Purification of glutathione reductase from chicken liver and investigation of kinetic properties

Glutathione reductase was purified from chicken liver and some characteristics of the enzyme were investigated. The purification procedure was composed of four steps: preparation of homogenate, ammonium sulfate precipitation, 2′,5′-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. Owing to the four consecutive procedures, the enzyme was purified 1714-fold, with a yield of 38%. Specific activity at the final step was 120 enzyme unit (EU)/mg of protein. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme was found to be 100 kDa by Sephadex G-200 gel filtration chromatography, and the subunit molecular weight was found to be 43 kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, and optimum temperature were 7.0, 7.4, 0.75 M Tris-HCl buffer including 1 mM EDTA, and 50°C, respectively. K _M and V _max values for NADPH and glutathione disulfide (GSSG) substrates were also determined for the enzyme.     

Characterization of aldose reductase and aldehyde reductase from the medulla of rat kidney

Aldose reductase and aldehyde reductase from the medulla of the rat kidney have been purified to homogeneity by using affinity chromatography, gel filtration and chromatofocusing. The molecular weights of aldose reductase and aldehyde reductase by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were found to be 37000 and 39000, respectively. The isoelectric points of aldose reductase and aldehyde reductase were found to be 5.4 and 6.2 by chromatofocusing, respectively. The major differences of amino acid compositions between both enzymes were found in serine, alanine and aspartic acid. Substrate specificity studies showed that aldose reductase utilized aldo-sugars such as D-glucose and D-galactose, but aldehyde reductase did not use them. The Km values of aldose reductase for various substrates were lower than those of aldehyde reductase. Aldose reductase utilized both reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduced nicotinamide adenine dinucleotide (NADH) as coenzymes, whereas aldehyde reductase utilized only NADPH. The presence of the sulfate ion resulted in a dramatic activation of aldose reductase whereas it did not affect aldehyde reductase activity. These enzymes were strongly inhibited by the known aldose reductase inhibitors. However, aldose reductase was more susceptible than aldehyde reductase to inhibition by the aldose reductase inhibitors.     

A molecular dynamics study of the inhibition of chicken dihydrofolate reductase by a phenyl triazine

Molecular dynamics simulations have been used to investigate the ternary complex formed between chicken liver dihydrofolate reductase, a phenyl triazine inhibitor, and reduced nicotinamide adenine dinucleotide phosphate (NADPH). The solvent was represented by a sphere of water molecules encompassing the system. We report the results of quantum mechanical calculations of the rotational barrier in the pyrophosphate link and the barrier to inversion of the triazine ring. AMBER parameters for NADPH and the triazine are provided. Over the course of a 300-ps molecular dynamics simulation of the ternary complex in water, the triazine inhibitor maintains the same hydrogen bonding and hydrophobic interactions with the enzyme that are observed in the X-ray crystal structure. Despite the low calculated barrier to inversion of the triazine ring, a single puckered conformation is observed throughout the simulation. It is proposed that this is primarily due to interactions with Phe34, which maintains an approximately parallel orientation to the triazine ring. The nicotinamide portion of NADPH maintains the interactions observed in the crystal structure, but more conformational change is observed at the adenine end together with associated changes in the protein. Two conformations for the sidechain of Tyr31 are present in the X-ray structure. The main simulation reported here corresponds to the conformation characterized by (χ₁ = ? 161°, χ₂ = ? 103°). A separate simulation was also performed in which the sidechain of Tyr31 was initially set to the other conformation present in the crystal structure (χ₁ = 139°, χ₂ = ?99°). During this simulation, χ₁ of this sidechain gradually changed until it occupied the region characterized by χ₁ = ?160°, thereby suggesting that this is the preferred conformation for this residue. The simulation required 200 ps to reach structural equilibrium (as measured by the root mean square, rms, deviation from the initial crystal structure), thus reinforcing the view that simulations of at least several hundreds of picoseconds are desirable when studying such systems. © 1995 John Wiley & Sons, Inc.     

Analysis of iron storage proteins in chicken liver and spleen tissues in comparison with human liver ferritin by Mössbauer spectroscopy

Summary Characterization of iron storage proteins in liver and spleen from normal chicken and chicken with lymphoid leukemia in comparison with human liver ferritin were considered by M?ssbauer spectroscopy (preliminary results). Small differences in M?ssbauer hyperfine parameters for both normal and lymphoid leukemia chicken liver and spleen were observed. The value of quadrupole splitting for human liver ferritin was higher than those for chicken tissues. A decrease of iron content in lymphoid leukemia chicken tissues was also found, however, the reason of this fact (pathology or feeding) was not clear yet.     

Kinetics and mechanism of cyclohexane oxidation with molecular oxygen in the presence of propionic aldehyde

The kinetics and mechanism of the liquid-phase oxidation of cyclohexane with molecular oxygen in the presence of the additives of propionic aldehyde are studied at 303.0, 322.5, and 341.5 K by measuring the rates of oxygen and propionic aldehyde consumption and the yields of the main reaction products (cyclohexanol (COL), cyclohexanone (CON), cyclohexyl hydroperoxide, and propionic acid and peracid). A kinetic scheme is proposed and rate constants of elementary reactions are estimated based on the analysis of their rates and the yields of the main cyclohexane products. The key reactions of the main steps (including chain initiation, propagation, and termination) are determined. An increase in the rate of cyclohexane oxidation and the yield of the target products (cyclohexanol, cyclohexanone, and cyclohexyl hydroperoxide) in the presence of propionic aldehyde suggests that highly active acylperoxy radicals participate in chain propagation. The [CON]/[COL] ratio indicates that these products are mainly formed in chain propagation. The strong effect of the Baeyer-Villiger rearrangement on both the rate of oxygen consumption and the yield of the target products at the initial stages of the process and at high propionic aldehyde concentrations is explained.     

Separation of different forms of transferrin by isoelectric focusing to detect effects on the liver caused by xenobiotics

Several genetic variants and also isoforms of transferrin differing in carbohydrate structure can be separated by polyacrylamide or agarose gel isoelectric focusing. Numerous blood plasma or serum samples can be analyzed in parallel in each gel. Studies of the heterogeneity of transferrin have already revealed many results of importance to different fields of human medicine. Gene typing can give important and useful information for paternity determination and in forensic medicine. The gene type C 2 seems to have increased frequency in certain malfunctions. Futhermore, functional abnormalities of liver cells can be revealed by determination of the concentrations of transferrin isoforms differing mainly in their carbohydrate parts. The isoforms can be quantified with zone immunoelectrophoresis assay. Thus valuable information can be obtained about important modulated regulations of cell and membrane functions, even when these are disturbed by disease and xenobiotics. The information may be useful e.g. in the detection of individuals suffering from toxic effects, to identify toxic agents and exposure conditions. Studies of house painters revealed that exposure to different types of paints had an effect on transferrin. Determination of the concentration of the isotransferrin with pI 5.7 in blood samples from alcoholics can be used as a marker for the detection of liver dysfunction and for the monitoring of therapy treatments. In addition, by analyzing the isotransferrins a rare genetic abnormality can be detected.     

Quantum-chemical study of the molecular forms of vanillin

Equilibrium structural parameters, dipole moments, relative energies, vibration spectra, and ionization potentials of various conformers of benzoid (B), p- and o-quinoid (Q), and deprotonated anionic forms of vanillin in a vacuum and in a polarizable medium with dielectric properties of water were calculated by the quantum-chemical method (U)B3LYP/cc-pVTZ. The intramolecular hydrogen bond in free vanillin benzoid molecule B1 is characterized by the energy of 4 kcal mol⁻¹ and by the barrier of cleavage 8 kcal mol⁻¹ that correspond to the conformational transition B1 → B2. The energy of the optimal p-quinoid conformer (Q1) is 22 kcal mol⁻¹ larger than the energy of B1 in a vacuum and 15 kcal mol⁻¹ in water. The p-quinoid forms greatly exceeds the benzoid forms by dipole moment, electrophilicity, and nucleophilicity. The structure calculated for the anion is intermediate between benzoid and p-quinoid forms, and the calculated energy of polarization stabilization in water is 57 kcal mol⁻¹.     

Determination of the liver cytosolic proteins that bind to p-hydroxyacetophenone

The purpose of the present study was to determine the proteins that bind to acetophenones in the liver. Immobilized p-hydroxyacetophenone (p-HAP) was used as a ligand of affinity chromatography. Analysis using sodium dodesyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that five polypeptides in the liver cytosolic fraction specifically bound to the p-HAP matrix. These polypeptides were digested with Lys-specific protease and used to generate peptide maps by reversed-phase high-performance liquid chromatography. Consequently, identification from a data base of protein sequences revealed that the five polypeptides were glycogen phosphorylase, cytosolic aldehyde dehydrogenase, adenosine kinase, class I alcohol dehydrogenase and glutathione S-transferase A2. In addition to p-HAP, acetylsalicylic acid also displayed a prominent ability to elute these five enzymes from the p-HAP affinity column loaded with the cytosolic fraction of the liver. Thus, p-HAP has affinities to the above liver enzymes and is a useful ligand for analysis of them.     

Separation and determination of protocatechuic aldehyde and protocatechuic acid in Salivia miltorrhrza by capillary electrophoresis with amperometric detection

Capillary electrophoresis with amperometric detection was applied to separate and determine protocatechuic aldehyde and protocatechuic acid in Salivia miltorrhrza preparations. The electrode used was a 0.3 mm diameter carbon disk electrode fixed in a wall-jet with amperometric detection. Under the optimum conditions, the two analytes were separated completely within 8 min. Excellent linearity was obtained in the concentration ranges of 0.25-100.0 microg ml(-1) and 0.50-100.0 microg ml(-1) for protocatechuic aldehyde and protocatechuic acid, respectively. The detection limits were 0.10 microg ml(-1) of protocatechuic aldehyde and 0.25 microg ml(-1) of protocatechuic acid, which were found to be lower than those of other methods that determine protocatechuic aldehyde (3,4-dihydroxybenzaldehyde) and protocatechuic acid (3,4-dihydrobenzoic acid) simultaneously. The mean recoveries of protocatechuic aldehyde and protocatechuic acid were 97.4% and 103.3%. This method has been successfully applied to monitor these two components in real samples such as Salivia miltorrhrza and its two traditional Chinese medicinal preparations.     

Evidence against in vitro modulation of rat liver cholesterol 7 alpha-hydroxylase activity by phosphorylation-dephosphorylation: comparison with hydroxymethylglutaryl CoA reductase

The activity of cholesterol 7 alpha-hydroxylase in rat liver microsomes was investigated under conditions favourable for phosphorylation-dephosphorylation. The enzyme activity was similar in the presence or absence of sodium fluoride during preparation. Preincubation with ATP and magnesium did not affect the enzyme activity. Cholesterol 7 alpha-hydroxylase was inhibited by alkaline phosphatase, but this inhibition was similar also after inactivation of the phosphatase. Under similar conditions, rat hepatic hydroxymethylglutaryl CoA reductase activity was clearly modulated in agreement with phosphorylation-dephosphorylation. The absence of such a modulation of cholesterol 7 alpha-hydroxylase argues against involvement of phosphorylation-dephosphorylation in the regulation of this enzyme.     

The further crystallographic refinement of trichosanthin at 2.7A resolution and the comparison among the three molecular structures in two crystal forms

The three-dimensional structure of trichosanthin at 2.7A resolution has been improved further, by refitting one of the C-terminal tails, adjusting 16 residues in the molecular surface regions, discarding some water molecules with high B values, and adjusting weights during the further refinement. The R-factor has been reduced to 18.5% and the r.m.s deviations from ideal geometry are also improved. The structures of the two molecules in the monoclinic asymmetric unit and the only molecule in the orthorhombic asymmetric unit are compared with one another. The main-chain structures for most of the residues in the three molecules are substantially the same. However, the courses of the three C-terminal tails are completely different, and the intermolecular interactions resulting from the particular packing of the molecules in the crystals account for the differences. The strand Be-2 and the preceding B-turn in small domain show large r.m.s. deviations among the three molecules and they are also involved in i     

蛋白质的微芯片电泳分离及其与脱氧核糖核酸迁移规律的比较

在自制的聚二甲基硅氧烷(PDMS)微芯片上,使用十二烷基磺酸钠(SDS)无胶筛分电泳分离体系(10 g/L的羟乙基纤维素(HEC), 1 g/L的SDS, 40 mmol/L磷酸盐缓冲溶液,pH 7.0),采用在线自校正激光诱导荧光检测方法,在6.4 min内高效分离了异硫氰酸荧光素(FITC)衍生的6种蛋白质标准样品,连续6次电泳所得迁移时间的相对标准偏差(RSD)均小于10%。用自主建立的脱氧核糖核酸(DNA)定量分离模型对蛋白质迁移数据进行拟合,发现SDS-蛋白质复合物迁移规律与DNA相似,但迁移淌度与相对分子质量及电场强度之间的线性关系明显变差,可见原DNA分离模型要扩展到蛋白质范围必须对一些参数进行校正。     

Ground and excited state isomeric forms of the anthracene-diethylaniline molecular exciplex

The potential energy surfaces for the molecular complex formed between anthracene (the electron acceptor) andN ,N-diethylaniline (DEA) (the electron donor) were computed as the quasi-adiabatic states resulting from the configuration interaction between the ground (AD), locally excited (A^*D) and charge-transfer (A⁻D⁺) excited electronic configurations. The results clearly indicate the existence of three geometrically and energetically different isomeric forms of the complex in the ground state. In the excited state, the potential energy surfaces reveal the existence of five well-defined equilibrium configurations separated by energy barriers and characterized by different admixtures of the (A^*D) and (A⁻D⁺) electronic configurations. Such a variety of equilibrium configurations in the ground and excited states is, in part, accounted for by the existence of two different conformational forms of DEA that can form complexes with anthracene, and are characterized by different balances between steric effects and interactions of electronic charge distributions in the complex components. The energies of transitions between the relevant ground and excited state equilibrium configurations were calculated and compared with spectroscopic data of a jet-cooled complex obtained in supersonic beam experiments. These transitions were successfully assigned to the observed resonance-like and exciplex-like spectra, and this enabled interpretation of observed changes in the fluorescence excitation and fluorescence spectra of the complex upon excess excitation energy.     

Purification and characterization of befunolol reductase from rabbit liver

An enzyme (befunolol reductase) which catalyzes the reduction of befunolol to dihydrobefunolol was purified from the cytosolic fraction of rabbit liver to homogeneity by various chromatographic techniques. Befunolol reductase had molecular weights of 29000 on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and 34000 on gel filtration. The enzyme required reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor and showed an optimal pH of 6.5. The apparent Km and Vmax values of the enzyme for the reduction of befunolol were 1.7 mM and 4.4 units/mg, respectively. Flavonoids, sulfhydryl reagents, heavy metals and coumarins strongly inhibited the enzyme. The enzyme catalyzed the reduction of a variety of aromatic ketones. In addition to befunolol, some ketone-containing drugs such as daunorubicin and levobunolol were efficiently reduced by the enzyme. On the basis of substrate specificities for steroids, befunolol reductase purified from the cytosolic fraction of rabbit liver appeared to be a 3 alpha-hydroxysteroid dehydrogenase.     

Determination of adduct forms of antitumor ruthenium(III) complex with cytosolic components by capillary electrophoresis with mass spectrometry

Using capillary electrophoresis with inductively coupled plasma mass spectrometry, the transformation of adduct forms of indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] with apo-transferrin and albumin under the effect of active intracellular reducing agents, glutathione and ascorbic acid, and citric acid as a complexant was studied under conditions simulating a cytosolic environment. These adducts of ruthenium with transport proteins are forms in which the anticancer drug exists after intravenous administration. Two modes of interaction of adducts with glutathione, ascorbic acid, and citric acid were studied: in a capillary using a background electrolyte containing a cytosolic active ingredient and upon incubation of the reaction components in the off-line mode, followed by their separation and determination by capillary electrophoresis. Incubation with intracellular components and separation were carried out in a 10 mM phosphate buffer solution (pH 6.0) containing 4 mM NaCl. The effect 1–10 mM of glutathione, 10 mM of ascorbic acid, and 50 mM of citric acid on the adducts was studied. It is shown that under the selected model conditions, new forms of ruthenium do not emerge.