Effect of inhibitors of alpha-naphthylisothiocyanate-induced hepatotoxicity on the in vitro metabolism of alpha-naphthylisothiocyanate

The role of S-oxidation in the toxic bioactivation of alpha-naphthylisothiocyanate (ANIT) was investigated. The effects of several thione compounds, inhibitors and an inducer of the cytochrome P-450-dependent mixed function oxidase systems on the in vitro metabolism of ANIT and aminopyrine were determined. Ethionamide, sodium diethyldithiocarbamate (Na-DDTC) and S-methyl diethyldithiocarbamate (Me-DDTC), three agents known to undergo metabolism by an S-oxidative pathway and diminish ANIT's toxicity, inhibited the in vitro enzymatic metabolism of ANIT by rat liver microsomes. Methimazole failed to alter either the hyperbilirubinemic response of ANIT or the in vitro metabolism of ANIT. All four thione compounds (i.e., ethionamide, Me-DDTC, Na-DDTC and methimazole) inhibited the enzymatic metabolism of aminopyrine by rat liver microsomes. Me-DDTC was the most potent, whereas methimazole was the least potent inhibitor of aminopyrine metabolism. Phenobarbital, which potentiates, and SKF-525A, which inhibits the hepatotoxicity of ANIT in vivo, correspondingly stimulated or inhibited the NADPH-dependent metabolism of ANIT and aminopyrine by liver microsomes. N-Decylimidazole (NDI), another classical inhibitor of cytochrome P-450-dependent monooxygenase system, inhibited both the in vivo toxicity and in vitro metabolism of ANIT. NDI also diminished the enzymatic metabolism of aminopyrine by liver microsomes. Thus the results of this study indicate that metabolism of ANIT is intimately related to its toxicity and that ANIT probably undergoes its toxic bioactivation via a cytochrome P-450-dependent S-oxidative pathway.     

ANIT toxicity toward mouse hepatocytes in vivo is mediated primarily by neutrophils via CD18

Alpha-naphthylisothiocyanate (ANIT) is a hepatotoxicant that causes acute cholestatic hepatitis with infiltration of neutrophils around bile ducts and necrotic hepatocytes. The objective of this study was to determine whether the beta2-integrin CD18, which plays an important role in leukocyte invasion and cytotoxicity, contributes to ANIT-induced hepatic inflammation and liver injury. Mice with varying levels of leukocyte CD18 expression were treated with ANIT and monitored for hepatic neutrophil influx and liver injury over 48 h. Mice that were partially deficient in CD18 (30% of normal levels) developed periportal inflammation and widespread hepatic necrosis after ANIT treatment in a pattern identical to that in wild-type (WT) mice. In contrast, mice that completely lack CD18 (CD18 null) were resistant to ANIT toxicity. Forty-eight hours after ANIT, CD18-null mice displayed 60% lower serum alanine aminotransferase (ALT) levels and 75% less hepatic necrosis, as shown by morphometry, than WT mice. This was true despite evidence that ANIT still provoked hepatic neutrophil influx in CD18-null mice. WT mice could also be protected from ANIT-induced hepatocellular necrosis, by depleting the animals of neutrophils. Notably, neither CD18-null mice nor neutrophil-depleted WT mice exhibited any attenuation of bile duct injury or cholestasis due to ANIT. We conclude from these experiments that neutrophils invade ANIT-treated livers in a CD18-independent fashion but utilize CD18 to induce hepatocellular cytotoxicity. The results emphasize that neutrophil-mediated amplification of ANIT-induced liver injury is directed toward hepatocytes rather than cholangiocytes. In fact, the data indicate that the majority of ANIT toxicity toward hepatocytes in vivo is neutrophil driven.     

Limited role for CXC chemokines in the pathogenesis of alpha-naphthylisothiocyanate-induced liver injury

Alpha-naphthylisothiocyanate (ANIT) is a hepatotoxin that causes severe neutrophilic inflammation around portal tracts and bile ducts. The chemotactic signals that provoke this inflammatory response are unknown. In this study, we addressed the possibility that ANIT upregulates CXC chemokines in the liver and that these compounds mediate hepatic inflammation and tissue injury after ANIT treatment. Mice treated with a single dose of ANIT (50 mg/kg) exhibited rapid hepatic induction of the CXC chemokine macrophage inflammatory protein-2 (MIP-2). MIP-2 derived primarily from hepatocytes, with no apparent contribution by biliary cells. In ANIT-treated mice, the induction of MIP-2 coincided with an influx of neutrophils to portal zones; this hepatic neutrophil recruitment was suppressed by 50% in mice that lack the receptor for MIP-2 (CXCR2(-/-)). Interestingly, despite their markedly reduced degree of hepatic inflammation, CXCR2(-/-) mice displayed just as much hepatocellular injury and cholestasis after ANIT treatment as wild-type mice. Moreover, after long-term exposure, ANIT CXCR2(-/-) mice developed liver fibrosis that was indistinguishable from that in wild-type mice. In summary, our data show that CXC chemokines are responsible for some of the hepatic inflammation that occurs in response to ANIT but that these compounds are not essential to the pathogenesis of either acute or chronic ANIT hepatotoxicity.     

Effect of liposomal antitumor preparation 5,6-benzocoumarin-5-uracil on intensity of free-radical processes in the liver microsomes and tumor cells of rats with transplanted Guerin's carcinoma

The contents of primary and secondary (TBA-active) products of lipid peroxidation were investigated in microsomal fraction of the liver and tumor cells of rats with transplanted Guerin's carcinoma and under the condition of antitumor liposomal preparation 5,6-benzcumarine-5-uracil (BCU) action. High level of lipid peroxidation process in the microsomal fraction is shown in the rat liver and tumor cells under the condition of BCU action in the period of intensive carcinoma growth. It remains till the period of tumor growth braking. This fact testifies to the prooxidation action of the preparation. Liposomal antitumor preparation BCU raises the process of lipid peroxidation in microsomal fraction of tumor cells and its action increases according to the malignant growth. The processes of lipid peroxidation in microsomal rat liver fraction approach the control data under the condition of the mentioned preparation. The investigated liposomal form of BCU possesses directed prooxidation action on the malignant tissue.     

Regulation of squalene epoxidase activity and comparison of catalytic properties of rat liver and Chinese hamster ovary cell-derived enzymes

Squalene epoxidase activity has been studied in cell-free preparations of Chinese hamster ovary (CHO) cells and rat liver. In contrast to rat liver microsomal squalene epoxidase, the enzyme of CHO cells is only slightly activated by the autologous cytosolic fraction, whereas phosphatidylglycerol or rat liver cytosolic preparations are potent stimulators of this enzyme. Triton X-100, a known stimulator of the hepatic squalene epoxidase, has no activating effect on the enzyme of CHO cells. The squalene epoxidase activity of both rat liver and CHO cells varies significantly according to the lipid content of the growth medium or diet. The changes in enzyme activity are shown to be entirely due to altered microsomal enzyme per se and not to changes in the activating properties of the soluble fraction. These results further support the proposed regulatory role of squalene epoxidase in cholesterogenesis.     

Characterization of the protective effects of melatonin and related indoles against alpha-naphthylisothiocyanate-induced liver injury in rats

The protective effect of melatonin, 6-hydroxymelatonin and N-acetylserotonin against alpha-naphthylisothiocyanate (ANIT)-induced liver injury was investigated and compared in rats injected once with the hepatotoxicant (75 mg/kg body weight). In rats injected with ANIT alone, liver injury with cholestasis developed within 24 h, as indicated by both serum levels of alanine aminotransferase (SGPT) and aspartic acid aminotransferase (SGOT) activities and serum total bilirubin concentration. The administration of melatonin or 6-hydroxymelatonin (10 mg/kg body weight) to ANIT-injected rats reduced significantly the serum levels of both SGPT and SGOT and the serum total bilirubin concentration. For all hepatic biochemical markers, melatonin was more effective that 6-hydroxymelatonin. By comparison, the administration of N-acetylserotonin (10 mg/kg body weight) to ANIT-injected rats did not reduce the serum levels of either hepatic enzymes or the serum total bilirubin concentration. In ANIT-injected rats, hepatic lipid peroxidation (LPO) was significantly higher than in control animals and this increase was significantly reduced by either melatonin, 6-hydroxymelatonin or N-acetylserotonin. Furthermore, ANIT treatment caused a significant reduction in liver microsomal membrane fluidity and this reduction was completely reversed by the three indoles. The liver from ANIT-injected rats showed several histopathological alterations; above all there was an acute infiltration of polymorphonuclear neutrophils and an increase in the number of apparent apoptotic hepatocytes. The concurrent administration of melatonin reduced the severity of all morphological alterations, specially the neutrophil infiltration and the number of presumed apoptotic cells. On the contrary, the administration of 6-hydroxymelatonin or N-acetylserotonin did not provide any protective effect in terms of the histopathological alterations. These results indicate that melatonin protects against ANIT-induced liver injury with cholestasis in rats, and suggests that this protective effect is likely due to its antioxidant properties and above all to its capacity to inhibit liver neutrophil infiltration, a critical factor in the pathogenesis of ANIT-induced liver injury. 6-hydroxymelatonin, although able to provide partial protection against the ANIT-induced hepatic injury, probably through its antioxidant properties by mechanisms that are unclear, was unable to reduce neutrophil infiltration. Finally, N-acetylserotonin in the experimental conditions of this study, only exhibited some antioxidant protection but had no protective effect against ANIT-induced hepatic damage.     

Digitoxin metabolism by liver microsomal cytochrome P-450 and UDP-glucuronosyltransferase and its role in the protection of rats from digitoxin toxicity by pregnenolone-16 alpha-carbonitrile

The aim of the present study was to investigate whether the mechanism by which pregnenolone-16 alpha-carbonitrile (PCN) protects rats from digitoxin toxicity was dependent on the induction of liver microsomal cytochrome P-450p and/or the UDP-glucuronosyltransferase active toward digitoxigenin monodigitoxoside (UDP-GT-dt1). Evidence is presented that suggests troleandomycin is a selective inhibitor of cytochrome P-450p in vivo, based on the pattern of inhibition observed when zoxazolamine paralysis time and hexobarbital sleeping time were measured in rats treated with different cytochrome P-450 inducers. A single dose of troleandomycin completely reversed the ability of PCN to protect rats from digitoxin toxicity, establishing the importance of cytochrome P-450p induction in the protective effect of PCN. The postpubertal decline in constitutive cytochrome P-450p levels in female but not male rats was paralleled by a female-specific, age-dependent decline in the rate of digitoxin sugar cleavage (i.e., digitoxosyl oxidation of digitoxin to 15'-dehydrodigitoxin and digitoxosyl cleavage to digitoxigenin bisdigitoxoside). This resulted in a marked sex difference in the rate of digitoxin sugar cleavage catalyzed by liver microsomes from mature rats (male/female approximately 6). However, no sex difference in digitoxin toxicity was observed in either immature or mature rats. In contrast to cytochrome P-450p, liver microsomal UDP-GT-dt1 activity increased dramatically with age in both male and female rats (mature/immature approximately 10). However, no age differences in digitoxin toxicity were observed in rats of either sex. The results indicate that cytochrome P-450p and UDP-GT-dt1 can be independently regulated in rat liver and that large changes in the constitutive levels of these microsomal enzymes have no effect on digitoxin toxicity. This suggests that the induction of cytochrome P-450p and UDP-GT-dt1 does not fully account for the mechanism by which PCN protects rats from digitoxin toxicity.     

Intracellular distribution of 5' nucleotidase in rat liver

The intracellular distribution of 5' nucleotidase was investigated in rat liver by biochemical analysis of cell fractions obtained by differential centrifugation. The enzymatic activity was measured by determination of the inorganic phosphorus liberated from 5' nucleotides. The 5' nucleotidase activity was mainly found in the nuclear and microsomal fractions. An attempt to extract the enzyme from these fractions with Mg(++) ion solutions was unsuccessful. It is concluded that 5' nucleotidase is actually present in the nuclear and microsomal fractions of rat liver cells.     

Intracellular Distribution of 5' Nucleotidase in Rat Liver

The intracellular distribution of 5' nucleotidase was investigated in rat liver by biochemical analysis of cell fractions obtained by differential centrifugation. The enzymatic activity was measured by determination of the inorganic phosphorus liberated from 5' nucleotides. The 5' nucleotidase activity was mainly found in the nuclear and microsomal fractions. An attempt to extract the enzyme from these fractions with Mg⁺⁺ ion solutions was unsuccessful. It is concluded that 5' nucleotidase is actually present in the nuclear and microsomal fractions of rat liver cells.     

Differences in naphthalene-induced toxicity in the mouse and rat

Following the intraperitoneal administration of naphthalene (200 mg/kg) to mice, the lung, in comparison with other organs, was selectively damaged. Histological examination of the lungs showed that it was the non-ciliated, bronchiolar epithelial cells (Clara cells) which were damaged. At higher doses (400 mg/kg and 600 mg/kg, i.p.), there was also damage to the cells in the proximal tubules of the kidney. In contrast to the effect in mice, doses of naphthalene as high as 1600 mg/kg (i.p.) caused no detectable pulmonary or renal damage in the rat. This difference in toxicity between the mouse and rat was reflected by the ability of naphthalene to more severely deplete the non-protein sulphydryls in the mouse lung and kidney than in those organs in the rat. In order to investigate the species difference in toxicity, the metabolism of naphthalene by lung and liver microsomes of the mouse and rat was studied. In all cases, naphthalene was metabolised to a covalently bound product(s) and to two major methanol-soluble products, which co-chromatographed with 1-naphthol and 1,2-dihydro-1,2-dihydroxynaphthalene. However, both the covalent binding and metabolism were approximately 10-fold greater in microsomes prepared from mouse lung compared with those from the rat. This observation may in part explain the difference in toxicity of naphthalene to the mouse and rat lung. As 1-naphthol is a major metabolite of naphthalene and previous work had suggested that most of the microsomal catalysed binding of naphthalene was due to further oxidation of 1-naphthol, the role of 1-naphthol in mediating the naphthalene-induced toxicity was investigated. In neither the mouse nor the rat did 1-naphthol cause a depletion of non-protein sulphydryl levels or tissue damage in the liver, lung or kidney. Thus the toxicity of naphthalene does not appear to be mediated via 1-naphthol.     

Regulation of human leukocyte microsomal hydroxymethylglutaryl-CoA reductase activity by a phosphorylation and dephosphorylation mechanism

The activity of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), obtained from cultured human IM-9 lymphoid cells or freshly isolated human peripheral blood leukocytes, is modulated by a phosphorylation/dephosphorylation mechanism. Addition of MgATP + ADP to IM-9 cell microsomal reductase leads to a time-dependent loss of enzyme activity. Inactivated reductase is reactivated by rat liver reductase phosphatase. Kinase-dependent IM-9 cell microsomal reductase, prepared by heating IM-9 microsomes for 15 min at 50°C, is inactivated in the presence of MgATP and ADP only after addition of cytosolic reductase kinase from either IM-9 cells, freshly isolated leukocytes or rat liver. Inactivation is time-dependent and dependent on the cytosolic protein concentration. Inactivated reductase is reactivated by rat liver reductase phosphatase. For cultured IM-9 cells and freshly isolated leukocytes incubated with culture medium for 2 h, the ratios of active (unphosphorylated) to total (phosphorylated + unphosphorylated) reductase activity are 0.22 and 0.43, respectively. Thus, in addition to its regulation by changes in the amount of total enzyme protein, human leukocyte reductase activity is also modulated by a phosphorylation/dephosphorylation mechanism.     

AM1, a glycoprotein from the submandibular glands of the mouse, has esterolytic and amidolytic activities

The activity of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), obtained from cultured human IM-9 lymphoid cells or freshly isolated human peripheral blood leukocytes, is modulated by a phosphorylation/dephosphorylation mechanism. Addition of MgATP + ADP to IM-9 cell microsomal reductase leads to a time-dependent loss of enzyme activity. Inactivated reductase is reactivated by rat liver reductase phosphatase. Kinase-dependent IM-9 cell microsomal reductase, prepared by heating IM-9 microsomes for 15 min at 50 degrees C, is inactivated in the presence of MgATP and ADP only after addition of cytosolic reductase kinase from either IM-9 cells, freshly isolated leukocytes or rat liver. Inactivation is time-dependent and dependent on the cytosolic protein concentration. Inactivated reductase is reactivated by rat liver reductase phosphatase. For cultured IM-9 cells and freshly isolated leukocytes incubated with culture medium for 2 h, the ratios of active (unphosphorylated) to total (phosphorylated + unphosphorylated) reductase activity are 0.22 and 0.43, respectively. Thus, in addition to its regulation by changes in the amount of total enzyme protein, human leukocyte reductase activity is also modulated by a phosphorylation/dephosphorylation mechanism.     

Characterization and partial purification of microsomal NAD(P)H:quinone oxidoreductases

Quinone oxidoreductases are flavoproteins that catalyze two-electron reduction and detoxification of quinones. This leads to the protection of cells against toxicity, mutagenicity, and cancer due to exposure to environmental and synthetic quinones and its precursors. Two cytosolic forms of quinone oxidoreductases [NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH:quinone oxidoreductase 2 (NQO2)] were previously identified, purified, and cloned. A role of cytosolic NQO1 in protection of cells from oxidative stress, cytotoxicity, and mutagenicity of quinones was established. Currently, we have characterized and partially purified the NQO activity from rat liver microsomes. This activity was designated as microsomal NQO (mNQO). The mNQO activity showed significantly higher affinity for NADH than NADPH as electron donors and catalyzed reduction of 2,6-dichlorophenolindophenol and menadione. The mNQO activity was insensitive to dicoumarol, a potent inhibitor of cytosolic NQO1. Western analysis of microsomal proteins revealed 29- and 18-kDa bands that cross-reacted with polyclonal antibodies raised against cytosolic NQO1. The mNQO activity was partially purified by solubilization of microsomes with detergent Chaps, ammonium sulfate fractionation, and DEAE-Sephacel column chromatography. The microsomal mNQO proteins are expected to provide additional protection after cytosolic NQOs against quinone toxicity and mutagenicity.     

Studies of microsomal glucose-6-phosphatase on liver of irradiated rats

The activity of microsomal glucose-6-phosphatase (EC 3.1.3.9) on male rat liver was measured 1-9 days after whole-body gamma-irradiation. A marked fall of activity, expressed per whole liver, was observed reaching a minimum on the 4th day following irradiation. The enzyme activity is partially and momentarily restored (on day 7), before a new decrease occurred. Furthermore, when the results are expressed per milligram of microsomal proteins, there was no change. Cysteamine, when injected in vivo, kept up the glucose-6-phosphatase of whole liver. On day 4, a histochemical demonstration of the enzyme in liver cells is in accordance with enzyme measures. These observations suggested that the enzyme quantity was altered during the acute radiation syndrome in the rat.     

Does a calmodulin-dependent Ca2+-regulated Mg2+-dependent ATPase contribute to hepatic microsomal calcium uptake?

Solubilization of microsomal proteins followed by calmodulin affinity chromatography resulted in the separation of two distinct Ca2+-Mg2+-ATPases (Ca2+-regulated Mg2+-dependent ATPases), one being insensitive to calmodulin (ATPase-1), the other being stimulated about 5-fold by calmodulin (ATPase-2). ATPase-2 accounts for only 8% of total microsomal Ca2+-Mg2+-ATPase-activity. ATPase-1 and -2 can also be distinguished by different pH optima, different sensitivity towards inhibition by vanadate and LaCl3, and different apparent Mr values of the phosphoenzyme intermediates (115,000 and 150,000 for ATPase-1 and ATPase-2 respectively). ATPase-1 from liver co-migrated with Ca2+-Mg2+-ATPase from rat skeletal-muscle sarcoplasmic reticulum, whereas ATPase-2 from liver co-migrated with calmodulin-dependent Ca2+-Mg2+-ATPase derived from rat skeletal-muscle sarcolemma. After separation of parenchymal and nonparenchymal liver cells, a calmodulin-dependent Ca2+-Mg2+-ATPase of Mr 150,000 was found only in the non-parenchymal cells. The kinetic parameters of ATPase-2 and the similarity of the apparent Mr of its phosphoenzyme intermediate to that of skeletal-muscle sarcolemma Ca2+-Mg2+-ATPase makes it likely that the calmodulin-sensitive Ca2+-Mg2+-ATPase found in rat liver microsomal fractions reflects a contamination with plasma membranes (possibly from non-parenchymal cells) rather than a true location in the endoplasmic reticulum of parenchymal liver cells.     

Glutathione depletion in a liver microsomal assay as an in vitro biomarker for reactive metabolite formation

Glutathione (GSH) plays a major role in cytoprotection, acting as a nucleophile trap for reactive species derived from xenobiotics. This has led to the development of an assay for the detection of reactive species generated by liver microsomal metabolism of xenobiotics. This assay has been used extensively to study reactive metabolites which initiate toxicity through a direct (non-immunological) mechanism, but there are few data on its ability to detect reactive metabolites that initiate toxicity through neo-antigen formation, or to detect xenobiotics that cause GSH loss by oxidation mediated by a redox cycling process. Accordingly, the ability of rat and human liver microsomes to metabolize xenobiotics to GSH-depleting metabolites has been investigated further. Of the five neo-antigen-forming xenobiotics tested, four (amodiaquine, phenobarbitone, procainamide, and sulphanilamide) displayed GSH reactivity that was either dependent or independent (amodiaquine) on metabolism. The other neo-antigen-forming xenobiotic (carbamazepine) was inactive in all microsomal samples tested. Four quinones believed to exert toxcity through arylation (1,4-benzoquinone) and/or redox cycling (duroquinone, menadione, mitomycin c) displayed GSH reactivity, as did nitrofurantoin and diquat, two other redox cycling xenobiotics. Induction of the mixed function oxidase system with Aroclor afforded little advantage when using rat liver microsomes, whilst there was considerable inter-individual variation in the ability of human liver microsomes to mediate metabolism-dependent GSH depletion. It is concluded that the liver microsome GSH depletion assay may be of general utility as a screen for a number of xenobiotic-derived reactive species.     

Glutathione depletion in a liver microsomal assay as an in vitro biomarker for reactive metabolite formation

Glutathione (GSH) plays a major role in cytoprotection, acting as a nucleophile trap for reactive species derived from xenobiotics. This has led to the development of an assay for the detection of reactive species generated by liver microsomal metabolism of xenobiotics. This assay has been used extensively to study reactive metabolites which initiate toxicity through a direct (non-immunological) mechanism, but there are few data on its ability to detect reactive metabolites that initiate toxicity through neo-antigen formation, or to detect xenobiotics that cause GSH loss by oxidation mediated by a redox cycling process. Accordingly, the ability of rat and human liver microsomes to metabolize xenobiotics to GSH-depleting metabolites has been investigated further. Of the five neo-antigen-forming xenobiotics tested, four (amodiaquine, phenobarbitone, procainamide, and sulphanilamide) displayed GSH reactivity that was either dependent or independent (amodiaquine) on metabolism. The other neo-antigen-forming xenobiotic (carbamazepine) was inactive in all microsomal samples tested. Four quinones believed to exert toxcity through arylation (1,4-benzoquinone) and/or redox cycling (duroquinone, menadione, mitomycin c) displayed GSH reactivity, as did nitrofurantoin and diquat, two other redox cycling xenobiotics. Induction of the mixed function oxidase system with Aroclor afforded little advantage when using rat liver microsomes, whilst there was considerable inter-individual variation in the ability of human liver microsomes to mediate metabolism-dependent GSH depletion. It is concluded that the liver microsome GSH depletion assay may be of general utility as a screen for a number of xenobiotic-derived reactive species.     

Cytosolic and microsomal epoxide hydrolases are immunologically distinguishable from each other in the rat and mouse

Antibodies raised to homogeneous rat liver microsomal epoxide hydrolase were used to distinguish microsomal epoxide hydrolase from epoxide hydrolase of cytosolic origin in mice and rats. Using double diffusion analysis in agarose gels, we show that anti-rat liver microsomal epoxide hydrolase forms a single precipitin line with solubilized microsomes from rat and mouse liver, but no reaction is seen with the corresponding cytosolic fractions. Rat or mouse microsomal epoxide hydrolase activity (using benzo[a]pyrene 4,5-oxide as substrate) can be completely precipitated out of solubilized preparations by the antibody, which is equipotent against rat and mouse microsomal epoxide hydrolase. No precipitation of cytosolic hydrolase activity (using trans-beta-ethyl styrene oxide as substrate) is seen with any concentration of the antibody tested. Thus, in the case of microsomal epoxide hydrolase, extensive immunological cross-reactivity exists between the two species, rat and mouse. In contrast, no cross-reactivity is detectable between cytosolic and microsomal epoxide hydrolase, even when enzymes from the same species are compared. We conclude that microsomal and cytosolic epoxide hydrolase activities represent distinct and immunologically non-cross-reactive protein species.     

Purification and characterization of two isoforms of T-kininogens from rat liver microsomes.

T-Kininogen is one of the acute phase proteins, and is a precursor of T-kinin and a cysteine protease inhibitor. Two homologous T-kininogens (TI- and TII-kininogens) were isolated from microsomal fraction of inflamed rat liver, by chromatographies on columns of DEAE-Sepharose CL-6B and DEAE-5PW and by affinity chromatography on a column of anti T-kininogen monoclonal antibody. The amino terminal amino acid sequences of the two microsomal pyridylethylated T-kininogens after pyroglutamyl aminopeptidase treatment were identical with those of TI- and TII-kininogens from inflamed rat plasma. Microsomal T-kininogens moved faster on SDS-PAGE after treatment with endoglycosidase H. The amounts of microsomal TI- and TII-kininogens in inflamed and non-inflamed rat liver were quantitated by immunoblotting of homogenates of liver microsomes using anti T-kininogen rabbit antiserum. The amounts of microsomal T-kininogens were increased in inflamed rat liver, but the ratio of the amounts of TI-kininogen to TII-kininogen was not different in the inflamed and non-inflamed rat liver. On the other hand, TII-kininogen was not significantly detected in non-inflamed rat plasma. These results indicate that the secretion of one of the T-kininogens, TII-kininogen, into plasma may be prevented by some unknown mechanism.