Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Certain toxic effects of phenytoin are thought to result from its cytochrome P-450-catalyzed bioactivation to a reactive arene oxide intermediate that binds covalently to proteins. Using an in vitro system, we examined an alternative hypothesis based upon the cooxidation of phenytoin to a reactive free radical intermediate by prostaglandin synthetase (PGS), horseradish peroxidase, or thyroid peroxidase. Microsomes from hepatic, thyroid, seminal vesicular, or pulmonary tissues, or PGS or horseradish peroxidase, were incubated with the appropriate enzymatic cofactors to study activities of cytochromes P-450 (NADPH), PGS (arachidonic acid), thyroid peroxidase (guiaicol, H2O2), and horseradish peroxidase (H2O2). The production of potentially teratogenic, reactive phenytoin intermediates during in vitro incubations was estimated by the amount of radiolabeled phenytoin bound covalently to microsomal protein or bovine serum albumin and by the detection of a free radical intermediate using ESR spectrometry. Arachidonic acid-dependent bioactivation of phenytoin was demonstrated for purified PGS and ram seminal vesicles (RSV), as well as for liver, lung, and kidney. Optimal arachidonate concentrations varied substantially for different tissues. Arachidonate-dependent binding of phenytoin with PGS and RSV was reduced to baseline levels by coincubation with the cyclooxygenase inhibitor indomethacin. Hydrogen peroxide-dependent covalent binding of phenytoin was observed with thyroid peroxidase and horseradish peroxidase, and binding was significantly reduced in these systems and in PGS and RSV by coincubation with the peroxidase inhibitor methimazole. Glutathione, the antioxidants caffeic acid and butylated hydroxyanisole, and the free radical trapping agent alpha-phenyl-N-t-butylnitrone (PBN) all significantly reduced arachidonate-dependent phenytoin binding. Oxygen uptake was increased in a dose-dependent manner by the arachidonate-dependent bioactivation of phenytoin by PGS. ESR spin-trapping techniques using PBN indicated the generation of a free radical intermediate during the metabolism of phenytoin by PGS. These results suggest that the hydroperoxidase component of PGS, as well as thyroid peroxidase and other peroxidases, can bioactivate phenytoin to a reactive free radical intermediate, which may be toxicologically relevant.
Mol Pharmacol 1989 Apr
PMID:In vitro bioactivation of phenytoin to a reactive free radical intermediate by prostaglandin synthetase, horseradish peroxidase, and thyroid peroxidase. 253 58

The amino acid sequence of D2 protein was compared with those of calcium binding proteins and receptor for calcium channel blockers in connection with the data showing the participation of Ca2+ in photosystem 2 electron transport and the inhibition of this process by calmodulin antagonists, calcium channel blockers and local anesthetics. Protein D2 possesses a pattern analogous to the "EF-hand" sites of the calcium binding proteins. Comparison of the amino acid sequence of the calmodulin fragment binding the phenothiazine type calmodulin antagonists with the amino acid sequence of D2 protein and calcium channel protein revealed a high degree of sequence identity. Common structural features take place also between the membrane spanning segment III of D2 protein, which contains the tyrosine residue (161), responsible for ESR-signal IIS, and the membrane segment IVS5 of calcium channel protein. A model explaining the mechanism of calcium function in the oxygen-evolving system is proposed.
Mol Biol (Mosk)
PMID:[Comparative analysis of sequences of protein D2 from photosystem 2 reaction center and various Ca 2+-binding proteins]. 255 91

The characteristics of vesicles formed from Dipalmitoyl Phosphatidyl Choline (DPPC) are sensitive to the presence of perturbing molecules such as drugs, peptides, hormones and vitamins. We have used ESR spin labeling and NMR techniques for studying interaction of such molecules with lipid bilayers. ESR spin labeling has been used to monitor thermotropic behaviour of model membranes. Different NMR probes such as 1H, 31P, 13C have been used to gather information regarding the mode of interaction. It has been observed that the model membrane systems respond differently depending upon the localization of the perturbing molecules in the lipid bilayer. Small molecules such as neurotransmitters epinephrine and norepinephrine decrease gel to liquid crystalline phase transition temperature significantly even when present in small amounts. Vitamin E acetate having a hydrophobic hydrocarbon tail orients parallel to the lipid molecule and thereby exhibits dynamics similar to palmitate chain. When the acetate group is replaced by hydroxyl group (alpha-tocopherol), the phase transition becomes broad and the lipid molecules loose freedom of lateral diffusion. This can be attributed to formation of hydrogen bond between the hydroxyl group of alpha-tocopherol and phosphate moiety of lipid. The conformation of antidepressants nitroxazepine and imipramine is significantly altered when embedded in lipid bilayer. Anaesthetic etomidate not only modifies thermotropic characteristics but also induces polymorphism. The normal bilayer arrangement of lipids gets transformed into hexagonal packing. Amino acid tryptophan induces cubic phases in the normal bilayer arrangement of DPPC dispersions. Peptide gonadoliberin shows a reduced internal motion due to the lipid peptide interaction. The major consequences of binding of lipids with externally added molecules are changes in the fluidity and permeability properties of membranes. It has been shown that permeability is effected by the presence of molecules such as propranolol, alpha-tocopherol and its analogue, neurotransmitters, etc. The magnetic resonance methods have thus evolved as power techniques in the study of membrane structure and function.
Mol Cell Biochem
PMID:Effect of incorporation of drugs, vitamins and peptides on the structure and dynamics of lipid assemblies. 269 35

Membranes of human spleen cells were hydrolyzed by papain and the extracellular portions of HLA antigen molecules isolated by monoclonal antibodies fixed on Sepharose. The isolated proteins were spin-labeled by TEMPO-dichlorotriazine. The values of rotational correlation times (tau) of spin-labeled proteins were calculated using dependencies of magnetic parameters found from ESR spectra vs viscosity at constant temperature. The tau-values were equal to 8 nsec for class I molecules and 14 nsec for class II molecules. These values were significantly lower than those predicted for a rigid sphere with dimensions equal to the extracellular portions of HLA molecules (20 nsec). This fact suggests the existence of flexibility in poly-functional HLA molecules, which seems to be important for their biological activity. In this respect, extracellular portions of HLA molecules resemble flexible Fc fragments (tau = 12 nsec) and differ from rigid Fab fragments (tau = 20 nsec) of immunoglobulins G. The rotation of the oligosaccharide chains attached to HLA molecules is restricted.
Mol Immunol 1987 Jul
PMID:Extracellular portions of HLA antigens are not compact globulae. 282 87

Membranes of human splenocytes were hydrolyzed by papain and extracellular portions of class I and class II HLA antigen molecules were isolated by monoclonal antibodies fixed on Sepharose 4B. The isolated proteins were spin-labeled by TEMPO-dichlorotriazine and the values of rotational correlation times (tau) of labeled proteins were found using dependencies of ESR spectra parameters vs viscosity at constant temperature. The tau-values were equal to 8 ns for class I molecules and 14 ns for class II molecules. These values are 2-3 times lower than predicted for a rigid ellipsoid with mol wt. 50 kDa (about 20 ns). This fact suggests the existence of flexibility of HLA molecules which seems to be important for their biological activity. In this respect extracellular portions of HLA antigen molecules resemble flexible Fc fragments (tau = 12 ns) and differ from rigid Fab fragments (tau = 20 ns) of immunoglobulins G. The values of tau of spin-labeled proteins adsorbed from membrane hydrolysates on IgG-column was equal to 6.5 ns. The proteins adsorbed on lentil lectin column (after isolation of HLA proteins) have the tau-values equal to 9 ns.
Mol Biol (Mosk)
PMID:[Intramolecular mobility of human major histocompatibility complex protein. A spin-label study]. 282 88

The reaction of oxyhemoglobin with phenylhydrazine has received considerable attention for many decades. The basis for this interest stems from the ability of phenylhydrazine and hydrazine-based drugs to induce hemolytic anemia. Considerable evidence obtained from in vitro ESR experiments implicates free radicals in the events leading to red blood cell hemolysis. However, until this report, no corroborating ESR evidence for in vivo free radical formation has been presented. We have successfully employed ESR to detect the formation of a radical adduct in the blood of rats which received an intragastric dose of phenylhydrazine followed by an intraperitoneal injection of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). An immobilized radical adduct was detected by ESR when phenylhydrazine was administered in a dosage comparable to that prescribed for currently employed hydrazine-based drugs. We were also able to detect this immobilized DMPO adduct when hydrazine was employed in place of phenylhydrazine in the rat studies. The results of a series of experiments led us to ascribe this DMPO radical adduct to the trapping of a hemoglobin-derived thiyl free radical.
Mol Pharmacol 1988 Mar
PMID:In vivo rat hemoglobin thiyl free radical formation following phenylhydrazine administration. 283 22

CCl4 has been shown previously to be metabolized to the trichloromethyl radical (.CCl3) and to a novel oxygen-containing carbon dioxide anion radical (.CO2-) in the perfused rat liver and in vivo. Since the role of free radicals in CCl4-induced hepatotoxicity is unclear, these studies were designed to determine if a relationship between .CO2- formation and halocarbon-induced hepatotoxicity exists. CCl4 or bromotrichloromethane (CBrCl3) was infused into livers from control or phenobarbital-treated rats perfused with either nitrogen- or oxygen-saturated Krebs-Henseleit bicarbonate buffer. Samples of effluent perfusate and chloroform/methanol extracts of liver were analyzed by ESR spectroscopy for free radical adducts following infusion of halocarbon and the spin trap, phenyl-t-butylnitrone (PBN). Hyperfine coupling constants and 13C-isotope effects observed in the ESR spectra of organic extracts of liver demonstrated the presence of the PBN radical adduct of .CCl3 from both halocarbons. Radical adducts in aqueous extracts of liver and effluent perfusate had hyperfine coupling constants and 13C-isotope effects identical to those of PBN/.CO2- generated chemically from formate. The PBN/.CO2- radical adduct was also observed in urine following the intragastric administration of CBrCl3 and PBN. Detection of PBN/.CO2- adducts in the effluent perfusate was decreased 3- to 4-fold by DIDS (0.2 mM), an inhibitor of the plasma membrane anion transport system. The rate of formation of PBN/.CO2- was decreased 2- to 3-fold following inhibition of cytochrome P-450-dependent monooxygenases by metyrapone (0.5 mM) and was increased about 2-fold by induction of cytochrome P-450 by phenobarbital pretreatment. Toxicity of halocarbons in the perfused liver was assessed by measuring the release of lactate dehydrogenase (LDH) into the effluent perfusate in livers from phenobarbital-treated rats under conditions identical to those employed to detect radical adducts (i.e., during the infusion of CCl4 or CBrCl3 into livers perfused with either nitrogen- or oxygen-saturated perfusate). Under all conditions studied, PBN/.CO2- was detected in the effluent perfusate within 2-4 min. Metabolism of halocarbons to PBN/.CO2- was 6- to 8-fold faster during perfusion with nitrogen-saturated rather than with oxygen-saturated perfusate. Concomitantly, liver damage detected from LDH release occurred much sooner during halocarbon infusion in the presence of nitrogen-saturated rather than oxygen-saturated perfusate. A good correlation between the rate of formation of PBN/.CO2- and the time of onset of LDH release following halocarbon infusion was observed.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1988 Mar
PMID:The carbon dioxide anion radical adduct in the perfused rat liver: relationship to halocarbon-induced toxicity. 283 23

ESR and microcalorimetry methods were employed to investigate the thermotropic properties and structure of proteoliposomes that incorporate cytochrome P450 and DMPC-DMPG binary mixtures depending on cytochrome P450 content and phospholipid composition. The microcalorimetry data demonstrated that the incorporation of cytochrome P450 into the phospholipid mixture resulted in bilayer thermal stabilization. The maximum shift of the temperature and proteoliposome transition enthalpy were achieved at the protein/lipid molar ratio of 1:1000 in almost equimolar phospholipid mixture. Using fatty acids that were spin-labeled at different positions (C5, C12, C16), it has been shown that the incorporation of cytochrome P450 into lipid mixtures containing 0-100% DMPG decreases C12 and C16 mobility and increases the C5 order parameter at transition phase (30 degrees C) and liquid crystal phase (37 degrees C) of bilayer. The maximum alteration amplitude of the probes used was not characteristic for the separate DMPC and DMPG but rather for the mixture at the molar ratio close to equimolar value. It is proposed that cytochrome P450 incorporation into the binary mixture initiated the formation of the bilayer crystal-like phase.
Mol Biol (Mosk)
PMID:[Interaction of cytochrome P-450 with phospholipids in dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol mixtures]. 283 88

The spin-labeled bovine serum albumin and IgG were studied in search of an experimental approach for comparison of different models of rotational mobility of spin label. These models are: the model of isotropic motion of spin label together with the macromolecule (IM); the model of highly anisotropic motion of spin label (HAM); and the model of slow isotropic motion of label around the binding site (SIML). The experimental spectra were measured on a common X-band ESR spectrometer and on the unique 140 GHZ (lambda = 2 mm) ESR spectrometer under the same conditions. Theoretical spectra were computer-calculated according to Freed's theory. We have found, that the results of temperature-viscosity experiments in X-band are contradictory to the model of IM both for the BSA and IgG species. The models of HAM and SIML for the BSA give identical X-band spectra. The bovine serum albumin spectra in the 2 mm region strongly contradict to the assumptions of the HAM model. Also, the SIML model fails to describe the experimental spectra in terms of isotropic motion of the spin label around the binding site. X-band spectra of IgG can not be explained by the SIML model, while the same spectra in the 2 mm region can not be explained by the HAM model.
Mol Biol (Mosk)
PMID:[Analysis of existing models of spin label movement during spin labeling of biological macromolecules]. 283 90

Prostaglandin H synthase (PHS) hydroperoxidase-mediated metabolism of phenylbutazone and the relationship of this metabolism to the inhibition of PHS cyclooxygenase by phenylbutazone was investigated. Phenylbutazone was metabolized to several intermediates and metabolites. A phenylbutazone carbon-centered radical (aN = 14.6 G) formed by PHS hydroperoxidase was trapped by 2-methyl-2-nitrosopropane and detected by ESR in incubations with ram seminal vesicle microsomes. 4-Hydroperoxy- and 4-hydroxyphenylbutazone were isolated from incubations of phenylbutazone with either ram seminal vesicle microsomes or horseradish peroxidase. Phenylbutazone (100 microM-2 mM) inhibited PHS cyclooxygenase in incubations of PHS apoenzyme reconstituted with hematin. Phenylbutazone (5-250 microM) did not inhibit PHS cyclooxygenase in incubations of PHS apoenzyme reconstituted with manganese protoporphyrin IX, which lacks hydroperoxidase activity. Thus, metabolism of phenylbutazone by PHS hydroperoxidase is required for it to inhibit PHS cyclooxygenase. 4-Hydroperoxy- and 4-hydroxyphenylbutazone were ineffective inhibitors of PHS cyclooxygenase. Other hydroperoxides that easily rearrange to peroxyl radicals were potent inhibitors of PHS cyclooxygenase, suggesting that the phenylbutazone peroxyl radical may be the inhibitor. 4-Hydroperoxyphenylbutazone was not reduced to 4-hydroxyphenylbutazone by PHS hydroperoxidase. We propose that 4-hydroxyphenylbutazone formation occurs by a nonenzymatic reaction of two phenylbutazone peroxyl radicals and their subsequent rearrangement to alkoxy radicals, which abstract hydrogen atoms. Our data indicate the importance of PHS hydroperoxidase in the inactivation of PHS cyclooxygenase by peroxides.
Mol Pharmacol 1988 Aug
PMID:Prostaglandin hydroperoxidase-dependent oxidation of phenylbutazone: relationship to inhibition of prostaglandin cyclooxygenase. 284 54


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