NADPH-generating system: Influence on microsomal mono-oxygenase stability during incubation for the liver-microsomal assay with rat and mouse S9 fractions

Activity levels of 7-ethoxycoumarin O-deethylase (ED), aminopyrine N-demethylase (APD), p-nitroanisoleO-demethylase (p-NAD) and glucose-6-phosphate dehydrogenase (G-6-PDH) were determined in incubation mixtures for the liver-microsomal assay (LMA) at time 0 and after 1 and 2 h incubation under conditions for mutagenic assay. The experiments were performed with S9 liver fractions from mice (induced with Na-phenobarbital and β-naphthoflavone) and rats (induced with Aroclor 1254) with and without G-6-PDH in the incubation mixtures.

In the absence of G-6-PDH the activities were significantly lower at time 0 in the mouse. The pattern of stability, however, was similar for the activities, with an increase of stability after 1 and 2 h of pre-incubation (an exception for p-NAD).

Only ED activity showed a similar behaviour in the rat. No differences were present for APD and p-NAD activities at time 0 in the rat, but the enzyme stabilities were significantly decreased after 2 h of incubation (about 15% and 10% for APD and p-NAD respectively) in the absence of G-6-PDH.

At time 0, the amounts of G-6-PDH differed between mouse and rat fractions; however, during the incubations for LMA they decreased by about 57% and 53% for the two species, respectively. In addition to the above biochemical results, the presence of exogenous G-6-PDH in the incubations for the mutagenic assay, significantly increased the mitotic gene conversion and mitotic crossing-over of dimethylnitrosamine (DMN) and AR2MNFN (a nitroimidazo[2,1-b]thiazole) in the D₇ strain of Saccharomyces cerevisiae.     

Study of the optimal temperature for the liver microsomal assay with mice S9 fractions

The effect of temperature on enzymatic activity and stability was studied with respect to the monooxygenase activities of aminopyrine-N-demethylase (APD) and p-nitroanisole O-demethylase (pNAD) under incubation conditions for the liver microsomal assay. The activities of S9 liver fractions of mice induced with sodium phenobarbital and beta-naphthoflavone were determined during a period of preincubation in a range of temperatures from 30 to 44 degrees C. The greatest value of the mean specific activity was found at 40-42 degrees C for both APD and pNAD. The rapid increase of lipid peroxidation after 1 h of incubation at temperatures higher than 42 degrees C can provide an explanation of the enhancement of the rate of inactivation. In order to determine whether biological response is affected by the modifications induced by temperature in the metabolic activating system, tester strain D7 of Saccharomyces cerevisiae was used to assay the genetic activity of the well known premutagenic agent cyclophosphamide by incubating the mixtures both at the traditional temperature of 37 degrees C and at 42 degrees C. We suggest that the use of more favourable conditions for LMA with respect to enzymatic activity, than the traditional ones could improve the reliability and the sensitivity of such tests.     

Genetic and biochemical investigation on chloral hydrate in vitro and in vivo

Chloral hydrate (CH), a metabolite of trichloroethylene (TCE), was studied in vitro using the D7 diploid strain of Saccharomyces cerevisiae, with and without a mammalian microsomal activation system (S9 fraction), and in vivo by intrasanguineous host-mediated assay (HMA). The in vivo effects on the hepatic microsomal monooxygenase induced by CH in mice pretreated with beta-naphthoflavone (beta-NF) and Naphenobarbital (PB) were also investigated. Chloral hydrate induced a significant increase of mitotic gene conversion in D7 strain both in vivo and in vitro. The enzymatic determinations in mice showed a decrease in aminopyrine N-demethylase (APD) and p-nitroanisole O-demethylase (p-NAD) activities (about 37% and 29% respectively) after one acute dose of CH. Moreover, stability experiments, carried out in the conditions of the liver microsomal assay (LMA), showed an increase of residual activity, after 1 h of preincubation with respect to the control (about 22% and 9% for APD and p-NAD respectively).     

Stability of microsomal mono-oxygenase during incubations for the liver microsomal assay with S9 fractions of mouse liver under various inductions

Aminopyrine-N-demethylase and p-nitroanisole-O-demethylase activities were determined in incubation mixtures for the liver microsomal assay at time zero and after 1 h of incubation in the conditions for the mutagenic assay. The experiments were performed with the S9 liver fraction of mice in the basal state and induced with sodium phenobarbital, β-naphthoflavone or both. Lipid peroxidation was also determined.

The experiments were repeated with female mice and also in the presence of styrene, as an example of a xenobiotic substance. The activity of pNAD was much more stable than that of APD in all the conditions tested. The pattern of stability, however, was similar for the two activities: more stable than controls with S9 fractions from β-NF-induced mice, less stable than controls in PB-induced mice, intermediate between controls and PB-induced mice in those with combined induction by PB + βNF. Styrene 50 mM in the incubation mixtures led to a marked inactivation of enzymic activity, similar in all cases and reaching about 90% in 1 h. S9 fractions from female mice gave enzymes slightly more stable in almost all cases. Lipid peroxidation was appreciably more elevated in basal than in induced animals.

It was concluded that, for a mutagenesis test on an unknown xenobiotic, S9 fractions from mice following PB and β-NF induction are to be preferred from the point of view of activation.     

Studies on the incubation mixtures for the in vitro mutagenesis test with metabolic activation (microsomal assay). Behaviour of mixed function oxidase and lipid peroxidation in the presence of some xenobiotics.

The effect of some xenobiotics on microsomal mixed function oxidase and lipid peroxidation, in mice, in incubation mixtures for the in vitro mutagenesis test with metabolic activation was studied. Aniline 1 or 2 mM and aminopyrine 0.38 or 8.33 mM completely inhibited the lipid peroxidation with small protection of the monooxygenase. Styrene 50 or 100 mM inhibited to a lesser extent the lipid peroxidation with marked increase in the inactivation of the monooxygenase. By a technique based on successive additions of fresh microsomes it was possible to evaluate the part of the inactivation due to enzyme denaturation and that due to inhibition. EDTA 40 mM was not able to protect from inactivation in the presence of aniline 1mM. Data of this type could be utilized to obtain more reliable results of in vitro mutagenesis tests with metabolic activation by suitably managing the enzyme activity in the incubation mixtures in order to keep it as constant as possible.     

Regulation of triacylglycerol synthesis in the liver: a decrease in diacylglycerol acyltransferase activity after treatment of isolated rat hepatocytes with glucagon

Isolated rat hepatocytes were used to investigate the possibility of a short-term effect of glucagon on the synthesis of triacylglycerols in the liver. Incubation of hepatocytes in the presence of glucagon, followed by homogenization in a buffer containing F- (50 mM) and EDTA (2.5 mM), resulted in a 53% decrease in activity of microsomal diacylglycerol acyltransferase (EC 2.3.1.20), the only enzyme that is exclusively involved in the synthesis of triacylglycerols. The activity of cholinephosphotransferase (EC 2.7.8.2), which also uses diacylglycerols as substrate, was not decreased after exposure of the hepatocytes to glucagon. This may imply that triacylglycerol synthesis can be regulated independently of phosphatidylcholine synthesis. The activity of diacylglycerol acyltransferase in microsomes isolated from a homogenate of whole liver could be reduced by preincubating the microsomes with Mg2+ (5 mM), ATP (1 mM) and 105 000 X g supernatant. The enzyme could be reactivated by incubation of the washed microsomes with a 105 000 X g supernatant in the presence of dithiothreitol (5 mM). Fluoride (50 mM) inhibited this reactivation. It is concluded that the activity of diacylglycerol acyltransferase is subject to hormonal short-term control, possibly via a phosphorylation-dephosphorylation mechanism.     

Pulmonary phosphatidic acid phosphatase. Properties of membrane-bound phosphatidate-dependent phosphatidic acid phosphatase in rat lung.

1. The membrane-bound phosphatidate-dependent phosphatidic acid phosphatase activity of rat lung has been investigated in cytosol and microsomal fractions using as a substrate [32P]phosphatidate bound to heat inactivated rat liver microsomes. Both activities demonstrated broad pH optima with a maximum of 7.4--8 for the cytosol and a maximum of 6.5--7.5 with microsomal preparations. 2. At low concentrations (0--5 mM) Mg2+ produced a slight stimulation of the cytosol activity but at higher concentrations an inhibition was observed. Low concentrations (1.0--2.0 mM) of EDTA abolished the cytosol activity and reduced the microsomal activity to half. In both cases, the addition of Mg2+ in the presence of EDTA resulted in an activity which was more than 2-fold greater than that observed in the absence of chelator or divalent cation. 3. The cytosol activity was relatively resistant to the addition of ionic and nonionic detergents. In general, the addition of a number of phosphate esters increased rather than decreased the release of 32Pi, indicating a relative specificity for phosphate groups associated with a hydrophobic environment. The addition of aqueous dispersions of phosphatidate, lysophosphatidic acid or phosphatidylglycerophosphate markedly reduced the hydrolysis of membrane-bound [32P]phosphatidate. The cytosol activity was slightly inhibited by the addition of phosphatidylcholine. 4. In an attempt to estimate the relative contributions of the cytosol and microsomal activities in vivo, these activities were assayed using [32P]phosphatidate endogenously generated on rat lung microsomes. With the 32P-labelled microsomes, the hydrolysis remained linear over the 45 min of the experiment. Addition of high speed supernatant produced a rapid release of 32Pi during the first 10 min followed by a more gradual release similar to that oberved with the microsomes alone. The cytosol activity remained greater than the microsomal activity at all times studied. 5. When [14C]phosphatidate-labelled microsomes were incubated in the presence of nonradioactive CDPcholine, the addition of cytosol markedly stimulated the incorporation of radioactivity into phosphatidylcholine. This observation suggests that the phosphatidic acid phosphatase activity associated with the cytosol has a role in phosphatidylcholine (and presumably surfactant) biosynthesis in rat lung.     

A comparative study of the microsomal S6 phosphatase and phosphorylase phosphatase activities in rat liver.

Rat liver microsomes contain type-1 S6 phosphatase (acting on the serine residues phosphorylated by protein kinase A) and type-1 phosphorylase phosphatase activities. The main aim of this study has been to characterize the microsomal S6 phosphatase activity and to compare its properties with those of the phosphorylase phosphatase activity in the same microsomal preparation. The specific activities of both microsomal S6 phosphatase and phosphorylase phosphatase were 1.6- to 1.7-fold higher in the smooth endoplasmic reticulum than in the rough sarcoplasmic reticulum. Both phosphatase activities were inhibited to a similar extent by MgCl2 (10 mM) and NaF (22 mM), were completely suppressed by glycerophosphate (80 mM) and ZnCl2(10 mM), and were stimulated by MnCl2(1 mM). When analyzed by gel filtration on Sephadex G-100 superfine, both phosphatase activities eluted as broad peaks, stretching from the void volume to 45-60 kDa. The microsomal S6 phosphatase and phosphorylase phosphatase activities also displayed the following distinct characteristics: (a) Mn2+ stimulated the S6 phosphatase activity 2.9-fold more than the phosphorylase phosphatase activity, (b) limited trypsin digestion of microsomal preparations increased the phosphorylase phosphatase activity by 1.5- to 2-fold, but decreased the S6 phosphatase activity by 50%, (c) a synthetic peptide analog of S6 (S6229-239) (200 microM), which did not act as a substrate for the microsomal S6 phosphatase and did not affect its activity, inhibited the microsomal phosphorylase phosphatase activity by about 50%, and (d) the elution profile of the phosphorylase phosphatase activity was markedly broader than that of the S6 phosphatase activity. A series of in vivo studies showed that streptozotocin-diabetes and insulin replacement therapy as well as ip injection of insulin or vanadate, which modified the microsomal S6 phosphatase activity, had no statistically significant effects on the microsomal phosphorylase phosphatase activity. Taken together, these results suggest that the microsomal S6 phosphatase and phosphorylase phosphatase activities are due to two distinct enzyme populations.     

Synthesis, antifungal and antioxidant screening of some novel benzimidazole derivatives

Some novel benzimidazole derivatives were synthesized and their in vitro effects on rat liver microsomal NADPH-dependent lipid peroxidation (LP) level, ethoxyresorufin O-deethylase (EROD) and antifungal activities were determined. A significant decrease in male rat liver microsomal LP level was noted by compounds 4c (52%), 4e (58%) and 4h (43%) at 10(-3) M concentration. Compounds 4c (100.0%), 4h (100.0%), 5c (98.0%) and 5h (100.0%) inhibited the microsomal ethoxyresorufin O-deethylase (EROD) enzyme activity better than that of the specific inhibitor caffeine (85%). Among these compounds, only compounds 4b and 4h exhibited moderate activity against C. albicans whereas the others had weak effects.     

Biosynthesis of estradiol-17 beta fatty acyl esters by microsomes derived from bovine liver and adrenals

A fatty acyl coenzyme A:estradiol-17 beta acyl transferase activity has been detected in bovine hepatic and adrenocortical microsomes. It is thoroughly increased when adenosine triphosphate (5 mM) and coenzyme A (1 mM) are added to incubation buffer. Using a substrate concentration of 185 microM, the hepatic and adrenocortical microsomal activities have been found to be to 2.4 +/- 0.1 and 5.5 +/- 0.2 nmol/h/mg prot., respectively. Five major estradiol-17-esters have been isolated by reverse phase high performance liquid chromatography from both microsomal incubations, the fatty acid moieties being: arachidonate, linoleate, oleate, palmitate and stearate. However, the distribution of hepatic metabolites is quite different from that obtained with adrenocortical membranes, this is well explained by the corresponding differences between the endogenous contents of free fatty acids. With any of the two types of microsomal membranes used, the results show that estradiol is more susceptible to be esterified to polyunsaturated fatty acids than saturated ones. The possible physiological implications of such an activity in liver and adrenals are discussed.     

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%.     

In vivo and in vitro destruction of rat liver cytochrome P-450 by a monoterpene ketone, pulegone

In vivo administration of pulegone once daily decreased the levels of liver microsomal cyt. P-450 to the extent of 32 and 76% at the end of 24 and 96 hrs respectively. However, cyt. b5 and NAD(P)H-cyt. c reductase activities remained unchanged. In vitro incubation (15 min) of liver microsomes from phenobarbitol (PB)-treated rats with pulegone (10 mM), aerobically or anaerobically resulted in the loss (approximately 60%) of cyt. P-450 in the presence or absence of NADPH. Destruction of cyt. P-450 was more in PB-treated microsomes as compared to 3-methylcholanthrene (MC)-treated and control microsomes. The loss of cyt. P-450 was accompanied by a concomitant loss of microsomal heme. In contrast, menthone or carvone upon incubation with PB-induced microsomes resulted in the conversion (25-40%) of cyt. P-450 to cyt. P-420 without any loss of microsomal heme. The destructive process is irreversible, time dependent, linear upto a substrate concentration of 10 mM and follows first order kinetics.     

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.     

Cholesterol 7α-hydroxylase in rat liver microsomal preparations

Subcellular fractions containing microsomes prepared from rat livers homogenized in the absence of EDTA catalysed the oxidation of cholesterol to 7alpha-hydroxycholesterol, 7-oxocholesterol, 7beta-hydroxycholesterol and 5alpha-cholestane-3beta,5,6beta-triol. These reactions required native protein, molecular oxygen and NADPH. It is suggested that these compounds are formed by a peroxidation analogous to the peroxidation of fatty acids catalysed by liver microsomal preparations. Incubations of [4-(14)C]cholesterol with microsomal preparations from rat liver homogenized in the presence of EDTA gave 7alpha-hydroxy[(14)C]cholesterol as the main product. This reaction required molecular oxygen and NADPH, and was inhibited by CO. The mass of 7alpha-hydroxycholesterol formed during the incubation was measured by a double-isotope-derivative dilution procedure. This procedure was used to assay the activity of cholesterol 7alpha-hydroxylase and to measure low concentrations of endogenous 7alpha-hydroxycholesterol in liver.     

Thiol-protein disulphide oxidoreductases. Assay of microsomal membrane-bound glutathione-insulin transhydrogenase and comparison with protein disulphide-isomerase.

1. Inhibition of endogenous microsomal NADPH oxidase by CO enables membrane-bound glutathione-insulin transhydrogenase (EC 1.8.4.2) to be assayed conveniently by a linked assay involving NADPH and glutathione reductase (EC 1.6.4.2). 2. The specific activity of the enzyme in rat liver microsomal preparations is of the order of 1 nmol of oxidized glutathione formed/min per mg of membrane protein. 3. The specific activity of the enzyme is comparable in rough and smooth microsomal fractions, and the activity is not affected by treatment with EDTA and the removal of ribosomes from rough microsomal fractions. 4. Membrane-bound glutathione-insulin transhydrogenase is not affected by concentrations of deoxycholate up to 0.5%, whereas protein disulphide-isomerase (EC 5.3.4.1) is drastically inhibited. 5. On these grounds it is concluded that, in rat liver microsomal fractions, glutathione-insulin transhydrogenase and protein disulphide-isomerase activities are not both catalysed by a single enzyme species.     

Evidence for the presence of distinct flavin-containing monooxygenases in human tissues

Catalytic activities and substrate specificity of flavin-containing monooxygenase were examined in human tissues. During incubation with imipramine, human hepatic microsomes efficiently carried out cytochrome P450-dependent reactions but not the formation of N-oxide, while in kidney imipramine N-oxide was the only metabolite formed during in vitro incubation. The production of imipramine N-oxide was essentially due to flavin-containing monooxygenase as shown by thermal inactivation. In contrast, thiobenzamide and dimethylaniline were actively transformed by both human liver and kidney flavin-containing monooxygenase. Neither the modification of pH nor the solubilization of microsomal membranes increased imipramine N-oxidation in human liver. Kinetic analysis indicated a poor affinity (about 7 mM) of human liver microsomes for imipramine versus 0.3 mM in kidney. Immunological studies were undertaken to support enzymatic data. Antibodies raised against rat liver flavin-monooxygenase reacted strongly with human kidney microsomes but extremely weakly with liver microsomes. The relative amount of immunochemically determined protein correlated well with imipramine N-oxidation activity. A dose-dependent inhibition of imipramine N-oxidation by anti-flavin-monooxygenase antibodies was observed in human kidney, as well as in rat kidney and liver. Taken together, the results can be interpreted by the possible existence in human tissues of distinct flavin-containing monooxygenases exhibiting a partial overlapping substrate specificity. The protein involved in imipramine N-oxidation is missing from human liver but actively carries out the reaction in kidney, while another protein catalyzes the oxidation of thiobenzamide and dimethylaniline in both tissues.     

Characterization of tyrosylprotein sulfotransferase from rat liver and other tissues

The sulfoconjugation of tyrosyl residues is a widespread post-translational modification of biologically active peptides and proteins. In this paper we describe the characterization of a rat liver tyrosylprotein sulfotransferase that is capable of catalyzing the transfer of a sulfate moiety from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the synthetic polymer, poly-(Glu6,Ala3,Tyr1) (EAY; Mr 47,000) using a simple filter paper assay. Following sucrose density gradient centrifugation and comparison with known subcellular marker enzyme activities, rat liver tyrosylprotein sulfotransferase activity was shown to have a distribution similar to the Golgi enzyme, galactosyltransferase. Using the enriched Golgi preparation, rat liver tyrosylprotein sulfotransferase displayed a pH optimum of 6.7 and required the presence of 20 mM Mn2+ for maximal activity. Co2+ (20 mM) was able to produce 26% of the maximal stimulation observed with Mn2+, whereas other metal ions, such as Mg2+, Ca2+, and Co2+, were not effective in stimulating tyrosylprotein sulfotransferase activity. Whereas tyrosylprotein sulfotransferase activity was observed in the native membrane-bound state, EAY sulfation was maximally enhanced 3-fold when assayed in the presence of Lubrol Px. Under the optimal conditions for assaying the sulfation of EAY by a rat liver enriched Golgi fraction, significant degradation of the sulfate donor, PAPS, was observed. The addition of both NaF and 5'-AMP to the incubation mixture was found to effectively prevent PAPS degradation and increase the amount of product formed in the assay by 10-fold. Using the optimized conditions for the sulfation of EAY by rat liver tyrosylprotein sulfotransferase, membrane-bound sulfotransferase activity was also observed in the crude microsomal pellets of a variety of rat tissues, including lung, pituitary, and cerebellum, as well as in livers from different species.     

Acid and alkaline phosphatase activity in Trypanosoma cruzi epimastigotes

Phosphatase activity in intact Trypanosoma cruzi epimastigotes has been demonstrated. After subcellular fractionation three activities were characterized: (a) a membrane-bound microsomal acid activity with an optimum pH of 4.0 and a Km of 1.2 mM, strongly inhibited by tartrate and fluoride; (b) a soluble cytosolic acid activity with an optimum pH of 5.5 and a Km of 0.95 mM, strongly inhibited by p-hydroxymercuribenzoate, EDTA and copper ions and activated by cyanide, manganese and magnesium ions; and (c) a soluble cytosolic alkaline activity with an optimum pH of 8.0 and a Km of 3.8 mM, inhibited by p-hydroxymercuribenzoate, fluoride, EDTA, and copper, calcium and zinc ions. This activity was increased by magnesium and manganese ions.     

Galactose-specific receptors on liver cells. I. Hepatocyte and liver macrophage receptors differ in their membrane anchorage

Colloidal gold particles coated with asialoglycoproteins are bound by hepatocytes as well as by liver macrophages. Binding by both cell types is inhibited by N-acetylgalactosamine and related saccharides and is dependent on the presence of Ca2+. We have now performed an electron microscopic study on receptor anchorage in the plasma membranes. Cells with prebound ligand were treated with 20 mM EDTA at 4 degrees C, washed free of chelator and tested for residual galactose-specific receptor activity. Whereas hepatocytes preserve binding activity (73% of untreated control), liver macrophages lose galactose-specific receptor activity (12% of untreated control). Liver macrophages regain binding activity after a 2 min incubation at 37 degrees C allowing for receptor recycling. If the macrophages were fixed with low glutaraldehyde concentration prior to EDTA treatment they fully retained their receptor activity (74% of control). Ligands were also removed from both cell types by incubation with 80 mM N-acetylgalactosamine. After washing the cells free of the competing monosaccharide, both the hepatocytes as well as the macrophages show full binding activity (120% and 85% of untreated controls). Therefore, membrane anchorage sites of the macrophage receptors are not identical to ligand-binding sites. These results suggest a Ca2+-Mg2+-dependent receptor anchorage on the macrophage plasma membrane. As shown in the accompanying paper (Roos, P.H., Hartmann, H.J., Schlepper-Sch?fer, J., Kolb, H. and Kolb-Bachofen, V. (1985) Biochim. Biophys. Acta 847, 115-121), EDTA-induced dissociation from the membrane can be used for isolation of the galactose-specific receptors of liver macrophages.     

Methionine S-oxidation in human and rabbit liver microsomes: evidence for a high-affinity methionine S-oxidase activity that is distinct from flavin-containing monooxygenase 3.

Methionine has previously been shown to be S-oxidized by flavin-containing monooxygenase (FMO) forms 1, 2, and 3. The most efficient catalyst was FMO3, which has a Km value for methionine S-oxidation of approximately 4 mM, and exhibits high selectivity for formation of the d-diastereoisomer of methionine sulfoxide. The current studies provide evidence for an additional methionine S-oxidase activity in liver microsomes. Human and rabbit liver microsomes exhibited a biphasic response to methionine at concentrations ranging from 0.05 to 10 mM, as indicated by both Eadie-Hofstee plots and nonlinear regression. The low-affinity component of the biphasic response had Km values of approximately 3 and 5 mM for humans and rabbits, respectively, as well as high diastereoselectivity for methionine sulfoxide formation. The low-affinity activity in rabbit liver microsomes was inhibited by methimazole, S-allyl-l-cysteine, and by mild heat treatment, suggesting the activity is FMO3. The high-affinity component of the biphasic response had Km values of approximately 0.07 and 0.04 mM for humans and rabbits, respectively, as well as lower diastereoselectivity for methionine sulfoxide formation. Further characterization of the high-affinity activity in rabbit liver microsomes indicated lack of involvement of cytochrome P450s or reactive oxygen species. The high-affinity activity was inhibited 25% by potassium cyanide and greater than 50% by methimazole and S-allyl-l-cysteine. Mild heat treatment produced 85% inhibition of the low-affinity activity, but only 30% inhibition of the high-affinity activity. Both high- and low-affinity activities were decreased by 85% in flavin-depleted microsomes. Because these results suggested the additional S-oxidase activity has characteristics of an FMO, recombinant human FMO4 was evaluated as a potential catalyst of this activity. Recombinant FMO4 catalyzed S-oxidation of both methionine and S-allyl-l-cysteine, with similar diastereoselectivity to the high-affinity microsomal S-oxidase; however, the Km values for both reactions appeared to be greater than 10 mM. In summary, these studies provide evidence for two microsomal methionine S-oxidase activities. FMO3 is the predominant catalyst at millimolar concentrations of methionine. However, at micromolar methionine concentrations, there is an additional S-oxidase activity that is distinct from FMO3.