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PubMed1980

Hepatic drug metabolism during ethanol ingestion in riboflavin deficient rats.

Riboflavin deficiency was induced by feeding rats a riboflavin-deficient diet for 1 month. In order to find out if there are any combined effects of ethanol and riboflavin deficiency on drug metabolism, a group of riboflavin-deficient rats were also given ethanol in their drinking water. At the end of the feeding period, hepatic drug-metabolizing enzyme activities were determined. The hepatic phospholipid and protein contents were the same in rats receiving a standard diet and in those on a riboflavin-deficient diet. However, ethanol ingestion in both groups enhanced significantly the phospholipid content. Ethanol ingestion also markedly enhanced the hepatic cytochrome P-450 concentration in rats fed either a standard or riboflavin-deficient diet. Ethoxycoumarin O-deethylase activity was significantly lower in riboflavin-deficient rat livers than in those of the controls. In both groups ethanol ingestion nearly doubled the activities. Aryl hydrocarbon hydroxylase activity was also significantly decreased during riboflavin deficiency. However, ethanol administration did not change the activities of this enzyme. UDP glucuronosyltransferase activity was slightly lower in riboflavin-deficient rat livers than in those fed a standard diet. No significant decrease was found in the epoxide hydrase activity in the riboflavin-deficient rats. However, the riboflavin-deficient rats had enhanced activity after the ethanol ingestion.

Animals↗
PubMed1977

Preparation and characterization of total, rough and smooth microsomes from the lung of control and methylcholanthrene-treated rats.

Optimal conditions for the preparation of relatively pure microsomes and microsomal subfractions from rat lung have been determined. The most importnat of these conditions is homogenization of a 20% (w/v) suspension of lung tissue in 0.44 M sucrose/1% (w/v) bovine serum albumin with four up-and-down strokes at 440 rev./min in a Potter-Elvehjem homogenizer. The 10000 X g supernatant prepared from this homogenate can be centrifuged at 105000 X g to obtain total microsomes or subfractionated into rough and smooth microsomes on a Cs+-containing discontinuous sucrose gradient. The total, rough and smooth microsomes have been characterized in terms of their chemical composition, enzymatic activity, and morphology. These preparations should prove useful in studies of various enzymes in lung (e.g. benzpyrene monooxygenase, epoxide hydrase, enzymes of phospholipid and ascorbic acid synthesis) and in subfractionations designed to reveal heterogeneites in the lateral plane of the lung endoplasmic reticulum.

Animals↗
PubMed1980

Effects of chronic treatment with di-(2-ethylhexyl) phthalate on rat liver microsomal activities.

The effects of chronic di-(2-ethylhexyl)phthalate (DEHP) on liver microsomal activity were studied in rats. Daily doses of 50 and 500 mg/kg for 4 weeks did not affect O-demethylation, aromatic hydroxylation, N-demethylation, C3-hydroxylation, styrene monooxygenase, glutamic-oxalacetic and glutamic-pyruvic transaminases (GOT, GPT). Inhibition of glutathione-S-transferase A and C and induction of epoxide hydrase, glutathione-S-transferase B and nitroreductase activity were instead observed. Protein, cytochrome P-450 and reduced glutathione levels in liver did not appear to be affected by DEHP pretreatment.

Animals↗
PubMed1988

Effects of TCDD-estradiol interactions in three strains of mice.

Interactions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and estradiol were studied in three strains of mice: CD-1 and C57B/6 (TCDD sensitive) and DBA/2 (TCDD resistant at lower doses). Immature females were injected with 0-200 ng/kg/day estradiol for 2 weeks, s.c. On days 7, 9, 11, and 13, mice received 10 micrograms TCDD/kg by gavage. Relative uterine weight increased in mice of all three strains treated with estradiol alone. Uterine imbibition was suppressed by TCDD treatment, although this effect was antagonized in a saturable manner by coadministration of estradiol. TCDD induced aryl hydrocarbon hydroxylase (AHH) in liver microsomes of treated mice independent of estradiol dose and strain of mice (the dose of TCDD used was high enough to cause AHH induction in the resistant DBA/2 mice). Treatment of CD-1 mice, but not other strains, with TCDD and estradiol resulted in a decrease in the electrophoretic band of hepatic microsomal proteins comigrating with cytochrome P-450a and epoxide hydrase.

Age Factors↗
PubMed2005

Carbonic anhydrase inhibitors. Zonisamide is an effective inhibitor of the cytosolic isozyme II and mitochondrial isozyme V: solution and X-ray crystallographic studies.

The antiepileptic drug zonisamide was considered to act as a weak inhibitor of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) (with a K(I) of 4.3 microM against the cytosolic isozyme II). Here we prove that this is not true. Indeed, testing zonisamide in the classical assay conditions of the CO2 hydrase activity of hCA II, with incubation times of enzyme and inhibitor solution of 15 min, a K(I) of 10.3 microM has been obtained. However, when the incubation between enzyme and inhibitor was prolonged to 1 h, the obtained K(I) was of 35.2 nM, of the same order of magnitude as that of the clinically used sulfonamides/sulfamates acetazolamide, methazolamide, ethoxzolamide and topiramate (K(I)s in the range of 5.4-15.4 nM). The inhibition of the human mitochondrial isozyme hCA V with these compounds has been also tested by means of a dansylamide competition binding assay, which showed zonisamide and topiramate to be effective inhibitors, with K(I)s in the range of 20.6-25.4 nM. The X-ray crystal structure of the adduct of hCA II with zonisamide has also been solved at a resolution of 1.70 A, showing that the sulfonamide moiety participates in the classical interactions with the Zn(II) ion and the residues Thr199 and Glu106, whereas the benzisoxazole ring is oriented toward the hydrophobic half of the active site, establishing a large number of strong van der Waals interactions (<4.5 A) with residues Gln92, Val121, Phe131, Leu198, Thr200, Pro202.

Binding Sites↗
PubMedDate not supplied

Toxicity and microsomal enzyme induction effects of several polybrominated biphenyls of Firemaster.

Some toxicological and pharmacological effects of 2,4,5,2',5'-penta- (congener 1), 2,3,4,2',4',5'-hexa- (congener 5), 2,4,5,3',4',5'-hexa- (congener 6), 2,3,4,5,3',4',-hexa- (congener 7), and 2,3,4,5,2',3',4'-heptabromobiphenyl (congener 9) were evaluated in male rats given a single 90 mg/kg ip injection and killed seven days later. Only congener 7 depressed body weight gain, spleen and thymus weights, and caused severe histopathological changes in the thymus. Congener 7 caused the largest increase in liver weight and the most changes in liver pathology while congener 1 failed to enlarge this organ and caused the mildest ultrastructural changes. Liver microsomes were isolated and evaluated for enzyme induction from all treated rats except those administered congener 6, which was previously identified as a mixed-type enzyme inducer (Dannan et al., 1978b). All congeners increased the liver microsomal cytochrome P-450 content, but only congener 7 shifted the carbon monoxide difference spectrum absorption maximum to 448.0 nm. The microsomal ethyl isocyanide difference spectrum 455/430 nm ratio was increased the most by congener 7 (3 fold). All congeners increased cytochrome P-450 reductase and microsomal epoxide hydrase activities by nearly 1.5-3 fold. Congener 7 failed to induce aminopyrine-N-demethylase activity but the remaining congeners increased it by 2 fold. Congener 7 was the most effective inducer of benzo[a]pyrene hydroxylase and p-nitrophenol UDP-glucuronyl transferase. These results add to the suggestion that the presence of an ortho halogen on a polyhalogenated biphenyl does not completely abolish toxicity or 3-methylcholanthrene-type microsomal enzyme induction.

Animals↗
PubMed1978

Metabolism of benzo(a)pyrene in animals with high aryl hydrocarbon hydroxylase levels and high rates of spontaneous cancer.

Ambystoma tigrinum found in a sewage polluted pond had high levels of aryl hydrocarbon hydroxylase (AHH) activity that decreased to the basal level of control animals after being held several months in clean water. The qualitative formation of benzo(a)pyrene (BP) metabolites by salamander hepatic microsomes was similar to those seen for other species. Inhibition of epoxide hydrase activity did not alter the total metabolite production but did change the ratio of metabolites. A correlation appears to exist between high AHH induction, the presence of polycyclic hydrocarbon pollutants, and the high rate of spontaneous cancer in salamanders.

Ambystoma↗
PubMed1993

Engineering the zinc binding site of human carbonic anhydrase II: structure of the His-94-->Cys apoenzyme in a new crystalline form.

The structure of the His-94-->Cys variant of human carbonic anhydrase II (CAII) has been determined by X-ray crystallographic methods to a resolution of 2.3 A with a final crystallographic R factor of 0.155. This variant of CAII crystallizes in orthorhombic space group P2(1)2(1)2(1) which is the first example of a new crystal form for this important zinc hydrase (the wild-type enzyme crystallizes in monoclinic space group P21 under similar crystallization conditions). Although the overall structure of the enzyme in the orthorhombic crystal form is similar to that of the wild-type protein in the monoclinic crystal form, the rms deviation of C alpha atoms between the two structures is 0.5 A. Larger structural deviations occur in regions of the protein molecule involved in crystal lattice contacts, and significant structural changes are found in the polypeptide strand containing Cys-94. Surprisingly, no electron density corresponding to a zinc ion is found in the active site of crystalline His-94-->Cys CAII, even though the stoichiometry of zinc binding to this variant in solution is confirmed by atomic absorption spectroscopy. However, the KD for zinc dissociation from the variant is increased 10(4)-fold compared with wild-type enzyme; furthermore, under the crystallization conditions of high ionic strength (1.75-2.5 M ammonium sulfate), the observed KD is increased further, which leads to zinc dissociation. Spectroscopic analysis of Co(2+)-substituted His-94-->Cys CAII indicates that the metal binds in a tetrahedral geometry with a new thiolate bond.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Structure and energetics of a non-proline cis-peptidyl linkage in a proline-202-->alanine carbonic anhydrase II variant.

The crystal structure of a human carbonic anhydrase II (CAII) variant, cis-proline-202-->alanine (P202A), has been determined at 1.7-A resolution, indicating that the wild-type geometry, including the cis-peptidyl linkage, is retained upon substitution of proline by alanine. The CO2 hydrase activity and affinity for sulfonamide inhibitors of P202A CAII are virtually identical to those of wild type. However, the substitution of cis-alanine for cis-proline decreases the stability of the folded state by approximately 5 kcal mol-1 relative to both the unfolded state and an equilibrium intermediate in guanidine hydrochloride-induced denaturation. This destabilization can be attributed mainly to the less favorable cis/trans equilibrium of Xaa-alanine bonds compared to Xaa-proline bonds in the denatured state although other factors, including increased conformational entropy of the denatured state and decreased packing interactions in the native state, also contribute to the observed destabilization. The high catalytic activity of P202A CAII illustrates that unfavorable local conformations are nonetheless endured to satisfy the precise structural requirements of catalysis and ligand binding in the CAII active site.

Alanine↗
PubMed1991

Conformational mobility of His-64 in the Thr-200----Ser mutant of human carbonic anhydrase II.

The three-dimensional structure of the Thr-200----Ser (T200S) mutant of human carbonic anhydrase II (CAII) has been determined by X-ray crystallographic methods at 2.1-A resolution. This particular mutant of CAII exhibits CO2 hydrase activity that is comparable to that of the wild-type enzyme with a 2-fold stabilization of the E.HCO3- complex and esterase activity that is 4-fold greater than that of the wild-type enzyme. The structure of the mutant enzyme reveals no significant local changes accompanying the conservative T200S substitution, but an important nonlocal structural change is evident: the side chain of catalytic residue His-64 rotates away from the active site by 105 degrees about chi 1 and apparently displaces a water molecule. The displaced water molecule is present in the wild-type enzyme; however, the electron density into which this water is built is interpretable as an alternate conformation of His-64 with 10-20% occupancy. The rate constants for proton transfer from the zinc-water ligand to His-64 and from His-64 to bulk solvent are maintained in the T200S variant; therefore, if His-64 is conformationally mobile about chi 1 and/or chi 2 during catalysis, compensatory changes in solvent configuration must sustain efficient proton transfer.

Carbonic Anhydrases↗
PubMed1992

Purification and characterization of a carbonic anhydrase II inhibitor from porcine plasma.

Plasma from many vertebrates, including pigs, contains a soluble component that inhibits the CO2 hydrase activity of carbonic anhydrase (CA). This activity was purified to homogeneity (approximately 4000-fold) from porcine plasma using a combination of DEAE-Affi-Gel Blue chromatography and carbonic anhydrase II-affinity chromatography, yielding 16 mg of inhibitory protein/L of plasma. This protein, porcine inhibitor of carbonic anhydrase (pICA), is a monomeric protein with an apparent molecular mass of 79 kDa, as determined by electrospray mass spectrometry. As isolated, pICA contains about 3 kDa of N-linked glycosylation removable by peptide N-glycosidase F. pICA inhibits CA reversibly with a 1:1 stoichiometry. pICA is a potent and specific inhibitor of the CA II isozyme, with Ki < 0.1 nM for porcine CA II at pH 7.4. Although the Ki is dependent on the CA isozyme type (CA II << CA IV << CA III approximately CA I), it is relatively insensitive to the species source, as long as it is mammalian. The Ki is pH dependent with log Ki decreasing linearly as the pH decreases, implicating at least one ionizable group with the pKa < or = 6.5 in the binding interaction. The isozyme and species dependence of the inhibition suggest that pICA interacts with amino acids on the surface of CA II.

Animals↗
PubMed2007

Carbonic anhydrase inhibitors. DNA cloning, characterization, and inhibition studies of the human secretory isoform VI, a new target for sulfonamide and sulfamate inhibitors.

The secretory isozyme of human carbonic anhydrase (hCA, EC 4.2.1.1), hCA VI, has been cloned, expressed, and purified in a bacterial expression system. The kinetic parameters for the CO2 hydration reaction proved hCA VI to possess a kcat of 3.4 x 10(5) s-1 and kcat/KM of 4.9 x 10(7) M-1 s-1 (at pH 7.5 and 20 degrees C). hCA VI has a significant catalytic activity for the physiological reaction on the same order of magnitude as the ubiquitous isoform CA I or the transmembrane, tumor-associated isozyme CA IX. A series of sulfonamides and one sulfamate have been tested for their interaction with this isozyme. Simple benzenesulfonamides were rather ineffective hCA VI inhibitors, with inhibition constants in the range of 1090-6680 nM. Better inhibitors were detected among such derivatives bearing 2- or 4-amino-, 4-aminomethyl-, or 4-hydroxymethyl moieties or among halogenated sulfanilamides (KI values of 608-955 nM). Some clinically used compounds, such as acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, topiramate, sulpiride, and indisulam, or the orphan drug benzolamide, showed effective hCA VI inhibitory activity, with inhibition constants of 0.8-79 nM. The best inhibitors were brinzolamide and sulpiride (KI values of 0.8-0.9 nM), the latter compound being also a CA VI-selective inhibitor. The metallic taste reported as a side effect after the treatment with systemic sulfonamides may be due to the inhibition of the salivary CA VI. Some of the compounds investigated in this study might be used as additives in toothpastes for reducing the acidification produced by the relevant CO2 hydrase activity of enamel CA VI, which leads to the formation of protons and bicarbonate and may have a role in cariogenesis.

Amino Acid Sequence↗
PubMed1997

Expression of the acatalytic carbonic anhydrase VIII gene, Car8, during mouse embryonic development.

The carbonic anhydrase (CA)-like protein, CA VIII, lacks the typical carbon dioxide hydrase activity of the CA isozymes. However, the high degree of amino acid sequence similarity between the products of the mouse and the human CA VIII genes suggests an important biological function. We have attempted to investigate the function of this gene in mammalian development by conducting an in situ hybridization study on sagittal sections of mouse embryos at gestation days of 9.5-16.5 using a 35S-labelled riboprobe. Results indicate that this gene (called Car8 in mice) is expressed as early as day 9.5 in a variety of organs including liver, branchial arches, neuroepithelium and developing myocardium. Between days 10.5 and 12.5, it showed a widespread distribution of mRNA expression that became more restricted as development progressed. The level of expression of Car8 mRNA was relatively high in the brain, liver, lung, heart, gut, thymus and epithelium covering the head and the oronasal cavity.

Animals↗
PubMed1973

Induction of mutations in DNA-repair deficient bacteria by a liver microsomal metabolite of aflatoxin B1.

Certain strains of Salmonella typhimurium and Escherichia coli, particularly those which are very sensitive to u.v. light, are killed when incubated with rat liver mixed function oxidases and aflatoxin B(1). UvrA or recA strains of E. coli are more susceptible than the wild-type strain, while the double mutant uvrA recA is the most sensitive strain yet tested. The aflatoxin B(1) metabolite is also able to induce reverse mutations in 2 histidine auxotrophic strains of S. typhimurium, one strain of which is reverted specifically by frame shift mutagens and the other by agents inducing base pair substitutions.Pretreatment of rats with either 3-methylcholanthrene or benzo(a)pyrene, both inducers of liver microsomal mixed function oxidases, did not alter the amount of lethal aflatoxin B(1) metabolite formed, whereas an increase was observed after phenobarbitone pretreatment. Addition of the nucleophiles methionine, cysteine, glutathione, sodium thiosulphate or sodium sulphide, or the epoxide hydrase inhibitor, cyclohexene oxide to the toxicity assay medium did not alter bacterial killing by the aflatoxin B(1) metabolite. 2,3-Dimercaptopropanol had some protective action.Toxic metabolites were also formed when 5-methoxysterigmatocystin, O-methylsterigmatocystin, parasiticol or versicolorin A, but not vericolorin B, were incubated with mixed function oxidases. The relationship between the metabolite of aflatoxin B(1) lethal to bacteria and that which initiates liver cancer is discussed.

Aflatoxins↗
PubMed1989

Renal membrane-bound carbonic anhydrase. Purification and properties.

Microsomes from perfused human donor kidneys were separated by differential centrifugation in sucrose, and thoroughly washed before solubilization by the nonionic detergent nonyl-beta-D-glucoside. The solubilized material was first applied onto an affinity chromatographic column of acetazolamide-oxirane-SepharoseR-CL-4B to remove contaminating cytoplasmic carbonic anhydrase isozymes CA I and CA II. It was then added onto an affinity column of p-aminomethylbenzene sulfonamide coupled to CM Bio-gel AR to purify the membrane-bound carbonic anhydrase activity. This resulted in a 50% pure enzyme. It was then concentrated and fractionated on an anion-exchange column, and desalted and purified to homogeneity (SDS-PAGE and isoelectric focusing) by gel filtration. The enzyme was now purified 411-fold from extractable membrane protein. Its molecular weight was 34.4 kDa from gel filtration and SDS-PAGE, and 36.7 kDa from amino acid analysis. The amino acid composition differed from that of the cytoplasmic isozymes CA I, II, and III. Antisera, produced in rabbits against the purified SDS-treated enzyme, reacted with native nondenatured membrane enzyme protein but only weakly with CA II. Kinetically the enzyme was similar to CA II with respect to hydrase and esterase activities and to inhibition by various sulfonamides. Considered together, the data suggest that the human kidney contains a membrane-bound carbonic anhydrase protein that differs from the cytoplasmic isozymes CA I, II, and III and the secretory form (CA VI) in the saliva.

Carbonic Anhydrases↗
PubMed1976

Specific positions involved in enzyme catalyzed covalent binding of benzo[a]pyrene to poly(G).

Covalent binding of benzo[a]pyrene to poly(G) was studied with the use of a radioactive assay and specifically labeled substrates to define the role of the 1, 3- and 6-positions of the hydrocarbon during this process. Binding was shown to be dependent on microsomes, NADPH, O2 and poly(G). 7, 8-Benzoflavone and 2', 2'-diethylaminoethyl-2, 2-diphenyl valerate were inhibitory w.hereas modulators of epoxide hydrase activity had little effect. 3H and 14C studies suggested a possible loss of one to two protons. Incorporation of [6-3H1]benzo[a]pyrene provided evidence that the 6-position of the hydrocarbon was not metabolized during covalent attachment to poly(G) and, furthermore, results with [1, 3, 6-3H]benzo[a]pyrene suggest that the 1- and 3-positions may not be involved either. After scaling up of the standard assay 20-fold, characterization of the tritiated BaP-poly(G) complex was carried out by hydrolysis and subsequent chromatography. Thin-layer chromatography of the isolated hydrolysis products treated with HCl or alkaline phosphatase indicated that the complex formed between BaP and poly(G) was covalently linked and composed of hydrocarbon-nucleotide(s).

Animals↗
PubMed1977

Mutagenicity and cytotoxicity of benz[alpha]anthracene diol epoxides and tetrahydro-epoxides: exceptional activity of the bay region 1,2-epoxides.

Three diastereomeric pairs of diol epoxides, two tetrahydro-epoxides, and the K-region oxide of the polycyclic aromatic hydrocarbon benz[a]anthracene were evaluated for mutagenic activity in strain TA 100 of Salmonella typhimurium and in line V79-6 of Chinese hamster lung cells. The two diastereomeric 1,2-epoxides of the trans-3,4-dihydrodiol of benz[a]anthracene are 15 to 35 times more mutagenic to the bacteria and 65 to 125 times more mutagenic to the mammalian cells than are the diastereomeric pairs of benz[a]anthracene-8,9-diol-10,11-epoxides or benz[a]anthracene-10,11-diol-8,9-epoxides. 1,2-Epoxy-1,2,3,4-tetrahydrobenz[a]anthracene is the most mutagenic and cytotoxic of the nine derivatives and is 5 and 25 times more mutagenic than 3,4-epoxy-1,2,3,4-tetrahydrobenz[a]anthracene in bacterial and mammalian cells, respectively. In either test system, benz[a]anthracene 5,6-oxide (K-region oxide) has less than 10% of the activity of any of the 1,2-epoxides derived from benz[a]anthracene. The relative stabilities of the derivatives in aqueous solution do not account for the differences in mutagenic activity because the more mutagenic derivatives tend to be less stable. The benz[a]anthracene diol epoxides, like the benzo[a]pyrene diol epoxides, are refractory to the action of epoxide hydrase. The exceptional mutagenic activity of the 1,2-epoxide derivatives of benz[a]anthracene is consistent with and supportive of the hypothesis that bay region epoxides on saturated, angular benzo-rings of unsubstituted polycyclic aromatic hydrocarbons are ultimate carcinogens.

Benz(a)Anthracenes↗
PubMed1995

Structure-assisted redesign of a protein-zinc-binding site with femtomolar affinity.

We have inserted a fourth protein ligand into the zinc coordination polyhedron of carbonic anhydrase II (CAII) that increases metal affinity 200-fold (Kd = 20 fM). The three-dimensional structures of threonine-199-->aspartate (T199D) and threonine-199-->glutamate (T199E) CAIIs, determined by x-ray crystallographic methods to resolutions of 2.35 Angstrum and 2.2 Angstrum, respectively, reveal a tetrahedral metal-binding site consisting of H94, H96, H119, and the engineered carboxylate side chain, which displaces zinc-bound hydroxide. Although the stereochemistry of neither engineered carboxylate-zinc interaction is comparable to that found in naturally occurring protein zinc-binding sites, protein-zinc affinity is enhanced in T199E CAII demonstrating that ligand-metal separation is a significant determinant of carboxylate-zinc affinity. In contrast, the three-dimensional structure of threonine-199-->histidine (T199H) CAII, determined to 2.25-Angstrum resolution, indicates that the engineered imidazole side chain rotates away from the metal and does not coordinate to zinc; this results in a weaker zinc-binding site. All three of these substitutions nearly obliterate CO2 hydrase activity, consistent with the role of zinc-bound hydroxide as catalytic nucleophile. The engineering of an additional protein ligand represents a general approach for increasing protein-metal affinity if the side chain can adopt a reasonable conformation and achieve inner-sphere zinc coordination. Moreover, this structure-assisted design approach may be effective in the development of high-sensitivity metal ion biosensors.

Binding Sites↗