Separation and partial characterization of multiple forms of rat liver alcohol dehydrogenase

Rat liver alcohol dehydrogenase was purified and four isoenzyme forms, demonstrated by starch gel electrophoresis, were separated by O-(carboxymethyl)-cellulose chromatography. Each of the isoenzymes had a distinct isoelectric point. All isoenzymes were active with both ethanol (or acetaldehyde) and steroid substrates, and had similar Michaelis-Menten constants for each of the substrates and coenzymes studied. The three isoenzymes with the lowest migration toward the cathode exhibited the same pH optimum of 10.7 for ethanol oxidation, a greater activity with 5 beta-androstan-3 beta-ol-17-one than with ethanol as a substrate, and an unchanged electrophoretic mobility following storage in the presence of 100 microM dithiothreitol. By contrast the isoenzyme with the highest mobility toward the cathode exhibited a pH optimum of 9.5 for ethanol oxidation, a low steroid/ethanol ratio of activity, and converted to the migrating pattern of the two isoenzymes with intermediate mobility when stored. The similarities between the isoenzymes of rat liver alcohol dehydrogenase differ considerably from differences in substrate specificity exhibited by isoenzymes of horse liver alcohol dehydrogenase.     

芳香烃受体在非酒精性脂肪性肝脏疾病中的作用

非酒精性脂肪性肝脏疾病(nonalcoholic fatty liver diseases,NAFLD)是目前备受关注的一种肝脏疾病。肥胖、2型糖尿病、高脂血症等是NAFLD的重要危险因素,但其发病机理仍不十分清楚。芳香烃受体(aryl hydrocarbon receptor,AHR)是由配体激活的转录因子,其在多种重要疾病活动中发挥了重要作用。近年来多项研究表明AHR激活促进了NAFLD的发病进展,对AHR参与NAFLD发病机制的探讨将有利于进一步阐明NAFLD的发病机理,为NAFLD的防治提出新的思路。本文就AHR与NAFLD关系的研究进展做一综述,以期为该领域的研究提供新的方向。     

NADH-dependent aryl hydrocarbon hydroxylase in rat liver mitochondrial outer membrane.

NADH-dependent 3,4-benzpyrene hydroxylase activity was detected in the purified mitochondrial outer membrane fraction from the livers of rats treated with 3-methylcholanthrene. The specific activity in the outer membrane fraction is nearly equal to that of microsomes, a level too high to be accounted for only by the microsomal contamination. On the other hand, the NADPH-dependent 3,4-benzpyrene hydroxylase activity in the outer membrane fraction is about 50% of that of microsomes. The ratio of the specific activity of NADPH- to NADH-dependent 3,4-benzpyrene hydroxylase in microsomal fraction was about 3.5, while that of the outer membrane fraction was about 1.5. Moreover, it was found that NADH-dependent 3,4-benzpyrene hydroxylase activity in mitochondrial outer membrane from control rat liver was cyanide-insensitive, while that in microsomes was cyanide-sensitive. These results suggest the presence in the mitochondrial outer membrane fraction of aryl hydrocarbon hydroxylase activity which uses as electron donor NADH nearly to the same extent as NADPH. The hydroxylase system is composed of cyanide-insensitive cytochrome P-450 and is inducible markedly by 3-methylcholanthrene treatment. The probable electron transfer pathways in the mitochondrial outer membrane cytochrome P-450 oxidase system are discussed.     

Induction by 9-hydroxyellipticine of aryl hydrocarbon hydroxylase in perinatal rat liver and in primary fetal hepatocytes in culture

N-nitrosodiethanolamine is converted to N-(2-hydroxyethyl)-N-(formylmethyl)nitrosamine (EFMN) and N-(2-hydroxyethyl)-N-carboxymethyl) nitrosamine (ECMN) by rat S9 liver preparation as a result of beta-oxidation. The beta-oxidized metabolites were isolated and identified by gas chromatography-mass spectrometry (GC-MS) by comparison with authentic standards. An original gas chromatographic method with thermal energy detection was set up to measure both metabolites quantitatively. Under the experimental conditions described, when NAD+ was used as cofactor, about 1% of N-nitrosodiethanolamine (NDELA) was converted to EFMN and about half of the latter product was in turn converted to ECMN. The beta-nitrosamino aldehyde seems to transfer the nitroso moiety to other amino-compounds, even at physiological pH. The significance of the metabolic formation of EFMN in relation to the carcinogenicity of NDELA is discussed.     

Characterization of an inducible aryl hydrocarbon receptor-like protein in rat liver.

The individual pretreatment of Sprague-Dawley rats with either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 2,2',4,4',5,5'-hexachlorobiphenyl (HCB) has been previously shown to result in the "induction" of [3H]TCDD specific binding activity in hepatic tissue. In the present work, the coadministration of TCDD and HCB increased the concentration of hepatic proteins capable of binding [3H]TCDD specifically by at least 2-3-fold. This increase was shown not to be the result of activation, by HCB, of a form of the receptor having low affinity toward [3H]TCDD into a form with high affinity. Kinetic analysis of the time course of binding of [3H]TCDD to induced cytosol was consistent with the presence of an "inducible" binding protein in addition to the "constitutive" aryl hydrocarbon (Ah) receptor present in cytosol from untreated animals. The liganded ([3H]TCDD) form of the inducible binding component lost its ligand much faster than the liganded form of the constitutive Ah receptor at 37 degrees C; apparent first order rate constants for loss of [3H]TCDD were 0.55 min-1 and less than 0.0024 min-1, respectively. Conversely, the unliganded form of the induced binding component was slightly more stable (approximately 2-fold) toward thermal inactivation than the unbound constitutive Ah receptor. The [3H]TCDD-bound protein(s) in uninduced and induced cytosols behaved identically in a sucrose gradient; 8.7-8.9 S in the absence of salt, shifted to 5.5 S by 0.4 M KCl. They were also indistinguishable by gel permeation chromatography, and by photoaffinity labeling their TCDD-binding subunits, approximate molecular weights 105,000. These results show the hepatic TCDD-binding protein(s) induced upon pretreatment of Sprague-Dawley rats with TCDD/HCB to be kinetically distinct from the Ah receptor, but structurally very similar.     

Molecular characterization and developmental expression of the aryl hydrocarbon receptor from the chick embryo

The aryl hydrocarbon receptor (AhR) was cloned from the chick embryo and its function and developmental expression characterized. Chicken AhR cDNA coded for 858 amino acid protein and 396 bp of 3' UTR. The basic helix loop helix domain exhibited 87-100% amino acid identity to avian, mammalian, and amphibian AhR, and 69-74% to piscine AhR. The PAS (Per-ARNT-Sim) region was slightly less well conserved with (a) 97% identity to other avian sequences, (b) 81-86% to amphibian and mammalian AhR, and (c) 64-69% with piscine AhR. The carboxy terminus diverged the most among species with less than 53% amino acid identity between chicken and any available mammalian and piscine AhR sequences. The chicken AhR RNA and protein were 6.1 kb and 103 kDa, respectively. Chicken AhR dimerized with human AhR nuclear translocator and bound the mammalian dioxin-response element in a ligand-dependent manner. AhR protein was detected in neural ganglia; smooth, cardiac, and skeletal muscle; and epithelium involved in epithelial-to-mesenchymal transformations, such as pituitary, gastrointestinal tract, limb apical-ectodermal ridge, and kidney collecting ducts. AhR mRNA was detected in all tissues expressing protein, except myocardium. Cytochrome P4501A4 mRNA was highly induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a subset of tissues expressing AhR, including small intestine, liver, kidney, blood vessels, and outflow tract myocardium. In conclusion, the AhR sequence and function is highly conserved between birds and mammals, and although many tissues express AhR during chick embryo development, only a subset are responsive to TCDD induction of CYP1A4.     

Multiple forms of cytochrome P-450: resolution and purification of rabbit liver aryl hydrocarbon hydroxylase.

We describe the resolution and partial purification of two minor forms of cytochrome P-450 from liver microsomes of rabbits treated with 2,3,7,8-tetrachlorodibenzo-$\text{p}$-dioxin. Both forms have different electrophoretic mobilities when compared to the major form of cytochrome P-450 isolated from this source. The two cytochromes show different activities with several substrates. One form is very active in the hydroxylation of benzo($\text{a}$)pyrene when reconstituted with highly purified NADPH-cytochrome P-450 reductase.     

Binding of the rat liver 7-8 S dexamethasone receptor to deoxyribonucleic acid

The 7-8 S form of the [3H]dexamethasone (9 alpha-fluoro-11 beta,17,21-trihydroxy-16 alpha-methylpregna-1,4-diene-3, 20-dione) receptor from rat liver cytosol can be converted to the 3-4 S form by RNase treatment or high salt, suggesting a salt-sensitive association between the receptor protein and RNA. In DNA-cellulose column assays, the gradient-purified 3-4 S form bound DNA more efficiently than the 7-8 S form, though the 7-8 S form was also capable of binding to DNA-cellulose to a significant extent. Activated 7-8 S dexamethasone receptor could be released from its association with soluble DNA by treatment with DNase I. Sucrose gradient analysis showed that the released receptor sedimented as the 7-8 S form and was sensitive to RNase treatment, which induced a conversion to the 3-4 S form. Activated RNase-generated 3-4 S receptor again displayed a higher degree of binding to soluble DNA and was recovered in the 3-4 S form following DNase extraction. The fact that the 3-4 S form bound immobilized or soluble DNA more efficiently suggests that the associated RNA of the 7-8 S form interferes directly or indirectly with the receptor association with DNA. The observation that the receptor binds to DNA in its 7-8 S form suggests that the receptor complex is capable of binding RNA and DNA concurrently.     

Isolation and characterization of rat liver nuclear glucocorticoid receptor

The rat liver nuclear glucocorticoid receptor has a molecular weight of 90 000. Using antibody bound to the stationary matrix, the cytosol and nuclear glucocorticoid receptors from rat liver were purified. The translocation of glucocorticoid receptor from rat liver cytosol into the nucleus was studied using immunoaffinity chromatography. Immediately after the intraperitoneal injection of rats with the hormone, the receptor translocation started and was complete within 10 min. The 90 000 dalton nuclear receptor component is identical to the 90 000 dalton cytosol component. They have identical molecular weights in the same gel electrophoresis system and produce identical peptide fragments after digestion with Staphyolococcal aureus V8 protease. The receptor component enriched by immunoaffinity chromatography from cytosol of adrenalectomised rats contained mainly a 45 000 dalton component.     

Bis-aryloxadiazoles as effective activators of the aryl hydrocarbon receptor

Bis-aryloxadiazoles are common scaffolds in medicinal chemistry due to their wide range of biological activities. Previously, we identified a 1,2,4-bis-aryloxadiazole that blocks mammary branching morphogenesis through activation of the aryl hydrocarbon receptor (AHR). In addition to defects in mammary differentiation, AHR stimulation induces toxicity in many other tissues. We performed a structure activity relationship (SAR) study of 1,2,4-bis-aryloxadiazole to determine which moieties of the molecule are critical for AHR activation. We validated our results with a functional biological assay, using desmosome formation during mammary morphogenesis to indicate AHR activity. These findings will aid the design of oxadiazole derivative therapeutics with reduced off-target toxicity profiles.