Characterization of sheep liver and lung microsomal ethylmorphine N-demethylase

Ethylmorphine N-demethylase activity of the sheep liver and lung microsomes was reconstituted in the presence of solubilized microsomal cytochrome P-450, NADPH-cytochrome c reductase and synthetic lipid, phosphatidylcholine dilauroyl. The Km of the lung microsomal ethylmorphine N-demethylase was calculated to be 4.84 mM ethylmorphine from its Lineweaver-Burk graph and lung enzyme was inhibited by its substrate, ethylmorphine, when its concn was 25 mM and above, reaching to 67% inhibition at 50 mM concn. The Lineweaver-Burk and Eadie-Hofstee plots of the liver enzyme were found to be curvilinear. From these graphs, two different Km values were calculated for the liver enzyme as 4.17 mM and 0.40 mM ethylmorphine. Ethylmorphine N-demethylase activities of both liver and lung microsomes were inhibited by NiCl2, CdCl2 and ZnSO4. Ethylalcohol inhibited N-demethylation of ethylmorphine in lung and liver microsomes. Acetone (5%) slightly enhanced the N-demethylase activity of the liver enzyme, whereas 5% acetone completely inhibited the lung enzyme. Phenylmethylsulfonyl fluoride at 0.10 mM and 0.25 mM concn had no effect on liver enzyme activity, while at these concns, it inhibited the activity of the lung enzyme by about 35%.     

Differential response of cadmium toxicity towards Mg2(+)-ATPase activity in hepatic and renal nuclear membrane in rats

Treatment with cadmium chloride (40mg/kg body wt/day) for three days led to a marked inhibition of Mg2(+)-ATPase activity in rat liver nuclear membrane, whereas it stimulated the enzyme in renal nuclear membrane. On 30 days treatment (15 mg/kg body wt/day) the effect was totally different i.e. stimulation of enzyme activity in the liver and inhibition in the kidney tissue. Arrhenius plot analysis of the enzyme activity showed significant increase in phase transition temperature only in liver tissue of rats subjected to acute treatment. Lineweaver Burk plots also showed differential effect of cadmium toxicity on the enzyme activity, i.e. while both Km and Vmax were changed in the liver, there was change only in Km of enzyme from kidney.     

Determination of aniline and metabolites produced in vitro by liquid chromatography/electrochemistry

A method utilizing reverse-phase liquid chromatography/electrochemistry (LC/EC) was developed for the simultaneous determination of aniline and its hydroxylated derivatives, p-aminophenol, o-aminophenol, m-aminophenol, and N-phenylhydroxylamine. To achieve separation of these compounds, a mobile phase of 3.0% dimethylformamide and 97.0% 0.05 M piperazine acetate, pH 5.4, containing 0.05 M KNO3 was developed. A procedure is also presented for the determination of p-nitrophenol, nitrobenzene, and nitrosobenzene, possible aniline metabolites in higher N-oxidation states, using reductive amperometric detection. The hydroxylated compounds, including the hydroxylamine, and nitrosobenzene are easily detected as metabolites of aniline in mouse liver slice or microsomal preparations. No prior extraction, preconcentration, or derivatization steps are needed for the determinations, which can be accomplished by a direct injection of the incubation mixture. The Km value for the hepatic aniline 4-hydroxylase activity in male Cox-Swiss mice microsomal preparations has been determined to be 0.52 mM; the Vmax value is 2.90 +/- 0.64 nmol min-1 mg microsomal protein-1. Detection limits for all compounds of interest are in the picomole range.     

Effect of cadmium on pulmonary and renal microsomal drug metabolizing enzymes of the guinea-pig

1. The effect of acute cadmium (Cd) treatment on pulmonary and renal microsomal aniline 4-hydroxylase and ethylmorphine N-demethylase enzyme activities of adult male guinea-pigs were assessed 72 hr following a single dose of Cd ion (2 mg Cd2+/kg i.p.). Tissue and microsomal Cd levels were also determined. 2. There were no significant differences between either lung or kidney tissue weights, microsomal protein contents or enzyme activities of Cd treated and control animals. 3. The tissues and microsomes of Cd-treated animals were found to have significantly higher levels of Cd than those of control animals. In Cd treated animals, tissue and microsomal Cd levels of kidney were found to be higher than that of lung. 4. In vitro addition of cadmium chloride (CdCl2) to incubation mixtures produced concentration related inhibitions of microsomal drug metabolizing enzymes in each tissue. However, in vitro effect of CdCl2 was found to be stronger on drug metabolizing enzymes of kidney than those of lung. In addition, while the strength of Cd effect was more pronounced on the activity of ethylmorphine N-demethylase than that of aniline 4-hydroxylase in the lung, the opposite was observed in the kidney.     

Comparison of highly purified sheep liver and lung NADPH-cytochrome P-450 reductases by the analysis of kinetic and catalytic properties

1. Reductase was purified to electrophoretic homogeneity from sheep liver and lung microsomes. The specific activity of both enzymes ranged from 55 to 66 mumol cytochrome c reduced/min/mg protein. 2. Liver and lung reductases appeared to have similar kinetic and spectral properties. Km (NADPH) and Km (cytochrome c) values were calculated to be 14.3 +/- 1.23 microM and 22.2 +/- 2.78 microM for liver and 11.1 +/- 0.70 microM and 20.0 +/- 2.15 microM for lung reductase, respectively. Kinetic studies showed that cytochrome c can bind the oxidized form of the enzyme as well as its reduced form and both reductases operated through a ping-pong type mechanism. 3. These reductases cannot be distinguished on the basis of monomer molecular weights (Mr 78,000) except that the liver reductase was found to be more susceptible to proteolytic attack. 4. Both reductases supported aniline 4-hydroxylation and ethylmorphine N-demethylation reactions to the same extent in the reconstituted systems. However, sheep lung reductase appeared only 36.5 and 14.8% as effective in catalyzing benzo[a]pyrene reaction as an equivalent amount of reductase from liver in the presence of liver cytochrome P-450 and 3MC-treated rat liver cytochrome P-448, respectively.     

Effect of divalent metal ions on the microsomal aniline hydroxylase system of rainbow trout

The ability of trout to metabolize aniline in vitro in the presence of some divalent metal ions was investigated in the liver microsomes of rainbow trout, Salmo gairdneri. Trout liver microsomes were highly capable of catalyzing aniline hydroxylation to p-aminophenol with a specific activity of 0.068 nmoles/min per mg of microsomal protein in potassium phosphate buffer, pH 7.4 at 25°C. The activity of the aniline hydroxylase system was competitively inhibited by Hg⁺², Ni⁺², Cd⁺², and Zn⁺², while Cu⁺² and Fe⁺³ seemed to inhibit the activity noncompetitively at 1 mM aniline concentrations. IC₅₀ values at fixed aniline concentration were estimated to be 0.45 mM for Hg⁺², Ni⁺², and Cd⁺², 1.8 mM for Zn⁺² and Fe⁺³, and 1.3 mM for Cu⁺². Eadie-Hofstee plots gave identical V_max values of approximately 0.046 nmol/min per mg of protein while K_m values were increased in the presence of Hg⁺², Ni⁺², CD⁺², and Zn⁺², indicating competitive inhibition. Both K_m and V_max values were affected by Fe⁺³ and Cu⁺², suggesting noncompetitive inhibition. K_i values extracted from the Dixon plots were determined t be 0.23, 0.43, and 0.65 mM for Hg⁺², Ni⁺², and Cd⁺², respectively, providing the most effective inhibition on the aniline hydroxylase system among studied metal ions. The K_i values were much higher in the presence of others. The results indicate a selective inhibition of the aniline hydroxylase system of trout liver microsomes by divalent metal ions. © 1997 John Wiley & Sons, Inc.     

Lung microsomal p-nitrophenol hydroxylase -- characterization and reconstitution of its activity

1. Formation of catechols from benzene and nitrobenzene have been implicated in the carcinogenic activity of these chemicals. In liver, p-nitrophenol, an intermediate of p-nitrobenzene is enzymatically converted to 4-nitrocatechol. 2. For the first time in this study, the presence of a highly active enzyme catalyzing the formation of 4-nitrocatechol from p-nitrophenol was detected in lung microsomes. The average specific activity of lung p-nitrophenol hydroxylase was found to be 0.494 nmol 4-nitrocatechol formed mg prot-1 min-1. 3. The optimum conditions for sheep lung microsomal p-nitrophenol hydroxylase were established. The maximal activity was noted at pH 6.8. The rate of p-nitrophenol hydroxylation was linear up to 2 mg prot/ml of incubation mixture. The maximal rate of 4-nitrocatechol formation was observed with 0.25 mM p-nitrophenol. 4. The Lineweaver-Burk and Eadie-Hofstee plots were found to be curve-linear. Two different Km values were calculated as 11.6 and 71.4 microM from the Lineweaver-Burk plot and as 10.7 and 74.5 microM from the Eadie-Hofstee plot. This suggested that there were either two forms of enzyme or two different enzymes participating in ortho hydroxylation of p-nitrophenol in lung microsomes. 5. Lung microsomal p-nitrophenol hydroxylase activity of sheep was reconstituted in the presence of purified lung microsomal cytochrome P-450, NADPH dependent cytochrome P-450 reductase and synthetic lipid, phosphatidylcholine dilauroyl.     

In vivo effects of 3-methylcholanthrene, phenobarbital, pyrethrum and 2,4,5-T isooctylester on liver, lung and kidney microsomal mixed-function oxidase system of guinea-pig: a comparative study

The optimum conditions (pH, microsomal protein amount and substrate concentration) of guinea-pig liver, lung and kidney microsomal aniline 4-hydroxylase, ethylmorphine N-demethylase and benzo[a]pyrene hydroxylase activities were determined. Male guinea-pigs weighing 500-700 g were administered 3-methylcholanthrene (25 mg/kg, i.p. 3 days), phenobarbital (75 mg/kg, i.p. 3 days), pyrethrum (120 mg/kg, i.p. 2 days) and 2,4,5-T isooctylester (200 mg/kg, i.p. 3 days). 3-Methylcholanthrene treatment caused significant increases in liver microsomal benzo[a]pyrene hydroxylase and kidney microsomal aniline 4-hydroxylase activities. However, with phenobarbital treatment the only significant increase was observed in liver microsomal ethylmorphine N-demethylase activity. Pyrethrum treatment decreased kidney microsomal ethylmorphine N-demethylase activity significantly. 2,4,5-T isooctylester treatment increased liver microsomal aniline 4-hydroxylase and lung microsomal ethylmorphine N-demethylase activities significantly. Liver microsomal NADPH-cytochrome c reductase activity was increased significantly by phenobarbital and pyrethrum treatment. The other treatments did not cause any significant changes in microsomal NADPH-cytochrome c reductase activities of liver, lung and kidney. Cytochrome P-450 content of guinea-pig liver microsomes were increased significantly about 2.5-fold and 2-fold by treatment with 3-methylcholanthrene and phenobarbital, respectively. 3-Methylcholanthrene also caused 1 nm spectral shift in the absorption maxima of CO difference spectrum of the dithionite-reduced liver microsomal cytochrome P-450, forming P-449.     

Properties of diacylglycerol kinase in adult and fetal rat lung

Diacylglycerol kinase activity is found in both adult and fetal lung. Approximately 27 and 52% of the total activity is found in microsomes and cytosol, respectively. The activity is maximal at pH 7.4. The apparent Km for ATP is 0.11 mM and 0.21 mM for cytosol and microsomes, respectively. The apparent Km for dioleoylglycerol is 0.05 mM for cytosol and 0.14 for microsomes. Maximal activity in cytosol and microsomes is obtained with 2.0 mM dexoycholate. Other detergents cannot substitute for deoxycholate. Phosphatidylglycerol stimulates activity in the absence and in the presence of deoxycholate. Phosphatidylserine also stimulates activity, whereas phosphatidylethanolamine was inactive and phosphatidylcholine inhibited the reaction. Linoleic acid produced inhibition. The general properties of the enzyme were similar for fetal and adult lung. Diacylglycerol kinase from microsomes and cytosol fraction from both fetal and adult lung was most active with dioleoylglycerol and diacylglycerol from egg phosphatidylcholine. Significantly lower activity was obtained with dipalmitoylglycerol. Phosphatidylglycerol did not alter the relative substrate preferences. The activity in microsomes increased with development from 19 days gestation to a maximal activity at 21 days gestation. Maximal activity was about 2-fold higher than the adult. The activity dropped rapidly reaching adult values prior to birth (22 days gestation). The activity in cytosol fractions increased gradually from 19 days gestation, reaching adult values by 22 days gestation.     

Protein disulfide-isomerase retains procollagen prolyl 4-hydroxylase structure in its native conformation

Protein disulfide-isomerase was isolated as a homogeneous protein from 15-day-old chick embryos. The enzyme has a molecular weight of 56,000 in SDS-polyacrylamide gel electrophoresis. Its Km value for randomly cross-linked ribonuclease, a protein used as a substrate for the enzyme, was 0.3 microM, and the Km value for DTT was 1.0 microM. Its optimum pH was 7.5 and its optimum temperature, 33 degrees C. The maximal velocity of pure protein disulfide-isomerase from chick embryos under optimal conditions was about 29,000 units/g. Protein disulfide-isomerase was able to activate purified prolyl 4-hydroxylase 2- to 3-fold, the activation being higher for enzyme stored for a longer time. This activation is probably due to the repairing of disulfide exchanges occurring in the prolyl 4-hydroxylase structure during purification and storage. Prolyl 4-hydroxylase activity was very stable in microsomes, however, and protein disulfide-isomerase was unable to increase the microsomal prolyl 4-hydroxylase activity, suggesting that prolyl 4-hydroxylase retains its native conformation in microsomes. Protein disulfide-isomerase was able to reactivate prolyl 4-hydroxylase inactivated by mild H2O2 treatment. The activity obtained after this treatment and protein disulfide-isomerase incubation corresponded to the amount of prolyl 4-hydroxylase tetramer found after H2O2 treatment. The data suggest that protein disulfide-isomerase is able to activate only the tetramer part of the enzyme preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

A convenient assay for mephenytoin 4-hydroxylase activity of human liver microsomal cytochrome P-450

A simple and rapid method for the determination of (S)-mephenytoin 4-hydroxylase activity by human liver microsomal cytochrome P-450 has been developed. [Methyl-14C] mephenytoin was synthesized by alkylation of S-nirvanol with 14CH3I and used as a substrate. After incubation of [methyl-14C]mephenytoin with human liver microsomes or a reconstituted monooxygenase system containing partially purified human liver cytochrome P-450, the 4-hydroxylated metabolite of mephenytoin was separated by thin-layer chromatography and quantified. The formation of the metabolite depended on the incubation time, substrate concentration, and cytochrome P-450 concentration and was found to be optimal at pH 7.4. The Km and Vmax rates obtained with a human liver microsomal preparation were 0.1 mM and 0.23 nmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450, respectively. The hydroxylation activity showed absolute requirements for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH in a reconstituted monooxygenase system. Activities varied from 5.6 to 156 pmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450 in 11 human liver microsomal preparations. The basic system utilized for the analysis of mephenytoin 4-hydroxylation can also be applied to the estimation of other enzyme activities in which phenol formation occurs.     

Characterization of the forward and reverse reactions catalyzed by CDP-diacylglycerol:inositol transferase in rabbit lung tissue.

CDPdiacylglycerol:inositol transferase activity in rabbit lung tissue has been characterized and the optimum conditions for assaying this enzyme in vitro were determined. Rabbit lung tissue CDPdiacylglycerol:inositol transferase activity was found primarily in the microsomal fraction. The pH optimum of the enzyme activity was between 8.8 and 9.4, and the reaction was dependent on either Mn2+ or Mg2+. Detergents and Ca2+ inhibited the activity of the enzyme. The apparent Km values of the enzyme for CDPdioleoylglycerol and myoinositol were 0.18 mM and 0.10 mM, respectively. The reversibility of the reaction catalyzed by CDPdiacylglycerol:inositol transferase in microsomes prepared from rabbit lung tissue was demonstrated by the synthesis of [3H]CMPdiacylglycerol when [3H]CMP and phosphatidylinositol were present in the incubation mixture. The reverse reaction was characterized and its importance in the regulation of the acidic phospholipid composition of surfactant during lung development is discussed. The pH optimum for the reverse reaction was 6.2, and the reverse reaction was also dependent on Mn2+ or Mg2+. The apparent Km value of CDPdiacylglycerol:inositol transferase for CMP was found to be 2.8 mM.     

Comparative studies of sheep lung and liver nitrofurantoin reductase

1. Nitrofurantoin reductase which catalyzes the bioactivation of nitrofurantoin was purified to electrophoretic homogenity from sheep liver and lung microsomes, with a yield of 15% and 35%, respectively. The specific activity of both reductases was found to be similar (140 nmol/min/mg protein).2. The effects of nitrofurantoin and NADPH concentrations, pH, ionic strength, amount of enzyme and reaction period, on the enzyme activity were studied and the optimum conditions for maximum activity of purified liver and lung nitrofurantoin reductases were determined.3. The enzyme concentration was found proportional with the square root of the rate of nitrofurantoin reduction up to approximately 15 μg protein/ml and 25 μg protein/ml incubation mixture for liver and lung nitrofurantoin reductases, respectively.4. The plots of inverse of the nitrofurantoin concentration against the inverse of the square root of the velocity for the reduction of nitrofurantoin by liver and lung enzymes gave K_m values as 27.78 μM and 32.25 μM, respectively.5. The purified liver and lung enzymes were also saturated by NADPH at similar concentrations and the K_m values were calculated as 29.4 μM and 35.5 μM, respectively.6. The effects of magnesium, nickel, cadmium and copper ions on the nitrofurantoin reductase activity were examined. Magnesium ion was found to have almost no effect, whereas the other ions inhibited the activity of both liver and lung reductases.     

Accleration of autooxidation of human oxyhemoglobin by aniline and its relation to hemoglobin-catalyzed aniline hydroxylation.

Changes in the ultraviolet/visible spectrum of human oxyferrohemoglobin upon addition of aniline were indicative of a concentration-dependent interaction of aniline with hemoglobin, resulting in accelerated autooxidation of the hemoprotein. Oxygen was found to markedly inhibit this interaction of aniline with oxyhemoglobin. The dependence of the rate of autooxidation on aniline concentration followed saturation kinetics and showed a half-maximal response at 8 mM aniline. This value is equal to the value of Km for aniline as substrate for the O2-dependent, hemoglobin-catalyzed hydroxylation reaction which yields p-aminophenol (Mieyal, J. J., Ackerman, R.S., Blumer, J.L., and Freeman, L.S. (1976) J. Biol. Chem. 241, 3436-3441). Thus, an aniline-oxyhemoglobin complex is implicated in the overall catalytic reaction. No detectable p-aminophenol was formed when aniline was combined with oxyhemoglobin in the absence of an electron donor, but hydroxylation of aniline does occur when NADPH, NADPH plus P-450 reductase, or Na2S2O4 are also added.     

荔枝果皮过氧化酶的纯化与性质研究

荔枝果皮采后褐变是影响这一重要热带水果经济价值的主要问题,酚类物质的酶促氧化一直被认为是造成植物组织褐变的关键因素,其中多酚氧化酶被研究得最多.过氧化物酶在植物体中分布很广,能够氧化多种底物,在荔枝果皮中的含量也很高.非结合性过氧化物酶已经被证明在果实的采后成熟与老化过程中参与多种过程.在这项研究中,用磷酸缓冲液提取荔枝果皮的非结合性过氧化物酶,并通过硫酸铵沉淀,DEAFSephadex A-50离子交换柱层析以及Sephadex G-100凝胶过滤进行纯化.对得到的酶溶液进行了酶学性质的研究,发现荔枝果皮过氧化物酶具有较高的热稳定性和高的最适反应pH值(6.8),能够氧化许多底物尤其是单酚和各种多酚类物质,反应抑制剂专一性与其他植物来源的过氧化物酶略有不同显示了过氧化物酶参与荔枝果皮褐变过程的可能性,并为提高荔枝采后贮藏性提供了新的思路.     

Identification of human cytochrome P450 isoforms involved in the 7-hydroxylation of chlorpromazine by human liver microsomes

Studies to identify the cytochrome P450 (CYP) isoform(s) involved in chlorpromazine 7-hydroxylation were performed using human liver microsomes and cDNA-expressed human CYPs. The kinetics of chlorpromazine 7-hydroxylation in human liver microsomes showed a simple Michaelis-Menten behavior. The apparent Km and Vmax values were 3.4+/-1.0 microM and 200.5+/-83.7 pmol/min/mg, respectively. The chlorpromazine 7-hydroxylase activity in human liver microsomes showed good correlations with desipramine 2-hydroxylase activity (r = 0.763, p < 0.05), a marker activity for CYP2D6, and phenacetin O-deethylase activity (r = 0.638, p < 0.05), a marker activity for CYP1A2. Quinidine (an inhibitor of CYP2D6) completely inhibited while alpha-naphthoflavone (an inhibitor of CYP1A2) marginally inhibited the chlorpromazine 7-hydroxylase activity in a human liver microsomal sample showing high CYP2D6 activity. On the other hand, alpha-naphthoflavone inhibited the chlorpromazine 7-hydroxylase activity to 55-65% of control in a human liver microsomal sample showing low CYP2D6 activity. Among eleven cDNA-expressed CYPs studied, CYP2D6 and CYP1A2 exhibited significant activity for the chlorpromazine 7-hydroxylation. The Km values for the chlorpromazine 7-hydroxylation of both cDNA-expressed CYP2D6 and CYP1A2 were in agreement with the Km values of human liver microsomes. These results suggest that chlorpromazine 7-hydroxylation is catalyzed mainly by CYP2D6 and partially by CYP1A2.     

Estrogen-2/4-hydroxylase activities in rat brain and liver microsomes exhibit different substrate preferences and sensitivities to inhibition

NADPH-dependent estrogen-2/4-hydroxylase activities in rat brain and liver microsomes were compared with respect to the utilization of different estrogens as substrates and the inhibitory effects of alpha-naphthoflavone, metyrapone and steroids. Of 6 different estrogens used as substrates, only 17 beta- and 17 alpha-estradiol were transformed relatively effectively by brain microsomes. In contrast liver microsomes utilized these two estrogens as well as ethynyl estradiol, estrone and diethylstilbestrol effectively. Estriol was a poor substrate for estrogen-2/4-hydroxylase activity in both tissues. With 40 microM 17 beta-estradiol as substrate the estrogen-2/4-hydroxylase activities in brain and liver were inhibited by alpha-naphthoflavone, metyrapone, progesterone, 17 alpha-hydroxyprogesterone and testosterone. The brain enzyme activity appeared to be more sensitive than the liver enzyme to inhibition by alpha-naphthoflavone and metyrapone. Testosterone propionate (50-100 microM) stimulated the brain enzyme activity significantly. Progesterone and 17 alpha-hydroxyprogesterone were the most effective steroidal inhibitors of brain estrogen-2/4-hydroxylase activity. In the liver the inhibitory potencies of 3 different steroids varied, depending on the estrogen used as substrate. With 17 beta-estradiol, for example, progesterone was the most potent steroidal inhibitor, while corticosterone was the most potent inhibitor when diethylstilbestrol was used as substrate. These findings indicate that rat liver microsomes can utilize a wider range of different estrogens for catecholestrogen formation than brain microsomes and suggest that the profiles of catecholestrogen-forming P-450 isozymes in the two organs differ.     

Purification and characterization of a microsomal cytochrome P-450 with high activity of coumarin 7-hydroxylase from mouse liver

Phenobarbital-induced coumarin 7-hydroxylase is high in DBA/2J and low in C57BL/6N inbred mice; this genetic difference is encoded by the Coh locus on chromosome 7. The aim of this study was to develop an antibody specific for this cytochrome P-450 polymorphism. P-450 fractions, highly specific for phenobarbital-inducible coumarin 7-hydroxylase activity, were purified from DBA/2J and C57BL/6N mouse liver microsomes. Both proteins are 49 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Soret peaks of the reduced cytochrome . CO complexes are 451 nm. Reconstituted DBA/2J coumarin 7-hydroxylase activity exhibits a V twice as high as, and a Km value 10-fold less than, the reconstituted C57BL/6N activity. Antibodies were raised in rabbit. By Ouchterlony immunodiffusion, both antibodies show 100% cross-reactivity with DBA/2J and C57BL/6N microsomes and purified antigens. Yet, DBA/2J but not C57BL/6N 7-hydroxylase activity is inhibited by the antibody to DBA/2J P-450. Both DBA/2J and C57BL/6N activities are blocked by the antibody to C57BL/6N P-450. Neither antibody has any effect on liver microsomal d-benzphetamine N-demethylase, ethylmorphine N-demethylase, aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase, acetanilide 4-hydroxylase, or aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity. The DBA/2J protein most specific for phenobarbital-induced coumarin 7-hydroxylation is designated 'P-450Coh'. Anti-(P-450Coh) precipitates a relatively minor 49-kDa protein from detergent-solubilized microsomes and from in vitro translation of poly(A+)-enriched total RNA of phenobarbital-treated DBA/2J mouse liver, whereas the major phenobarbital-induced P-450 proteins exhibit a molecular mass of about 51 kDa. The immunoprecipitated translation products correspond to a messenger RNA of 2100 +/- 100 nucleotides.     

Subcellular distribution and properties of aldehyde dehydrogenase in the rat liver

The subcellular distribution and certain properties of rat liver aldehyde dehydrogenase are investigated. The enzyme is shown to be localized in fractions of mitochondria and microsomes. Optimal conditions are chosen for detecting the aldehyde dehydrogenase activity in the mentioned fractions. The enzyme of mitochondrial fraction shows the activity at low (0,03-0.05 mM; isoenzyme I) and high (5 mM; isoenzyme II) concentrations of the substrate. The seeming Km and V of aldehyde dehydrogenase from fractions of mitochondria and microsomes of rat liver are calculated, the acetaldehyde and NAD+ reaction being used as a substrate.     

Sex-related difference in vitamin D3 25-hydroxylase of rat liver microsomes

Cholecalciferol 25-hydroxylase was partially purified by polyethylene glycol fractionation and chromatographies on octylamino-Sepharose and hydroxylapatite columns starting from the liver microsomes of female rats, and compared with P-450cc25 purified from the liver microsomes of male rats (Hayashi, et al. (1986) J. Biochem. 99, 1753-1763). On octylamino-Sepharose 4B column chromatography, most of the activity was recovered in the fraction eluted with 0.08% Emulgen 913 in the case of the male enzyme, whereas the female enzyme was recovered in the fraction eluted with 0.2% Emulgen. Anti-cc25 antibodies against purified male P-450cc25 inhibited the 25-hydroxylation activity of male polyethylene glycol (PEG) fraction and partially purified male enzyme, but did not inhibit the activities of the corresponding female fractions. The antibodies formed a single precipitation line with male P-450cc25, but did not form a precipitation line with partially purified female 25-hydroxylase on immuno-diffusion. These observations indicated that the vitamin D3 25-hydroxylase in female rat liver microsomes is a different entity from that of male rats.