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PubMed1977

Metabolism of benzo(a)pyrene by guinea pig pancreatic microsomes.

The in vitro microsomal metabolism of the strain 13 guinea pig pancreas was investigated by determining the benzo(a)pyrene (BP) hydroxylase activity in the 9000 x g supernatant and microsomal pellet. BP hydroxylase activity in both 9000 x g supernatant and microsomal pellet of the pancreas was less than 1% of the activity in the respective liver fractions, However, pretreatment of animals with methylcholanthrene or BP at 20 mg/kg, for either 1 day or 3 consecutive days, markedly enhanced the BP hydroxylase activity of pancreatic microsomes over that of controls; the induction in the liver microsomes was less than 2-fold over that of controls. The hydroxylation of BP by pancreatic microsomes was linear with time over a 30-min period, with the rate of hydroxylation dependent on both the enzyme and substrate concentrations.

Animals↗
PubMed2001

Change of oligosaccharides of rat brain microsomes depending on dietary fatty acids and learning task.

We have analyzed oligosaccharide chains in brain microsomes of rats fed an n-3 polyunsaturated fatty acid-deficient (safflower oil group; S group) or -rich (perilla oil group; P group) diet before and after brightness-discrimination learning tasks. The amount of concanavalin A-binding sites (mainly mannoside) of the brain microsomes was found to be significantly less in the S group than the P group before the learning task. Detailed analysis of glycoprotein glycans demonstrated that high mannose type oligosaccharides were dominant in brain microsomes before the learning task in both dietary groups, whereas multiantennary complex-type oligosaccharides became dominant after the learning task and especially a tetra-antennary glycan, that had a core structure of the glycan of neural cell adhesion molecule, was more increased in the S-group than the P group. When polysialylated glycans were analyzed on serotonin-conjugated HPLC column, the glycans in the S-group microsomes before the learning task contained larger amount of higher affinity-polysialylated glycans to serotonin column than those in the P-group, and also contained larger amount of phosphoglycans that showed also high affinity to serotonin column than the P-group. Removal of mannoside from microsomes by alpha-mannosidase-treatment changed the membrane surface physical property, especially permittivity, as revealed by analysis of the interaction with 1-anilinonaphthalene-8-sulfonate. These results suggest that high mannose content and several multiantennary glycans including polysialylated and phospho-glycans were changed by dietary n-3 fatty acid deficiency and learning task in rat brain microsomal glycoproteins and that these changes may affect membrane functions through changes of membrane surface physical properties and reactivity against serotonin.

Animals↗
PubMed2002

Evaluation of Supermix as an in vitro model of human liver microsomal drug metabolism.

SUPERMIX is a commercially available formulation of insect cell-expressed human drug-metabolizing cytochrome P450 (CYP) isoforms, mixed in proportions that are optimized to parallel their relative activities in human liver microsomes. We have evaluated the apparent functional affinity and capacity of individual CYP isoforms in SUPERMIX in comparison with microsomes from a panel of 12 human livers, using enzyme kinetic studies of isoform-selective index reactions. In addition, we have measured the concentration of NADPH cytochrome P450 oxidoreductase (OR) in SUPERMIX and compared it with the concentrations of this accessory electron transfer protein in human liver microsomes. No important differences were evident in the catalytic activities of CYPs 1A2, 2C8, 2C9, 2C19, 2D6 and 3A4 between SUPERMIX and human liver microsomes. However, SUPERMIX lacks CYP2B6 activity and did not hydroxylate the antidepressant bupropion, a clinically relevant substrate of this enzyme. In addition, the concentration of OR in SUPERMIX (1198 pmol mg protein(-1)) is 17-fold higher than the mean value in human liver microsomes (70 pmol mg protein(-1)). In conclusion, SUPERMIX lacks CYP2B6 activity and contains supraphysiological concentrations of the accessory electron transfer protein OR. These factors should be considered when this formulation is used as an in vitro model in human liver microsomal drug metabolism studies.

Algorithms↗
PubMed1990

Enantioselective hydrolysis of oxazepam 3-acetate by esterases in human and rat liver microsomes and rat brain S9 fraction.

Rates of hydrolysis of racemic and enantiomeric oxazepam 3-acetates (OXA) by esterases in human and rat liver microsomes and rat brain S9 fraction were compared. When rac-OXA was the substrate, esterases in human and rat liver microsomes were highly enantioselective toward (R)-OXA. In contrast, esterases in rat brain S9 fraction were highly enantioselective toward (S)-OXA. Hydrolysis rates of rac-OXA were highly dependent on the amount of esterases used. At 0.05 mg protein equivalent of esterases and 150 nmol of rac-OXA per ml of incubation mixture, the (R)-OXA was hydrolyzed 3.6-fold and 18.5-fold faster than (S)-OXA by rat and human liver microsomes, respectively. The specific activities (nmol of OXA hydrolyzed/mg microsomal protein/min) of liver microsomes in the hydrolysis of enantiomerically pure (R)-OXA were approximately 120 (rat) and 1,980 (human), and in the hydrolysis of enantiomerically pure (S)-OXA were 4 (rat) and 7 (human), respectively. In the incubation of rac-OXA with rat brain S9 fraction, (S)-OXA was hydrolyzed approximately 6-fold faster than (R)-OXA. Results also indicated an enantiomeric interaction in the hydrolysis of rac-OXA by esterases in rat and human liver microsomes; the presence of (R)-OXA stimulated the hydrolysis of (S)-OXA, whereas the presence of (S)-OXA inhibited the hydrolysis of (R)-OXA. In rat brain S9 fraction, the presence of (R)-OXA inhibited the hydrolysis of (S)-OXA, whereas the presence of (S)-OXA appeared to have stimulated the hydrolysis of (R)-OXA.

Animals↗
PubMed1995

Capillary electrophoresis of rat liver microsomes in polymer solutions.

Rat liver microsome components were separated by capillary zone electrophoresis in buffers containing substituted agarose, agarose crosslinked polyacrylamide, polyacrylamide, polyethylene glycol and dextran of various molecular weights. The best resolution of the components was obtained with polymers of 10-21 nm geometric mean radius. Both the crude and the purified preparations of microsomes exhibit a single major peak. In a Tris-borate-EDTA (TBE) buffer, containing polyacrylamide of 5 x 10(6) molecular weight, it has a retardation coefficient, KR, of 0.77 +/- 0.02. Translation of the KR value to geometric mean radii, R, on the basis of the standard curve applicable to polymer solutions, KR vs. R, with polystyrene carboxylate size standards in both dextran and polyacrylamide solution allows one to estimate a value of R as 13-16 nm for the major microsome component. The value is smaller than expected from electron microscopic measurements (100-250 nm), possibly due to the chemical and geometric differences between microsomes and the polystyrene particles used as size standards. The crude preparation also contains a minor component which is smaller and less charged than the major component. Another component, apparently very much larger than the major one, is seen in TBE buffer but not in a potassium-N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) buffer and is therefore thought to be an artifact of interaction with borate. After a short incubation under conditions promoting delayed microsome fusion, i.e. in presence of GTP and Mg++ and in the absence of polyethylene glycol, the electrophoretic pattern changes dramatically: it now exhibits five unretarded, highly mobile and, therefore, presumably large components in addition to the two original retarded components of the microsome and a less highly charged species similar in KR to the smaller of the original two components.

Acrylic Resins↗
PubMedDate not supplied

Immunofluorescent determination of the lobular distribution of a constitutive form of hepatic microsomal cytochrome P-450.

We have used an immunohistochemical approach to study the lobular distribution of constitutive liver microsomal cytochrome P-450. Cytochrome P-450 isolated (10.3 nmoles per mg protein) from hepatic microsomes from untreated, mature male Sprague-Dawley rats was used to produce antisera in rabbits. The IgG fraction produced single precipitin lines of identity with liver microsomes after double immunodiffusion, precipitated 80% of the total microsomal cytochrome P-450 and inhibited three cytochrome P-450-dependent enzyme activities. By these criteria, the IgG appeared to be specific for a constitutive form (or immunochemically related family) of liver microsomal cytochrome P-450. The pattern of fluorescence after indirect immunofluorescent labeling of liver sections depended on the route of tissue preparation and the concentration of primary antibody. In frozen sections, the labeling was uniform throughout the lobule, whereas in "antigen-depleted" paraffin-embedded sections it was heaviest in the centrilobular and midzonal regions. Increasing the concentration of primary antibody to 500 micrograms per ml inhibited the centrilobular labeling in frozen sections in a concentration-dependent manner. When specific isozymes of cytochrome P-450 were induced with phenobarbital or 3-methylcholanthrene, the constitutive cytochrome P-450 was localized predominantly in the periportal region. Decreases in cytochrome P-450 in rats treated with carbon tetrachloride or 1,2-dibromo-3-chloropropane were associated with antigen loss only in necrotic cells. Regional differences in the loss of antigen in paraffin sections and the inhibition of fluorescence in frozen sections establish that the lobular distribution of constitutive hepatic microsomal cytochrome P-450 is qualitatively heterogeneous and may be altered during hepatocellular responses to chemical treatment.

Animals↗
PubMed1986

Vitamin-K-dependent proteins in microsomes of primary Lewis lung tumors.

Microsomes isolated from Lewis lung (LL) primary tumors raised in C57BL/6 mice have been shown to (i) contain a 4-hydroxycoumarin (warfarin)-sensitive cycle of vitamin K metabolism which is at least qualitatively similar to that of liver, and (ii) catalyze the incorporation of NaH14 CO3 into endogenous protein in a vitamin-K hydroquinone-dependent reaction to produce gamma-carboxyglutamate. As in liver microsomes, LL microsomal reduction of vitamin K 2,3-epoxide to vitamin K was greatly enhanced by exogenous dithiols such as dithiothreitol, but under identical conditions the former was 10-fold faster. The R(+) and S(-) warfarin enantiomers were highly and equally effective inhibitors of both the liver and tumor vitamin K 2,3-epoxide reductases-the average I50 against the tumor enzyme was 0.25 microM. Partially purified reductases isolated by centrifugation of sodium-cholate-treated liver and LL tumor microsomes over a discontinuous sucrose gradient were also inhibited by the sulfhydryl reagent N-ethylmaleimide following their reduction by dithiothreitol. Like the activity of the epoxide reductase, that of the gamma-carboxylase was much lower in tumor than in liver microsomes and was only detectable in microsomes isolated from tumor-bearing mice previously administered S(-) warfarin. In view of the reported inhibition of LL tumor metastasis by warfarin and diet-induced vitamin-K deficiency, vitamin-K-dependent proteins may play a role in the spread and/or subsequent growth of LL cells.

Animals↗
PubMed1992

Interaction of gangliosides with the methyltransferase-I of microsomes from normal and neoplastic human mammary gland.

The activity of the phospholipid methyltransferase-I, which catalyzes the rapid transmethylation of phosphatidylethanolamine to phosphatidylcholine, was found to be about 6-fold enhanced in microsomal membranes of breast cancer with respect to the level found in normal human mammary gland. Exogenous gangliosides GM1 and GM2 added to neoplastic breast microsomes induced progressive inhibition of the methyltransferase activity. In contrast, in microsomal membranes of non-neoplastic breast tissue treated with these gangliosides, the methyltransferase activity was markedly increased. The addition of cholesterol to these microsomes led to complete inhibition of the GM1-stimulated enzyme activity. The methyltransferase activity was not affected by GM3 alone in either type of tissue. Experiments carried out on non-neoplastic microsomes revealed that the phospholipid methyltransferase-I was affected by that portion of gangliosides which remained stable associated to microsomal membranes.

Breast↗
PubMed1989

Halothane-induced hepatic microsomal lipid peroxidation in guinea pigs and rats.

Halothane-induced hepatic microsomal lipid peroxidation in guinea pigs and rats was examined with respect to the mixed function oxidase system, anaerobic dehalogenation activity of halothane, and the antioxidant system. The levels of cytochrome P-450 and NADPH-cytochrome P-450 reductase were significantly higher in guinea pigs than in rats. There was no difference between the two animals in anaerobic dehalogenation activity of halothane per cytochrome P-450 in microsomes. Microsomal alpha-tocopherol was significantly lower in guinea pigs than in rats, and was increased by multiple exposure to halothane in guinea pigs but remained lower than in rats. Microsomal alpha-tocopherol was decreased in rats by multiple exposure. The concentration of reduced glutathione and ascorbic acid was decreased significantly by multiple exposure to halothane in guinea pigs but not in rats. These results suggest that the higher level of halothane-induced hepatic microsomal lipid peroxidation in guinea pigs is due to the large production of radical metabolites resulting from the large amounts of cytochrome P-450, the high activity of NADPH-cytochrome P-450 reductase, and the low concentration of microsomal alpha-tocopherol.

Animals↗
PubMed2001

Purification and characterization of rat hepatic microsomal low molecular weight fatty acid ethyl ester synthase and its relationship to carboxylesterases.

We reported purification of a high molecular weight (HMW) (ca. 180 kD) and a low molecular weight (LMW) (ca. 60 kD) protein fractions from digitonized rat liver microsomes using ammonium sulfate precipitation followed by ion exchange and gel filtration column chromatography. Both fractions expressed fatty acid ethyl ester (FAEE) synthase as well as p-nitrophenyl acetate (PNPA)-hydrolyzing (esterase) activities. The HMW fraction was found to be a trimer with subunit molecular weight ca. 60 kD and structurally and functionally similar to rat hepatic microsomal carboxylesterase (CE, pI 6.1) and adipose tissue FAEE synthase. In this article, we report further purification and characterization of the LMW (minor) fraction expressing FAEE synthase activity and its structural and functional relationship to hepatic microsomal CEs. Using isoelectric focusing (IEF) followed by gel filtration-high-performance liquid chromatography (GF-HPLC), five proteins were purified, which expressed FAEE synthase as well as PNPA-hydrolyzing activity. The isoelectric point values of 6.5, 5.8, 5.6, 5.3, and 5.0 were found for the purified LMW proteins by IEF and each showed a peak corresponding to ca. 60 kD molecular weight by GF-HPLC, which expressed FAEE synthase as well as PNPA-hydrolyzing activity. Sodium dodecyl sulfate-polyacrylamide gel elecrophoresis (SDS-PAGE) analysis of the GF-HPLC purified LMW proteins revealed that these proteins are monomers (ca. 60 kD). All the purified LMW proteins cross-reacted with antibodies to rat adipose tissue FAEE synthase. Coelution of PNPA-hydrolyzing and FAEE synthase activity at each step of purification and cross-reactivity with rat adipose tissue FAEE synthase antibodies suggest that the purified proteins are related to various hepatic microsomal CEs. This conclusion is further supported by the homology of N-terminal amino acid sequence of the purified LMW proteins to various hepatic microsomal CEs and protease precursors. Therefore, LMW FAEE synthase activity most probably is expressed by various isozymes of hepatic microsomal CEs, which are also involved in the biotransformation of xenobiotic alcohols and amines.

Acyltransferases↗
PubMed1990

Spectroscopic quantitation of cytochrome P-450 in human lung microsomes.

The cytochrome P-450 content of human lung microsomes was measured by difference spectroscopy of the carbon monoxide-complexed hemoprotein. These measurements were only possible after the microsome preparation had been subjected to centrifugation over a discontinuous sucrose gradient, to remove an opaque black contaminant. The specific concentration of total cytochrome P-450 in human lung microsomes is essentially identical to that of microsomes prepared under identical conditions from untreated baboon lungs, but is only 0.7% of the specific content found in lung microsomes from untreated rabbits. These measurements correspond well to the observed metabolic capacities of the various microsome samples.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1983

Role of cholesterol in the structure and function of gastric microsomal vesicles.

Digitonin was used as a tool to investigate the organization and function of cholesterol in gastric microsomes. Microsomal vesicles were treated with digitonin for different time at 0-4 degrees C under isotonic conditions. The effects of digitonin treatment of the vesicles on removal of cholesterol, ultrastructural changes, (H+ + K+)-ATPase activity, and gastric ATPase-dependent H+ uptake ability were investigated. Microsomal cholesterol was extracted in an exponential manner with a t1/2 of 32 min. There was no release of microsomal phospholipids by digitonin treatment during the same period. Digitonin treatment (30 min) produced visible "holes" in the vesicles; at the same time (H+ + K+)-ATPase-dependent H+ uptake was abolished. Under the same conditions the K+-stimulated ATPase activity, however, was moderately (about 35%) reduced, although the response of K+ stimulation to valinomycin was obliterated. Longer digitonin treatment resulted in gradual diffusion and eventual disappearance of the "holes" with the generation of distorted cup-shaped microsomes. The data strongly suggest that membrane lipids are freely mobile and that there is a certain degree of specialization in the organization of gastric microsomal cholesterol for the proper maintenance of the membrane structure and function.

Adenosine Triphosphatases↗
PubMed2005

Influence of protein binding on acrolein turnover in vitro by oxazaphosphorines and liver microsomes.

For a correct determination of acrolein amounts generated in in vitro turnover experiments with oxazaphosphorines, it is necessary to characterize the interaction of acrolein with liver microsomal proteins. Acrolein, a highly reactive metabolite of oxazaphosphorines, readily forms covalent adducts with proteins by electrophilic attack on nucleophiles, such as the sulfhydryl group of cysteine, imidazole group of histidine, and amino group of lysine. The current investigations were mainly directed toward determination of the degree of acrolein-protein binding under conditions of in vitro experiments with liver microsome preparations. The acrolein concentration in protein dilution was determined by a fluorescence method. Moreover, the influence of sucrose and glycerine on the extent of acrolein-protein binding commonly used for the stabilization of microsomal preparations during storage was investigated. The current investigations show evidence that the chemical reaction of acrolein with liver microsomal proteins strictly follows first order kinetics. The main part of the formed acrolein in the in vitro attempts is available as bound part. Results of these investigations indicate that the calibration should be carried out with mixtures from liver microsome preparations and known amounts of acrolein under the same conditions as the in vitro experiments to record the entirely formed acrolein part (free and bound) in oxazaphosphorine turnover experiments. Glycerine is recommended as a preservative to store liver microsomes instead of sucrose because the latter reacts with acrolein.

Acrolein↗
PubMed1993

Formation and reduction of ascorbate radicals by hog thyroid microsomes.

The formation of ascorbate radicals, identified by ESR experiments, was observed in the ascorbate peroxidase reaction by thyroid microsomes. The steady-state concentration of ascorbate radicals decreased in the presence of NADH. The oxidation of NADH was followed optically. Using the ascorbic acid oxidase system, NADH-dependent electron transport in thyroid microsomes was examined. Ascorbate radicals competed with bound cytochrome b5 for the reaction with reduced NADH-cytochrome b5 reductase. The NADH-ascorbate radical reductase activity of thyroid microsomes was calculated to be 0.17 mumol/mg.s at 3.3 microM ascorbate radicals. Kinetic results show that the properties of NADH-cytochrome b5 reductase in thyroid microsomes were similar to those of the enzyme in liver microsomes. The formation of ascorbate radicals by thyroid microsomes was stimulated by the addition of thyroxine, and the stimulation was decreased also by NADH. The thyroxine-mediated oxidation of ascorbate is explained in terms of consecutive one-electron transfers initiated by bound thyroid peroxidase. These results, along with those described in our previous paper (M. Nakamura, I. Yamazaki, and S. Ohtaki, 1990, J. Biochem. 108, 804-810), support the idea that ascorbate protects thyroid hormones from oxidative degradation through the NADH-cytochrome b5 reductase system.

Animals↗
PubMed1995

The oxidation of ebselen metabolites to thiol oxidants catalyzed by liver microsomes and perfused rat liver.

The oxidation of 2-(methylseleno)benzanilide and 2-selenylbenzanilide, metabolites of the antioxidant drug ebselen, was examined in reactions catalyzed by rat, pig, and guinea pig liver microsomes and in perfused rat liver. Microsomes from all three species catalyzed NADPH- and oxygen-dependent oxidation of the selenide and the selenol to thiol-reactive metabolites. The oxidation product of the selenide was similar in properties to the chemically synthesized selenoxide [2-(methylseleninyl)benzanilide]. The selenoxide oxidized GSH and thiocholine at rate constants of 1.2 x 10(2) and 7.2 x 10(2) M-1 s-1, respectively at pH 7.4, 37 degrees C. n-Octylamine stimulated the oxidation of the ring-opened metabolites of ebselen catalyzed by pig and guinea pig liver microsomes but it had essentially no effect on these activities in rat liver microsomes. The selenoxidase activity of microsomes from all three species was partially (30-50%) sensitive to N-benzylimidazole. The effects of n-octylamine and the imidazole suggest that the oxidation of the selenide was catalyzed primarily by enzymes with the properties of flavin-containing and P450-dependent monooxygenases, but the nature of enzymes responsible for a small fraction of the N-benzylimidazole-sensitive activity was not fully resolved. The 2-(methylseleno)benzanilide oxidase activity of pig liver microsomes sensitive to N-benzylimidazole was only partially sensitive to antisera to pig liver NADPH-cytochrome P450 reductase. While neither 2-(methylseleno)benzanilide nor ebselen affected bile flow, the biliary efflux of GSSG was stimulated about fourfold in rat liver perfused with either of these selenium compounds. The increased GSSG efflux produced by 5 microM ebselen or its methyl metabolite was abolished by N-benzylimidazole.

Anilides↗
PubMed1996

Substrate influence on interaction between cytochrome P450 and cytochrome b5 in microsomes.

We have been able to demonstrate that cytochrome b4 interacts closely with cytochrome P450 in the microsomal membrane, and that substrates can serve to order the interaction: Using the water-soluble carbodiimide EDC we could crosslink cytochrome b5 and CYP2B4 in microsomes from phenobarbital-treated rabbits and cytochrome b5 and CYP1A2 in microsomes from beta-naphthoflavone-treated animals. The substrate benzphetamine increased the specific interaction between cytochrome b5 and CYP2B4, decreasing the formation of higher molecular weight oligomeric complexes with cytochrome b5. In contrast, no ordering of the interactions were obtained on addition of 7-ethoxycoumarin, a substrate of CYP1A2, or of benzphetamine to microsomes of beta-naphthoflavone-treated animals in the presence of EDC. Of interest, although evidence could be shown for complementary charge-pairing between cytochrome b5 and a number of other microsomal proteins in the membranes, and while the extent of CYP1A2 and CYP2B4 interaction with cytochrome b5 each exceeded 30% in the presence of substrate, no significant complexation of the P450s was obtained with any other microsomal proteins.

Animals↗
PubMed1996

Endogenous 7-oxocholesterol is an enzymatic product: characterization of 7 alpha-hydroxycholesterol dehydrogenase activity of hamster liver microsomes.

Previously, we described a new metabolite derived from endogenous cholesterol in the presence of hamster liver microsomal protein and NADPH (Song et al., 1991, Biochem. Pharmacol. 41, 1439-1447). Through gas chromatography/mass spectral analysis of the metabolite and its methoxime-3-dimethyl-t-butylsilyl ether derivative, this metabolite has been definitively identified as 7-oxocholesterol. Isotope incorporation experiments using molecular 18O2 demonstrated that no oxygen atoms from molecular oxygen were incorporated into the product, 7-oxocholesterol, when 7 alpha-hydroxycholesterol was used as substrate. In contrast, one atom of 18O was incorporated into cholesterol from 18O2 during its metabolism to form 7 alpha-hydroxycholesterol. Formation of 7-oxocholesterol was dependent upon the presence of NADP+, 7 alpha-hydroxycholesterol, and hamster liver microsomes. This enzyme appears to be a membrane-bound protein and its activity was most abundant in liver microsomal fractions and to a lesser extent in mitochondrial fractions; little or no activity was observed in nuclei or cytosol. The enzyme activity was present in highest content in the livers of hamsters and was also observed in human and bovine liver microsomes, but not those of mouse, rabbit, or rat. The reaction was inhibited by 2'-AMP, but not by anti-NADPH:cytochrome-P450 oxidoreductase globulin, carbon monoxide, metyrapone, nor miconazole. In contrast to the previously characterized 3 beta-hydroxy-delta 5-C27-steroid oxidoreductase activity, NAD+ did not serve as an effective cofactor for 7-oxocholesterol formation. The ability of NADPH to partially serve as a cofactor in this reaction was shown to be due to a high NADPH-oxidase activity of hamster liver microsomes, thereby providing sufficient NADP+ to serve as the oxidizing pyridine nucleotide for the reaction. These results document the existence of a non-P450, NADP(+)-dependent 7 alpha-hydroxycholesterol dehydrogenase in liver microsomes which catalyzes this reaction. The product, 7-oxocholesterol, is produced enzymatically in the livers of hamsters and other mammals and may regulate bile acid metabolism or other processes due to its action as an oxysterol.

Animals↗
PubMed1996

Identification of CYP4A11 as the major lauric acid omega-hydroxylase in human liver microsomes.

Human liver microsomes are capable of oxidizing lauric acid (laurate), a model medium-chain fatty acid, at both the omega- and omega-1 positions to form 12- and 11-hydroxylaurate, respectively. These laurate hydroxylation reactions are apparently catalyzed by distinct P450 enzymes. While the P450 responsible for microsomal laurate omega-1 hydroxylation in human liver has been identified as CYP2E1, the enzyme catalyzing omega-hydroxylation remains poorly defined. To that end, we employed conventional purification and immunochemical techniques to characterize the major hepatic laurate omega-hydroxylase in humans. Western blotting with rat CYP4A1 antibodies was used to monitor a cross-reactive P450 protein (M(r) = 52 kDa) during its isolation from human liver microsomes. The purified enzyme (7.4 nmol P450/mg protein) had an NH2-terminal amino acid sequence identical to that predicted from the human CYP4A11 cDNA over the first 20 residues found. Upon reconstitution with P450 reductase and cytochrome b5, CYP4A11 proved to be a potent laurate omega-hydroxylase, exhibiting a turnover rate of 45.7 nmol 12-hydroxylaurate formed/min/nmol P450 (12-fold greater than intact microsomes), while catalyzing the omega-1 hydroxylation reaction at much lower rates (5.4 nmol 11-hydroxylaurate formed/min/nmol P450). Analysis of the laurate omega-hydroxylation reaction in human liver microsomes revealed kinetic parameters (a lone Km of 48.9 microM with a VMAX of 3.72 nmol 12-hydroxylaurate formed/min/nmol P450) consistent with catalysis by CYP4A11. In fact, incubation of human liver microsomes with antibodies raised to CYP4A11 resulted in nearly 85% inhibition of laurate omega-hydroxylase activity while omega-1 hydroxylase activity remained unaffected. Furthermore, a strong correlation (r = 0.89; P < 0.001) was found between immunochemically determined CYP4A11 content and laurate omega-hydroxylase activity in liver samples from 11 different subjects. From the foregoing, it appears that CYP4A11 is the principle laurate omega-hydroxylating enzyme expressed in human liver.

Animals↗