Fraction Of Rat Liver Research Articles

Characterization of p92, karyopherin beta, co-purified with N-acetylglucosamine-bearing nucleoporins from rat liver nuclear envelopes.

A 92k protein (p92) was purified from the wheat germ agglutinin-Sepharose (WGA-Sepharose) bound fraction of a rat liver nuclear envelope salt-extract by DEAE-5PW and hydroxyapatite HPLCs. Partial amino acid sequence analysis of p92 revealed that it is karyopherin beta, which was found recently in the cytosolic fraction. It was shown using anti-p92 antiserum that the protein is present in the nuclear envelope and cytosolic fractions, in almost the same amounts, but not in other subcellular fractions of rat liver. p92 bound to N-acetylglucosamine bearing nucleoporins (GNPs) on WGA-Sepharose, but not directly to WGA. The amount of p92 found in the rat liver nuclear envelope fraction corresponded to about 10% of the nuclear pore complex in mass, and to as much as 140 mol of p92 per mol of nuclear pore complex. Hydrodynamic analysis of the purified p92 suggested that the molecule is present as a monomer and that it is a rod-shaped molecule. The interaction of p92 and GNPs seemed to be hydrophobic and ionic. Based on these results, the participation of nuclear envelope p92 in protein nuclear transport is discussed.

In-vitro Metabolism of a Novel Monocrotophos Derivative by Rat and Japanese Quail

NADPH-dependent inhibition of hepatic microsomal carboxylesterase by a derivative of monocrotophos (coded as RPR-5) was studied in rat and Japanese quail as a measure of monooxygenase-catalysed activation of RPR-5. There was NADPH-dependent inhibition of hepatic microsomal α-naphthyl acetate esterase (carboxylesterase) both in rat and quail, indicating monooxygenase-catalysed formation of an oxon that subsequently phosphorylated α-NaE. The pattern of in-vitro metabolism of 14 C-labelled RPR-5 by 11 000g supernatant (11-S), microsomes and 105 000g supernatant (105-S) fractions of rat and quail livers suggested the involvement of microsomal monooxygenases and carboxylesterases. A radiolabelled metabolite (M2) was tentatively identified as an acid produced by carboxyl esterase attack. In rat, metabolism by microsomal and cytosolic (105-S) carboxylesterases appeared to predominate with relatively little oxidative metabolism. In quail, putative microsomal carboxylesterase hydrolysis of RPR-5 was much lower than in the rat with almost neglible hydrolysis by cytosolic fractions. Also, production of M 2 by quail microsomes was substantially reduced after addition of NADPH, suggesting inhibition of a carboxyl esterase by the oxon of RPR-5. Differences in this detoxification of RPR-5 between rat and quail may be an important factor in determining selective toxicity and the results underline the importance of relating metabolism to toxicity when selecting animal models for toxicity testing.

In‐vitro Metabolism of a Novel Monocrotophos Derivative by Rat and Japanese Quail

NADPH-dependent inhibition of hepatic microsomal carboxylesterase by a derivative of monocrotophos (coded as RPR-5) was studied in rat and Japanese quail as a measure of monooxygenase-catalysed activation of RPR-5. There was NADPH-dependent inhibition of hepatic microsomal α-naphthyl acetate esterase (carboxylesterase) both in rat and quail, indicating monooxygenase-catalysed formation of an oxon that subsequently phosphorylated α-NaE. The pattern of in-vitro metabolism of 14C-labelled RPR-5 by 11000g supernatant (11-S), microsomes and 105000g supernatant (105-S) fractions of rat and quail livers suggested the involvement of microsomal monooxygenases and carboxylesterases. A radiolabelled metabolite (M2) was tentatively identified as an acid produced by carboxyl esterase attack. In rat, metabolism by microsomal and cytosolic (105-S) carboxylesterases appeared to predominate with relatively little oxidative metabolism. In quail, putative microsomal carboxylesterase hydrolysis of RPR-5 was much lower than in the rat with almost neglible hydrolysis by cytosolic fractions. Also, production of M2 by quail microsomes was substantially reduced after addition of NADPH, suggesting inhibition of a carboxyl esterase by the oxon of RPR-5. Differences in this detoxification of RPR-5 between rat and quail may be an important factor in determining selective toxicity and the results underline the importance of relating metabolism to toxicity when selecting animal models for toxicity testing.

Subcellular Distribution and Phosphorylation of the Nuclear Localization Signal Binding Protein, NBP60

We previously purified a nuclear localization signal binding protein, NBP60, from rat liver (1993,J. Biochem.113, 308–313). In this study, the subcellular localization of NBP60 was examined using anti-NBP60. Most NBP60 was found to be localized in the nuclear envelope fraction of rat liver obtained on cell fractionation followed by immunoblotting. Staining of the nuclei of cultured cells by the antibody was observed on immunofluorescence microscopy. NBP60 was widely detected in rat nuclear fractions prepared from other tissues and also in nuclei of cultured cells derived from other species. It was shown by immunoelectron microscopy that most NBP60 is present in the nuclear envelope and at least some of that is present on nuclear pore complexes. Although NBP60 was localized in the nuclear envelope in interphase cells, it diffused into the cytoplasm in the mitotic phase. The purified NBP60 was highly phosphorylated by a cdc2 mitotic kinase, whereas nuclear pore proteins p144, p62, p60, and p54 were not phosphorylated by the kinase directly. NBP60 was also phosphorylated by protein kinase A, calmodulin-dependent protein kinase II, and casein kinase II. The phosphorylation of NBP60 by cdc2 kinase and/or the other kinases may be related to the change in the protein's location during the mitotic phase.

Characterization of brimonidine metabolism with rat, rabbit, dog, monkey and human liver fractions and rabbit liver aldehyde oxidase.

1. In vitro metabolism of 14C-brimonidine by the rat, rabbit, dog, monkey and human liver fractions was studied to assess any species differences. In vitro metabolism with rabbit liver aldehyde oxidase and human liver slices, and in vivo metabolism in rats were also investigated. The hepatic and urinary metabolites were characterized by liquid chromatography and mass spectrometry. 2. Up to seven, six, 11 and 14 metabolites were detected in rat liver S9 fraction, human liver S9 fraction, human liver slices and rat urine respectively. Rabbit liver aldehyde oxidase catalysed the metabolism of brimonidine to 2-oxobrimonidine and 3-oxobrimonidine, and further oxidation to the 2,3-dioxobrimonidine. Menadione inhibited the liver aldehyde oxidase-mediated oxidation. 3. Hepatic oxidation of brimonidine to 2-oxobrimonidine, 3-oxobrimonidine and 2,3-dioxobrimonidine was a major pathway in all the species studied, except the dog whose prominent metabolites were 4',5'-dehydrobrimonidine and 5-bromo-6-guanidinoquinoxaline. 4. These results indicate extensive hepatic metabolism of brimonidine and provide evidence for aldehyde oxidase involvement in brimonidine metabolism. The species differences in hepatic brimonidine metabolism are likely related to the low activity of dog liver aldehyde oxidase. The principal metabolic pathways of brimonidine are alpha(N)-oxidation to the 2,3-dioxobrimonidine, and oxidative cleavage of the imidazoline ring to 5-bromo-6-guanidinoquinoxaline.

Assessment of the potential in vivo genotoxicity of fluoranthene.

Fluoranthene is a ubiquitous environmental pollutant. Although fluoranthene is mutagenic in bacterial and mammalian in vitro cell systems following metabolic activation by rat liver fraction, information on in vivo mutagenicity is lacking and studies on tumour initiating activity in mice are equivocal. In the present study, the potential genetic hazard to man was assessed using the mouse bone marrow micronucleus and rat liver unscheduled DNA synthesis test systems. Fluoranthene did not show any evidence of genotoxicity in either of the in vivo assays following acute oral administration at levels of up to 2000 mg/kg b.w.

Inhibitory and noninhibitory monoclonal antibodies to human cytochrome P450 2E1.

A panel of 17 hybridomas producing (MAbs) against human cytochrome P450 2E1 (h2E1) was generated by immunizing mice with baculovirus-expressed h2E1. All 17 hybridoma clones gave positive ELISA or immunoblots with either baculovirus-or vaccinia virus-expressed h2E1. Two of the latter were further developed due to their desirable characteristics. MAb 1-73-18 was found to be a powerful inhibitor of P450 h2E1; however, it did not yield a positive immunoblot. MAb 2-106-12 was found to be noninhibitory but formed a strong positive immunoblot with P450 h2E1. These MAbs to h2E1 were highly specific and did not recognize six other human P450s as tested with ELISA or immunoblot analyses. The MAbs to baculovirus-expressed h2E1 also reacted with h2E1 expressed from a vaccinia virus vector system as well as with microsomal fractions of human and acetone-treated rat liver. MAb 1-73-18 inhibited h2E1 enzyme activity catalyzing the metabolism of phenanthrene by 85%, p-nitroanisole by 90%, 4-methylanisole by 60-80%, toluene by 90%, and chlorzoxazone by 90%. The inhibitory MAb 1-73-18 is uniquely useful for determining the contribution of h2E1 to the metabolism of h2E1 substrates in human liver containing multiple P450s. The quantitatively determined contribution of h2E1 to the metabolism of the above substrates ranged from 25% to 75%. Thus, h2E1 was responsible for the following percentages of the total metabolism in human liver: p-nitroanisole (35%), phenanthrene (23%), methylanisole to cresol (25%), methylanisole to methoxybenzyl alcohol (12%), toluene (40%), and chlorzoxazone (72%). The MAb 2-106-12 forming a strong immunoblot is useful for determining the amount of h2E1 protein in a tissue. Thus the utility of the inhibitory and immunoblot positive MAbs is complementary and can determine both the contribution of h2E1 to the metabolism of specific substrates and the amount of h2E1 protein in human tissue. The analyses of metabolism with the inhibitory MAb 1-73-18 can be generalized and applicable to all h2E1 substrates.

Characterization of brimonidine metabolism with rat, rabbit, dog, monkey and human liver fractions and rabbit liver aldehyde oxidase

Characterization of brimonidine metabolism with rat, rabbit, dog, monkey and human liver fractions and rabbit liver aldehyde oxidase

Induction of DT-Diaphorase in Riboflavin-Deficient Rats by Xenobiotic Treatment.

This paper describes the induction of DT-diaphorase by xenobiotic treatment of riboflavin-deficient rats. Upon injection of β-naphthoflavone once a day for 3 successive days into male Wistar rats fed a riboflavin-supplemented diet for 4 weeks, the activity of DT-diaphorase in the cytosolic, mitochondrial, and microsomal fractions of rat liver increased several fold compared with that of the vehicle control. In the case of rats fed a riboflavin-deficient diet for 4 weeks, DT-diaphorase activity in the cytosolic fraction was markedly decreased and that in microsomes was somewhat diminished, whereas the activity in mitochondrial fraction did not change, compared with that of the above control. However, when the deficient rats were injected with β-naphthoflavone intraperitoneally, DT-diaphorase activity in the cytosolic, mitochondrial, and microsomal fractions increased in spite of a significant decrease in flavin levels in the body. These results indicate that DT-diaphorase is actually induced even under conditions of riboflavin deficiency and that the apoprotein of the enzyme takes up FAD to become holoenzyme, suggesting a redistribution of FAD among various flavoproteins in the liver of riboflavin-deficient rats upon administration of a xenobiotic. Among the activities of other flavoenzymes examined, the occurrence of the apoenzyme of glutathione reductase, thioredoxin reductase or NADPH-cytochrome P450 reductase was found upon the injection of β-naphthoflavone in the riboflavin-deficient rats, which might be an indication of the above-mentioned redistribution.

Effects of starvation, diabetes and carbon tetrachloride intoxication on rat kidney cortex and liver pyruvate carboxylase levels.

Pyruvate carboxylase (PC) has been quantified in rat liver and kidney cortex under experimental conditions that modify the gluconeogenic response in both organs: fasting, carbon tetrachloride-induced liver degeneration and alloxan-induced diabetes. Enzymatic activity has been assayed by a 14CO2-fixation method. The amount of enzyme has been determined by competitive ELISA using antibodies raised against the purified rat kidney cortex enzyme. Purified fractions of rat-liver and rat-kidney cortex PC have been used as standards. Fasting and carbon tetrachloride administration induced a significant increase (25% to 30%) in the amount of enzyme in liver and kidney cortex. Alloxan-induced diabetes produced a nearly two-fold increase in the hepatic levels of enzyme without a significant modification in the content of the renal enzyme. These results are discussed on the basis of the different metabolic implications of both organs during the physiological or toxic treatments.

Molecular mechanisms of toxic effects of fotemustine in rat hepatocytes and subcellular rat liver fractions.

Fotemustine is a clinically used DNA-alkylating 2-chloro-ethyl-substituted N-nitrosourea, which sometimes shows signs of haematotoxicity and reversible liver and renal toxicity as toxic side-effects. Mechanistic data on these side-effects are scarce and incomplete. In this study, firstly the cytotoxicity of fotemustine in freshly isolated rat hepatocytes was investigated and secondly the metabolism of fotemustine and possible mechanisms involved in the observed cytotoxicity. Fotemustine caused concentration- and time-dependent cytotoxic effects in rat hepatocytes. Extensive GSH-depletion and formation of GSSG were first observed, followed by lipid peroxidation and finally by cell death measured as LDH-leakage. 2-Chloroethyl analogues of fotemustine, which in contrast to fotemustine have no carbamoylating potency, were not toxic to rat hepatocytes. The data suggest that the cytotoxicity of fotemustine is resulting from its reactive decomposition product, DEP-isocyanate. GSH-conjugation of DEP-isocyanate was shown to protect against the cytotoxicity of fotemustine, however, only temporary and not completely. Synthetical DEP-SG, the GSH-conjugate of DEP-isocyanate, was also toxic to rat hepatocytes, albeit to a significantly lesser extent than fotemustine. In rat liver microsomes, no fotemustine-induced LPO was observed, suggesting that reactive decomposition products of fotemustine do not directly cause peroxidation of cellular membranes. Fotemustine did not affect the antioxidant enzymes superoxide dismutase, catalase, GSH-peroxidase, GSSG-reductase and GSH S-transferases. Thus, direct effects on these antioxidant enzymes are not likely to explain the cytotoxic effects of fotemustine in hepatocytes. In conclusion, it is proposed that the cytotoxicity of fotemustine in rat hepatocytes is caused by rapid and extensive depletion of GSH by DEP-isocyanate, a reactive decomposition product of fotemustine, consequently hampering the endogenous protection against its own toxicity. Knowledge of molecular mechanisms of the cytotoxicity of fotemustine may contribute to a more rational design of selective protection against toxic side-effects which occur upon therapy of patients with fotemustine.

Isolation and Characterization of a Novel Perchloric Acid-soluble Protein Inhibiting Cell-free Protein Synthesis

We found a novel protein in the postmitochondria supernatant fraction of rat liver, which is soluble in 5% perchloric acid and strongly inhibits protein synthesis in a rabbit reticulocyte lysate system. The protein extracted from the supernatant fraction with 5% perchloric acid was purified by ammonium sulfate fractionation and CM-Sephadex chromatography. The protein was shown to consist of two identical subunits with a molecular mass of 14 kDa. By immunoscreening with the rabbit antisera against the protein, a cDNA encoding the protein was cloned and sequenced. The cDNA contained an open reading frame of 411 base pairs encoding a 136-amino acid protein with a molecular mass of 14,149 Da. The deduced amino acid sequence was completely identical with that constructed from all of the above peptides. Interestingly, the perchloric acid-soluble protein inhibited cell-free protein synthesis in the rabbit reticulocyte lysate system in a different manner from RNase A. The protein is likely to inhibit an initiation stage of cell-free protein synthesis. Among the rat tissues tested, the protein was located only in liver and kidney. These findings are the first report on a new inhibitor that may be involved in the regulation of protein synthesis in those tissues.

Inhibition of the Thiol-Specific Antioxidant Activity of Rat Liver MSP23 Protein by Hemin

Mouse stress-inducible 23-kDa protein (MSP23) belongs to a new type of antioxidative protein family and is highly expressed in livers. To examine its physiological role in the tissue, we have purified native MSP23 protein from the cytosolic fraction of rat liver. The purified protein showed the thiol-specific antioxidant activity and protected glutamine synthetase from inactivation by a mixed metal-thiol oxidation. We examined the effect of hemin on that activity since it was recently shown by others that the rat liver MSP23 protein had a high binding affinity to heme. We have found that hemin at low concentrations inhibits the antioxidative activity of MSP23. This result suggests that one type of oxidant damage caused by hemin to cells is due to inactivation of the antioxidative protein, MSP23.

Induction of cytochrome P‐450 in rat liver by a polyphenol‐rich extract from a bird‐resistant sorghum grain

AbstractA polyphenol‐rich extract of a bird‐resistant sorghum grain can induce protein in the microsomal fraction (cytochrome P‐450) of rat liver to the same extent as a standard induction technique, which uses an intraperitoneal injection of β‐naphthoflavone along with sodium phenobarbital in the drinking water. This microsomal protein can convert the promutagen 2‐aminofluorene to the N‐hydroxylated mutagenic derivative, as measured by an Ames test, though a little less efficiently than that induced by the standard technique.

CHANGES IN INOSITOL 1,4,5-TRIPHOSPHATE BINDING IN MICROSOMAL FRACTIONS FROM THE RAT LIVER DURING SEPSIS

Inositol 1,4,5-triphosphate has been proposed as a second messenger for calcium mobilization. The addition of inositol 1,4,5-triphosphate at a low concentration has been shown to cause calcium release from intracellular microsomal stores in rat hepatocytes. The effects of sepsis on the inositol 1,4,5-triphosphate binding from microsomal fractions of rat liver were investigated. Sepsis was induced by cecal ligation and puncture (CLP). Control rats were sham operated. Three microsomal fractions (rough, intermediate, and smooth I) were isolated from the rat liver. The study of inositol 1,4,5-triphosphate receptor binding was performed with tritium-labeled inositol 1,4,5-triphosphate. Our results showed that the Bmax of inositol 1,4,5-triphosphate binding in early septic, late septic, and control groups was 14.9 +/- .9 fmol/mg, 9.8 +/- 1.0 fmol/mg, and 17.2 +/- 1.3 fmol/mg, respectively. The binding activity was unaffected during early sepsis but was significantly depressed by 40-50% (p < .05, vs. control) during late sepsis (18 h after CLP) in all three subfractions of endoplasmic reticulum. Because the inositol 1,4,5-triphosphate binding plays an important role in the regulation of intra-cellular calcium homeostasis in hepatocytes, an impairment of the calcium release due to depressed inositol 1,4,5-triphosphate binding in the endoplasmic reticulum may have a pathophysiological significance in contributing to altered hepatic metabolism during septic shock.

Rat Liver Subcellular Folate Distribution Shows Association of Formyltetrahydropteroylpentaglutamates with Mitochondria and Methyltetrahydropteroylhexaglutamates with Cytoplasm

The ternary complex method for the determination of folylpolyglutamates was combined with procedures for interconverting folate derivatives to measure 28 different folate derivatives in the subcellular fractions of rat liver. Folates in the homogenate showed a typical distribution with nearly equal quantities of penta- and hexaglutamates and pteridine derivatives in decreasing order as follows: 1) methyl substituted folates [5-methyltetrahydropteroylglutamates], 2) unsubstituted folates [tetrahydropteroylglutamates + 5,10-methylenetetrahydropteroylglutamates], 3) formyl substituted folates [5-formyltetrahydropteroylglutamates + 10-formyltetrahydropteroylglutamates + 5,10-methenyltetrahydropteroylglutamates], and 4) oxidized folates [dihydropteroylglutamates]. In the homogenate the methyl substituted folates exhibited a higher hexa:pentaglutamate ratio than did the other pteridine derivatives. As the fractionation proceeded toward purer subcellular components, the methyl substituted folates were found almost exclusively in the soluble fraction, and this fraction also contained the higher hexa:pentaglutamate ratio characteristic of the methyl substituted folates. The plasma membrane, the microsomal and the nuclear fractions did not contain appreciable folate. The mitochondrial fraction contained primarily formyl substituted and unsubstituted folates, and these folates exhibited the lower hexa:pentaglutamate ratios. These data support the hypothesis that folate-dependent one-carbon metabolism is compartmentalized in the eukaryotic cell.

Alcohol:NAD+ Oxidoreductase Is Present in Rat Liver Peroxisomes

Alcohol:NAD+ oxidoreductase was found in the peroxisomes of animal liver for the first time as follows. The distribution of alcohol:NAD+ oxidoreductase activity with nonanol as substrate in the light mitochondrial fraction (peroxisome-enriched fraction) of rat liver was examined by centrifugation in a sucrose density gradient. Most of the enzyme activity was localized in the mitochondria, with some activity in the peroxisomes. The administration of clofibrate, a peroxisome proliferator, to rats resulted in a marked increase of the enzyme activity in the peroxisomes, but not in the mitochondria. The enzyme was found to be located in the matrix of the peroxisomes. The evidence was obtained that the enzyme differed from alcohol dehydrogenases and alcohol oxidizing systems found previously. The enzyme activity was not affected by pyrazole, an inhibitor of alcohol dehydrogenase and sodium azide, an inhibitor of catalase. The enzyme was NAD(+)-dependent and oxidized straight chain aliphatic alcohols with a variety of carbon chains (C2-C18), showing the maximum on nonanol. Km values toward these aliphatic alcohols decreased with increasing chain length. The major reaction product was identified as the carboxylic acid by using high performance liquid chromatography.

The influence of glutathione and detoxifying enzymes on DNA damage induced by 2-alkenals in primary rat hepatocytes and human lymphoblastoid cells.

The reaction of 2-alkenals with GSH to form GSH conjugates by Michael addition is a major detoxification pathway. The reaction proceeds at a much higher rate under catalysis by glutathione S-transferase (GST) than the non-enzymatic reaction. Oxidation of 2-alkenals to the corresponding acids by cytosolic and microsomal fraction of rat liver also contributes to detoxification. Primary rat hepatocytes rich in GSH and proficient for GST and other metabolizing enzymes consume much more alkenal than human lymphoblastoid cells (Namalva cells), that are poor in GSH and in metabolic activities. In Namalva cells DNA single strand breaks were induced by much lower concentrations of acrolein, crotonaldehyde and (E)-2-hexenal than in primary rat hepatocytes. In both cell systems intracellular GSH depletion by 2-alkenals proceeds in a dose dependent manner, approaching about 20% of pretreatment level before DNA damage becomes detectable. GSH conjugates of (E)-2-hexenal and (2E,6Z)-2,6-nonadienal induce DNA damage in Namalva cells at high concentrations (1.5 mM). In the absence of GSH these conjugates decompose slowly into aldehyde and GSH. Although the rate of decomposition is only about 10(-4) times that of Michael adduct formation, such GSH conjugates could potentially function as transport molecules for 2-alkenals, if they reach tissues low in GSH and GST.

Involvement of microsomal vesicles in part of the sensitivity of carnitine palmitoyltransferase I to malonyl-CoA inhibition in mitochondrial fractions of rat liver.

Liver mitochondrial fractions as normally isolated contain only 10-20% of total mitochondria and may not be representative of the whole mitochondrial population. This study was designed to evaluate the dependence of the sensitivity of carnitine palmitoyl-transferase I (CPT I) to malonyl-CoA inhibition in mitochondrial fractions that are not normally studied. Four fractions prepared from rat liver were found to be contaminated to different extents by microsome vesicles, on the basis of marker-enzyme activities and micrographic data. Purification of mitochondrial fractions on a Percoll gradient decreased to some extent the microsomal contamination, which was due in part to the existence of close bonds between microsomes and the outer membranes of mitochondria. A greater degree of contamination of mitochondrial fractions by microsomes was correlated with a greater sensitivity of CPT I to malonyl-CoA inhibition. Attempts were made to enhance the sensitivity of CPT I to malonyl-CoA with the use of microsomes. Measurements performed by adding mitochondria and microsomes in the same CPT I assay failed to demonstrate any significant enhancement of malonyl-CoA inhibition. However, addition of ATP to a mixture of mitochondria and microsomes was shown to trigger the binding of both particles, as assessed by enzymic and micrographic data, and to increase the sensitivity of CPT I to malonyl-CoA inhibition. These results demonstrated that the binding of microsomes to mitochondria, unlike the simple mixing of both particles, was capable of altering the sensitivity of CPT I to malonyl-CoA. The data also suggest that this process could be of physiological importance, owing to the frequency of contiguous zones between mitochondria and endoplasmic reticulum observed in sections of intact liver cells.

Differences in the kinetic properties, effect of calcium and sensitivity to inhibitors of paraoxon hydrolase activity in rat plasma and microsomal fraction from rat liver

The properties of a rat hepatic microsomal enzyme that hydrolyses O, O-diethyl- p-nitro-phenylphosphate (paraoxon) were studied and compared to the paraoxon hydrolase activity found in rat plasma. The pH stability for both enzyme activities was optimum between pH 6.0 and 9.0. An overall analysis of the data showed that the microsomal fraction was less resistant to the effect of the pH than plasma. The kinetic constants for heat inactivation evaluated for paraoxonase in rat plasma and liver microsomal fraction indicate that paraoxonase tends to inactivate faster in rat liver microsomes than in rat plasma. The apparent activation energies of the heat inactivation process were 77.7 and 61.1 kcal/mol for rat plasma and microsomal fraction, respectively. Enzyme activity was lost after both dialysis and incubation with EDTA and partially restored by the addition of calcium. In rat plasma samples the requirement for calcium was absolute (essential activator) while in the microsomal fraction the reaction may occur, to a minimum extent, in the absence of the activator (non-essential activator). Calcium restored 85% activity when added immediately after EDTA; restored activity decreased when the time interval between addition of EDTA and calcium was increased. Other metals were not able to restore activity previously inhibited by EDTA or dialysis. The response to several inhibitors (EDTA, Mn, Co, Zn, Ba, Mg, Cu, La, Hg and p-hydroxy-mercuribenzoate) of rat plasma and microsomal fraction was studied, determining the type of inhibition and the inhibition constants. Plasma enzyme was always more resistant than liver sample to the effect of the inhibitors and showed different types of inhibition than the liver microsomal fraction. In general we found more differences than analogies between the rat plasma and liver enzymes which suggests the presence of two enzymes or two different forms of the same enzyme. Furthermore the existence of an EDTA-resistant fraction in rat liver microsomes suggests that more than one enzyme capable of hydrolysing paraoxon is present in the microsomal fraction of rat liver.