-450 isozymes, 2, 3b, 3c, 4, and 6 did not crossreact with anti-3a IgG as judged by Ouchterlony double diffusion, and radioimmunoassays indicated a crossreactivity of less than 1%. Greater than 90% of the activity of purified form 3a toward aniline, ethanol, n-butanol, and n-pentanol was inhibited by the antibody in the reconstituted system. The catalytic activity of liver microsomes from control or ethanol-treated rabbits was unaffected by the addition of either desferrioxamine (up to 1.0 mM) or EDTA (0.1 mM), suggesting that reactions involving the production of hydroxyl radicals from H2O2 and any contaminating iron in the system did not make a significant contribution to the microsomal activity. The addition of anti-3a IgG to hepatic microsomes from ethanol-treated rabbits inhibited the metabolism of ethanol, n-butanol, n-pentanol, and aniline by about 75, 70, 80, and 60%, respectively, while the inhibition of the activity of microsomes from control animals was only about one-half as great. The rate of microsomal H2O2 formation was inhibited to a lesser extent than the formation of acetaldehyde, thus suggesting that the antibody was acting to prevent the direct oxidation of ethanol by form 3a. Under conditions where purified NADPH-cytochrome P-450 reductase-catalyzed substrate oxidations was minimal, the P-450 isozymes other than 3a had low but significant activity toward the four substrates examined. The residual activity at maximal concentrations of the antibody most likely represents the sum of the activities of P-450 isozymes other than 3a present in the microsomal preparations. The results thus indicate that the enhanced monooxygenase activity of liver microsomes from ethanol-treated animals represents catalysis by P-450 isozyme 3a.

1-Butanol↗
PubMed1984

Cytoplasmic location of linoleoyl-CoA desaturase in microsomal membranes of rat liver.

The intramembrane localization of linoleoyl-CoA desaturase in rat liver microsomes was examined by various methods, such as digestion by proteases, effect of detergents, and inhibition by the antibodies against purified terminal desaturase. Exposure of the desaturase on the surface of microsomal vesicles was suggested by the fact that the enzyme activity in the intact microsomes was susceptible to tryptic digestion, and considerably inhibited by anti-desaturase antibodies. When microsomes were previously treated with trypsin, the enzyme became more susceptible to the antibodies. Furthermore, it was demonstrated that the protein fragments cleaved from microsomal membranes by tryptic digestion formed a single precipitin line with the antibodies by the double-immunodiffusion test. These findings suggest the presence of linoleoyl-CoA desaturase on the cytoplasmic surface in the endoplasmic reticulum, since tryptic digestion liberates only the protein components situated on the surface area of membranes. In addition, desaturase activity in the intact microsomes was not stimulated by addition of the detergent, indicating the further outside location of the active site of the enzyme in microsomal vesicles. The pretreatment of microsomes with a low concentration (0.05%) of sodium deoxycholate, which destroys the permeability barrier for macromolecules without membrane disassembly, did not increase the susceptibility to tryptic digestion and the antibodies. These results show that linoleoyl-CoA desaturase is not present in a latent state in the membrane.

Animals↗
PubMed1988

The effectiveness of inhibitors of soluble prolyl hydroxylase against the enzyme in the cisternae of isolated bone microsomes.

Inhibitors of purified, soluble prolyl hydroxylase (K. Majamaa et al. (1984) Eur. J. Biochem. 138, 239-245; K. Majamaa et al. (1986) J. Biol. Chem. 261, 7819-7823) were tested against isolated chick embryo bone microsomes containing intracisternal prolyl hydroxylase and its radiolabeled, unhydroxylated procollagen substrate. Two groups of inhibitors were used which consisted of pyridine-2-carboxylate and 1,2-dihydroxybenzene (catechol) derivatives. The 2,4- and 2,5-pyridine dicarboxylic acids, which are potent inhibitors of the soluble enzyme (Ki values 2 and 0.8 microM, respectively), were effective in the same concentration range against intracisternal prolyl hydroxylase, although their relative affinities were reversed. Inhibition by pyridine-2,4-dicarboxylate in the microsomal system was reversed by increasing the concentration of 2-oxoglutarate. Pyridine-2,4-dicarboxylic acid did not inhibit the uptake of 2-[14C]oxoglutarate into microsomes, so it appears likely that the inhibitor must traverse the microsomal membrane and act directly at the enzyme level. Pyridine-2-carboxylic acid was ineffective in the microsomal system at 1 mM whereas it is a relatively potent inhibitor of the soluble enzyme with a Ki of 25 microM. This finding suggests that the second carboxyl group of the pyridine carboxylate derivatives may be required for their transport into the microsomal lumen. In the soluble system, 3,4-dihydroxybenzoic acid and 1,2-dihydroxybenzene had been found to be competitive inhibitors with relatively low Ki values of 5 and 25 microM, respectively. In the microsomal system, half-maximal inhibition was obtained at approximately 50-100 microM and inhibition was not reversed by increasing the concentrations of either 2-oxoglutarate or ascorbate, alone or together. These results imply that in situ these compounds do not inhibit prolyl hydroxylase directly. Thus, the microsomal system can assess the accessibility of the intracisternal enzyme to potential inhibitors and offers an insight into the in cellulo potential of such compounds.

Animals↗
PubMed1988

Identification of P-450ALC in microsomes from alcohol dehydrogenase-deficient deermice: contribution to ethanol elimination in vivo.

Isozyme 3a of rabbit hepatic cytochrome P-450, also termed P-450ALC, was previously isolated and characterized and was shown to be induced 3- to 5-fold by exposure to ethanol. In the present study, antibody against rabbit P-450ALC was used to identify a homologous protein in alcohol dehydrogenase-negative (ADH-) and -positive (ADH+) deermice, Peromyscus maniculatus. The antibody reacts with a single protein having an apparent molecular weight of 52,000 on immunoblots of hepatic microsomes from untreated and ethanol-treated deermice from both strains. The level of the homologous protein was about 2-fold greater in microsomes from naive ADH- than from naive ADH+ animals. Ethanol treatment induced the protein about 3-fold in the ADH+ strain and about 4-fold in the ADH- strain. The antibody to rabbit P-450ALC inhibited the microsomal metabolism of ethanol and aniline. The homologous protein, termed deermouse P-450ALC, catalyzed from 70 to 80% of the oxidation of ethanol and about 90% of the hydroxylation of aniline by microsomes from both strains after ethanol treatment. The antibody-inhibited portion of the microsomal activities, which are attributable to the P-450ALC homolog, increased about 3-fold upon ethanol treatment in the ADH+ strain and about 4-fold in the ADH- strain, in excellent agreement with the results from immunoblots. The total microsomal P-450 content and the rate of ethanol oxidation were induced 1.4-fold and 2.2-fold, respectively, by ethanol in the ADH+ strain and 1.9-fold and 3.3-fold, respectively, in the ADH- strain. Thus, the total microsomal P-450 content and ethanol oxidation underestimate the induction of the P-450ALC homolog in both strains. A comparison of the rates of microsomal ethanol oxidation in vitro with rates of ethanol elimination in vivo indicates that deermouse P-450ALC could account optimally for 3 and 8% of total ethanol elimination in naive ADH+ and ADH- strains, respectively. After chronic ethanol treatment, P-450ALC could account maximally for 8% of the total ethanol elimination in the ADH+ strain and 22% in the ADH- strain. Further, cytochrome P-450ALC appears to be responsible for about one-half of the increase in the rate of ethanol elimination in vivo after chronic treatment with ethanol. These results indicate that the contribution of P-450ALC to ethanol oxidation in the deermouse is relatively small. Desferrioxamine had no effect on rates of ethanol uptake by perfused livers from ADH-negative deermice, indicating that ethanol oxidation by a hydroxyl radical-mediated mechanism was not involved in ethanol metabolism in this mutant.(ABSTRACT TRUNCATED AT 400 WORDS)

Alcohol Dehydrogenase↗
PubMed1989

Topographic studies of microsomal and pure prostaglandin H synthase.

Prostaglandin H synthase catalyzes the first step in the conversion of polyunsaturated fatty acids to prostaglandins, thromboxanes, and prostacyclins. The enzyme is normally bound to the endoplasmic reticulum membrane, but can be purified to homogeneity after solubilization with detergent. The topologies of the microsomal and the pure detergent-solubilized forms of the synthase were compared by an examination of their sensitivity to degradation by proteases, of the effect of heme on this protease sensitivity, and of the sizes of proteolytic fragments produced. For the microsomal synthase, the localization of proteolytic fragments was also determined. Analysis of the microsomal proteins after proteolytic digests involved separation by polyacrylamide gel electrophoresis and selective detection of the synthase-derived polypeptides with a polyclonal antibody against the pure synthase. With both the microsomal and the pure synthase, incubation with trypsin led to a progressive loss of cyclooxygenase activity and cleavage of the synthase subunit (70K Da) into two fragments of 38K and 33K Da. Incubation of the detergent-solubilized form of the synthase with proteinase K and chymotrypsin also produced a very similar pair of fragments (38K and 33K Da). After incubation of the microsomes with trypsin both the 38K and 33K Da fragments from the synthase remained bound to the membrane; no cyclooxygenase activity was released in soluble form from the microsomes by trypsin. Further, neither trypsin nor proteinase K released soluble radiolabeled peptides from microsomes whose synthase had been labeled with [acetyl-14C]-aspirin. With the microsomal synthase the sensitivity to protease (66% of the cyclooxygenase activity was lost after 90 min incubation with proteinase K) was enhanced by depletion of heme (84% of activity lost) and was decreased by addition of heme (only 20% of activity lost), just as had been previously demonstrated for the detergent-solubilized synthase. At each of several intervals during an incubation of the pure synthase with trypsin the extent of cleavage of the synthase polypeptide correlated reasonably well with the extent of loss of cyclooxygenase activity; a similar relation between proteolytic cleavage and loss of activity was observed in digests of the pure synthase supplemented with differing amounts of heme.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Purification of two isozymes of rat liver microsomal cytochrome P450 with testosterone 7 alpha-hydroxylase activity.

Cytochrome P450a was purified to electrophoretic homogeneity from liver microsomes from immature male Long-Evans rats treated with Aroclor 1254. Rabbit polyclonal antibody raised against cytochrome P450a cross-reacted with cytochromes P450b, P450e, and P450f (which are structurally related to cytochrome P450a). The cross-reacting antibodies were removed by passing anti-P450a over an N-octylamino-Sepharose column containing these heterologous antigens. The immunoabsorbed antibody recognized only a single protein (i.e., cytochrome P450a) in liver microsomes from immature male rats treated with Aroclor 1254 (i.e., the microsomes from which cytochrome P450a was purified). However, the immunoabsorbed antibody recognized three proteins in liver microsomes from mature male rats, as determined by Western immunoblot. As expected, one of these proteins (Mr 48,000) corresponded to cytochrome P450a. The other two proteins did not correspond to cytochromes P450b, P450e, or P450f (as might be expected if the antibody were incompletely immunoabsorbed), nor did they correspond to cytochromes P450c, P450d, P450g, P450h, P450i, P450j, P450k, or P450p. One of these proteins was designated cytochrome P450m (Mr approximately 49,000), the other cytochrome P450n (Mr approximately 50,000). Like cytochrome P450a, cytochrome P450n was present in liver microsomes from both male and female rats. However, whereas cytochrome P450a was detectable in liver microsomes from 1-week-old rats, cytochrome P450n was barely detectable until the rats were at least 3 weeks old. Furthermore, in contrast to cytochrome P450a, the levels of cytochrome P450n did not decline appreciably with age in postpubertal male rats. Cytochrome P450m was detectable only in liver microsomes from postpubertal (greater than 4 week-old) male rats. Cytochromes P450m and P450n were isolated from liver microsomes from mature male rats and purified to remove cytochrome P450a. When reconstituted with NADPH-cytochrome P450 reductase and lipid, cytochrome P450n exhibited little testosterone hydroxylase activity, whereas cytochrome P450m catalyzed the 15 alpha-, 18-, 6 beta-, and 7 alpha-hydroxylations of testosterone at 10.8, 4.6, 2.0, and 1.9 nmol/nmol P450/min, respectively. The ability of cytochrome P450m to catalyze the 7 alpha-hydroxylation of testosterone was not due to contamination with cytochrome P450a, which catalyzed this reaction at approximately 25 nmol/nmol P450a/min. Cytochrome P450m also converted testosterone to several minor metabolites, including androstenedione and 15 beta-, 14 alpha-, and 16 alpha-hydroxytestosterone.(ABSTRACT TRUNCATED AT 400 WORDS)

Age Factors↗
PubMed1992

Stimulation of microsomal production of reactive oxygen intermediates by rifamycin SV: effect of ferric complexes and comparisons between NADPH and NADH.

Rifamycins are antibacterial antibiotics which are especially useful for the treatment of tuberculosis. Reactive oxygen intermediates are produced in the presence of rifamycin SV and metals such as copper or manganese. Experiments were carried out to evaluate the interaction of rifamycin SV with rat liver microsomes to catalyze the production of reactive oxygen species. At a concentration of 1 mM, rifamycin SV increased microsomal production of superoxide with NADPH as cofactor 3-fold, and with NADH as reductant by more than 5-fold. Rifamycin SV increased rates of H2O2 production by the microsomes twofold with NADPH, and 4- to 8-fold with NADH. In the presence of various iron complexes, microsomes generated hydroxyl radical-like (.OH) species. Rifamycin SV had no effect on NADPH-dependent microsomal .OH production, irrespective of the iron chelate. A striking stimulation of .OH production was found with NADH as the reductant, ranging from 2- to 4-fold with catalyst such as ferric-EDTA and ferric-DTPA to more than 10-fold with ferric-ATP, -citrate, or -histidine. Catalase and competitive .OH scavengers lowered rates of .OH production (chemical scavenger oxidation) and prevented the stimulation by rifamycin. Superoxide dismutase had no effect on the NADH-dependent rifamycin stimulation of .OH production with ferric-EDTA or -DTPA, but was inhibitory with the other ferric complexes. In contrast to the stimulatory effects on production of O2-., H2O2, and .OH, rifamycin SV was a potent inhibitor of microsomal lipid peroxidation. These results show that rifamycin SV stimulates microsomal production of reactive oxygen intermediates, and in contrast to results with other redox cycling agents, is especially effective with NADH as the microsomal reductant. These interactions may contribute to the hepatotoxicity associated with use of rifamycin, and, since alcohol metabolism increases NADH availability, play a role in the elevated toxic actions of rifamycin plus alcohol.

Animals↗
PubMed1995

Arachidonic acid metabolism by human cytochrome P450s 2C8, 2C9, 2E1, and 1A2: regioselective oxygenation and evidence for a role for CYP2C enzymes in arachidonic acid epoxygenation in human liver microsomes.

The membrane-bound endogenous fatty acid arachidonic acid can be released from membranes by phospholipases and then metabolized to biologically active compounds by cyclooxygenases, lipoxygenases, and cytochrome P450 (CYP) enzymes. In the liver the CYP pathway is the most significant. Liver CYP arachidonate products include epoxyeicosatrienoic acids (EETs) and monohydroxylated products (HETEs). We examined metabolism of [1-14C]arachidonic acid by a panel of 10 human CYP enzymes expressed in HepG2 cells. In the absence of expressed CYP enzymes, control HepG2 cell microsomes generated only small amounts of omega- and omega--1-OH arachidonic acid (ratio 2:1). Microsomes from HepG2 cells expressing CYP2C8, 2C9, 1A2, and 2E1 were 7-21 times more active than microsomes from the HepG2 controls. CYP2C8, 2C9, and 1A2 principally generated epoxygenase products; 36 to 48% were in the form of EET-diols, reflecting host HepG2 microsomal epoxide hydrolase activity. CYP2C8 and 2C9 formed more 14,15- and 11,12-EET than did CYP1A2, while CYP1A2 formed more 8,9-EET. CYP2C9 also generated a peak with the retention time of 12-HETE. CYP2E1 generated omega--1-OH arachidonic acid and, to a lesser extent, omega-OH arachidonic acid (ratio 2:1). A small amount of epoxygenase activity was also detected for CYP2B6; its overall activity, however, was only about twice control levels. Activities of CYP2A6, 3A3, 3A4, and 3A5 were low and limited to the omega-/omega--1-OH arachidonic acid peak; CYP2D6 was inactive. Microsomes prepared from three individual human livers varied threefold in total arachidonic acid metabolism. For all three livers omega-OH arachidonic acid was the major product (up to 74% of total metabolites). Epoxygenase products constituted 14 to 28% of the total products; 60 to 83% of those were EET-diols, indicating that the human liver microsomes have substantial EET-epoxide hydrolase activity. 11,12-EET was the major EET for two livers and 14,15-EET for the third. The CYP2C inhibitor sulfaphenazole depressed human liver microsomal epoxygenase activity by 50% at 50 microM, while alpha-naphthoflavone inhibited arachidonic acid epoxygenase activity by 27% at 2 microM and by 32% at 10 microM. Collectively, these findings suggest that human liver microsomal arachidonic acid metabolism is catalyzed principally by CYP2C enzymes. CYP1A2, CYP2E1, and possibly CYP2B6 are likely to play more minor roles, though their contribution may be enhanced by exposure to inducers of those enzymes. CYP2A6, CYP2D6, and CYP3A enzymes are unlikely to make any significant contribution.(ABSTRACT TRUNCATED AT 400 WORDS)

Arachidonic Acid↗
PubMed1982

Intramembranous arrangement of the glycosylating systems in rough and smooth microsomes from rat liver.

The distribution of mannosyl-, glucosaminyl- and glucosyltransferases in rough and smooth microsomes isolated from rat liver homogenate has been investigated. Amphomycin and tunicamycin were used as inhibitors of dolichol-mediated glycosylation, and diazobenzene sulfonate and proteolytic enzymes were used as nonpenetrating surface probes. Under in vitro conditions only 20-30% of the proteins glycosylated are of the secretory type. Nonpenetrating surface probes, which interact with components on the outer surface of rough microsomal vesicles, decrease glycosylation of both secretory and membrane proteins to a great extent. Inhibitor sensitive glycosylation is present in both the outer and inner compartments of the microsomal membranes. In contrast, the surface probes and the inhibitors of dolichol-mediated glycosylation do not significantly affect protein glycosylation in smooth microsomes. When dolichol phosphate sugars were used as substrates, instead of nucleotide sugars, the probes used inhibited protein glycosylation in both subfractions. Glycosylation of externally added Lipidex-bound dolichol monophosphate and of ovalbumin were in agreement with the above results. It appears that both rough and smooth microsomes may possess several types of glycosylating pathways. The most prominent of these in rough microsomes under the conditions used is the dolichol mono- and pyrophosphate-mediated glycosylation of endogenous proteins, where the enzymes involved in the initial steps are distributed at the outer surfaces of the microsomal vesicles. The dominating pathway in smooth microsomes appears to function in completion of the oligosaccharide chain of the protein and this process does not involve lipid intermediates and cannot be influenced by nonpenetrating surface probes.

Animals↗
PubMed1983

A study on the topological distribution of phospholipids in microsomal membranes of chick brain using phospholipase C and trinitrobenzenesulfonic acid.

The transbilayer distribution of phospholipids in chicken brain microsomal membranes has been investigated using trinitrobenzenesulfonic acid and phospholipase C from Clostridium welchii. The exposure of intact microsomes to trinitrobenzenesulfonic acid showed that the labelling of aminophospholipids followed biphasic kinetics, indicating that these membranes contain a fast- and a slow-reacting pool of aminophospholipids. Use of microsomes radioiodinated on their surface led to the conclusion that the fast-reacting pool may be located on the outer leaflet of the microsomal vesicles. It contains about 35% of the phosphatidylethanolamine, 29% of the ethanolamine plasmalogens and 18% of the phosphatidylserine. The treatment of intact microsomes with the phospholipase C Cl. welchii produced the hydrolysis of 50% of the phospholipids without any loss of their permeability properties, indicating that they are not permeable to the hydrolase. Phospholipids extracted from the microsomes were hydrolyzed rapidly by the phospholipase C with the exception of phosphatidylserine and phosphatidylinositol. In intact microsomes about 90% of phosphatidylcholine, 32% of ethanolamine phospholipids and 60% of sphingomyelin were accessible to the phospholipase. These results suggest that the phospholipids have an asymmetric distribution in chicken brain microsomes, the external leaflet containing about 75% of the choline phospholipids and 25% of the aminophospholipids, whereas an opposite distribution is observed in the inner leaflet.

Animals↗
PubMed1979

Studies on the transverse localization of lysophospholipase in bovine liver microsomes using proteolytic enzymes.

1. Sonication of bovine liver microsomes completely solubilized the membrane-bound lysophospholipase II (EC 3.1.1.5). Co-chromatography with purified 125I-labelled lysophospholipase indicated that the enzyme was solubilized from microsomes in a lipid-free state. 2. In the presence of residual microsomal membranes, the solubilized lysophospholipase could only be partly degraded by trypsin (EC 3.4.21.4). Therefore, trypsin could not be used to study the transmembrane disposition of lysophospholipase in intact microsomes. 3. Chymotrypsin (EC 3.4.21.1) destroyed the solubilized lysophospholipase activity, even in the presence of residual microsomal membranes. 4. Lysophospholipase in intact microsomal vesicles was resistant to chymotrypsin digestion. 5. When microsomal vesicles were made leaky with lysophosphatidylcholine, chymotrypsin destroyed more than 95% of the lysophospholipase activity. 6. It is concluded from these experiments that at least the active center of lysophospholipase is located at the luminal side of the bovine liver microsomal membrane.

Animals↗