Thiol-dependent changes in the properties of rat liver sulphotransferases

1. Two enzymes (A and B) which catalyse the sulphation of p-nitrophenol and l-tyrosine methyl ester have been isolated from female rat livers. One of these enzymes (A) also catalyses the sulphation of dehydroepiandrosterone. 2. The K(m) values for the sulphation of p-nitrophenol and l-tyrosine methyl ester by enzyme B at pH7.5 are 1.5mum and 2.9mm respectively. 3. Enzyme B is oxidized on keeping at 0 degrees C when the K(m) and V(max.) values for the sulphation of p-nitrophenol are increased approx. 200-fold and fourfold respectively. This oxidized preparation of enzyme B fails to catalyse the sulphation of l-tyrosine methyl ester. 4. When the oxidized form of enzyme B is kept at 0 degrees C and low ionic strength then further forms of p-nitrophenol sulphotransferase are produced having even lower affinities for the sulphate acceptor. 5. The K(m) value for adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate is not affected during storage of the enzyme under these conditions. 6. Prolonged storage of enzyme B at low ionic strength leads to a considerable degree of polymerization of p-nitrophenol sulphotransferase and l-tyrosine methyl ester sulphotransferase. 7. The changes in the kinetic properties and molecular size of enzyme B during storage are reversed by dithiothreitol.     

Thiol-dependent degradation of protoporphyrin IX by plant peroxidases

Protoporphyrin IX (PP) is the last porphyrin intermediate in common between heme and chlorophyll biosynthesis. This pigment normally does not accumulate in plants because its highly photodynamic nature makes it toxic. While the steps leading to heme and chlorophylls are well characterized, relatively little is known of the metabolic fate of excess PP in plants. We have discovered that plant peroxidases can rapidly degrade this pigment in the presence of thiol-containing substrates such as glutathione and cysteine. This thiol-dependent degradation of PP by horseradish peroxidase consumes oxygen and is inhibited by ascorbic acid.     

The Effects of Alkali Metal Cations and Common Anions on the Frog Skin Potential

The effects on the potential difference across isolated frog skin (R. catesbeiana, R. pipiens) of changing the ionic composition of the bathing solutions have been examined. Estimates of mean values and precision are presented for the potential changes produced by substituting other alkali metal cations for Na at the outside border and for K at the inside border. In terms of ability to mimic Na at the outside border of bullfrog skin, the selectivity order is Li > Rb, K, Cs; at the outside border of leopard frog skin, Li > Cs, K, Rb. In terms of ability to mimic K at the inside border of bullfrog and leopard frog skin: Rb > Cs > Li > Na. Orders of anion selectivity in terms of sensitivity of the potential for the outside border of bullfrog skin are Br > Cl > NO₃ > I > SO₄, isethionate and of leopard frog skin are Br, Cl > I, NO₃, SO₄. An effect of the solution composition (ionic strength?) on the apparent Na-K selectivity of the outside border is described. The results of the investigation have been interpreted and discussed in terms of the application of the constant field equation to the Koefoed-Johnsen-Ussing frog skin model. These observations may be useful in constructing and testing models of biological ionic selectivity.     

Thiol-dependent peroxidases care little about homology-based assignments of function

Thiol-dependent peroxidase systems are reviewed with special emphasis on their potential use as drug targets. The basic catalytic mechanism of the two major thiol-peroxidase families, the glutathione peroxidases and the peroxiredoxins, are reasonably well understood. Sequence-based predictions of substrate specificities are still unsatisfactory. GPx-type enzymes are not generally specific for GSH but may specifically react with CXXC motifs as present in thioredoxins or tryparedoxins. Inversely, the peroxiredoxin family that was believed to be specific for CXXC-type proteins, also comprises glutathione peroxidases. Since structure-based predictions of function are also limited by small data bases, the increasing number of sequences emerging from genome projects require enzymatic characterization and genetic proof of relevance before they can be classified as drug targets.     

Effects of Sulfuroxy Anions on Degradation of Pentachlorophenol by a Methanogenic Enrichment Culture

We studied the degradation of pentachlorophenol (PCP) under methanogenic and sulfate-reducing conditions with an anaerobic mixed culture derived from sewage sludge. The consortium degraded PCP via 2,3,4,5-tetrachlorophenol, 3,4,5-trichlorophenol, and 3,5-dichlorophenol and eventually accumulated 3-chlorophenol. Dechlorination of PCP and metabolites was inhibited in the presence of sulfate, thiosulfate, and sulfite. A decrease in the rate of PCP transformation was noted when the endogenous dissolved H₂ was depleted below 0.11 μM in sulfate-reducing cultures. The effect on dechlorination observed with sulfate could be relieved by addition of molybdate, a competitive inhibitor of sulfate reduction. Addition of H₂ reduced the inhibition observed with sulfuroxy anions. The inhibitory effect of sulfuroxy anions may be due to a competition for H₂ between sulfate reduction and dechlorination. When cultured under methanogenic conditions, the consortium degraded several chlorinated and brominated phenols.     

Thiol-dependent peroxidases care little about homology-based assignments of function

Abstract

Thiol-dependent peroxidase systems are reviewed with special emphasis on their potential use as drug targets. The basic catalytic mechanism of the two major thiol-peroxidase families, the glutathione peroxidases and the peroxiredoxins, are reasonably well understood. Sequence-based predictions of substrate specificities are still unsatisfactory. GPx-type enzymes are not generally specific for GSH but may specifically react with CXXC motifs as present in thioredoxins or tryparedoxins. Inversely, the peroxiredoxin family that was believed to be specific for CXXC-type proteins, also comprises glutathione peroxidases. Since structure-based predictions of function are also limited by small data bases, the increasing number of sequences emerging from genome projects require enzymatic characterization and genetic proof of relevance before they can be classified as drug targets.     

Thiol-dependent protective systems in alimentary prevention of the toxic effect of carminomycin

The state of the thiol-dependent systems i.e. concentration of the SH-groups, activity of glutathione reductase and glutathione-S-transferase, carminomycin antitumor and toxic effects was studied under conditions of tumor growth and carminomycin therapy with the use of prophylactic rations (PR) aimed at stimulating the cell thiol-dependent and antioxidant systems for decreasing the drug toxic action. It was shown that addition of sulfur-containing amino acids, selenium and vitamin E to the ration of healthy and tumor-bearing rats (Walker carcinosarcoma 256) induced a decrease in the level of the SH-groups in the liver just likely promoting efficient extrahepatic usage of glutathione. After administration of carminomycin a long with the PR use, the liver showed the thiol-preserving capacity evidenced by a decrease or complete elimination of the above effect of the ration. The use of PR resulted in a marked increase in the glutathione-S-transferase activity in cytosol and to a lesser extent in the liver microsomes. A regulating effect of the PR on the activity of glutathione reductase was observed: its inhibition in the healthy animals and stimulation after carminomycin administration in the heart of the healthy animals and the liver of the tumor-bearing animals.     

Effects of Anions on the Capacity and Affinity of Copper Adsorption in Two Variable Charge Soils

Effects of nitrate,(NO₃⁻) chloride (Cl⁻), sulfate (SO₄^2-, and acetate (Ac⁻) on Cu²⁺ adsorption and affinity of the adsorbed Cu²⁺ were evaluated in two Fe and Al enriched variable charge soils from Southern China. The maximum adsorption of Cu²⁺ (M, a parameter from the Langmuir isotherm model) in the presence of different anions decreased in the order Cl⁻ > Ac⁻ > NO₃⁻ > SO₄^2- for both soils. The clayey loamy soil (mixed siliceous thermic Typic Dystrochrept, TTD), developed on the Arenaceous rock, adsorbed less Cu²⁺ than the clayey soil (kaolinitic thermic Plinthudults, KTP), derived from the Quaternary red earths, regardless of anion type present in the medium. The affinity of adsorbed Cu²⁺ to both soils could be characterized by the Kd (distribution coefficient) values and successive extraction of the adsorbed Cu²⁺ with 1-mol NH₄Ac L⁻¹. The log₁₀Kd value was smaller for the TTD soil than for the KTP soil and decreased in the order of Cl⁻ > NO₃⁻ > SO₄^2- > Ac⁻ at low initial Cu²⁺ concentrations (≤40 mg Cu²⁺L⁻¹), whereas at 80 mg Cu²⁺L⁻¹, the log₁₀Kd value was similar for NO₃⁻, SO₄^2-, and Ac⁻, but was slightly higher for Cl⁻. Complete extraction of Cu²⁺ adsorbed in the presence of Ac⁻ was achieved. Influence of NO₃⁻ and SO₄^2- on the affinity of adsorbed Cu²⁺ was similar, but the effects of Cl⁻ depended on the initial Cu²⁺ concentrations. The extracted percentage of the adsorbed Cu²⁺ in the presence of NO₃⁻ or SO₄^2- increased with increasing Cu²⁺ adsorption saturation. The presence of Cl⁻, NO₃⁻, or SO₄^2- markedly decreased the equilibrium solution pH for both soils with increasing initial Cu²⁺ concentrations, and the delta pH values at the highest Cu²⁺ level were 0.5, 0.63, and 0.55 U for the TTD soil and 0.79, 0.84, and 0.93 U for the KTP soil, respectively for the three anions. The presence of Ac⁻ had a minimal influence on the equilibrium solution pH because of the buffering nature of the NaAc/HAc medium which buffered the released protons. The effects of anions on Cu²⁺ adsorption and affinity of the adsorbed Cu²⁺ were dependent on anion types and were apparently related to the altered surface properties caused by anion adsorption and/or the formation of anion– Cu²⁺ complexes.     

Structural Effects of Cl and Other Anions on the Water Oxidizing Complex of Chloroplast Photosystem II

To further our understanding of the role of Cl⁻ and certain other monovalent anions in the oxygen evolving photosystem II of chloroplasts, dissociating and stabilizing anion effects on the extrinsic 17 and 23 kilodalton polypeptides of the photosynthetic water oxidizing complex were investigated. It was found that (a) the dissociation of the two polypeptides in Cl⁻ free media of pH ≈ 7 was enhanced by millimolar concentrations of the divalent anion SO₄²⁻ and also by divalent cations like Mg²⁺ and Ca²⁺; (b) the dissociation was opposed by relatively low concentrations of monovalent anions with an order of effectiveness Cl⁻ = Br⁻ > NO₃⁻ > F⁻ > ClO₄⁻; (c) at molar concentrations, SO₄²⁻ stabilized the binding of the 23 kilodalton polypeptide, while Cl⁻ and Br⁻ became dissociating agents, in agreement with studies by Blough and Sauer (1984 Biochim Biophys Acta 767: 377-381); (d) the binding of the polypeptides was strengthened at room temperature relative to 0°C, indicating an involvement of hydrophobic forces. It is suggested that a specific binding of Cl⁻, or certain substitutes, organizes the protein surfaces and/or the adjacent water layers in the water oxidizing complex in a way that not only stabilizes its assembly, but is essential for the catalytic mechanism as well. Binding of, or charge screening by, divalent ions interferes with this process. At high salt concentrations, all these effects are overridden by “lyotropic” actions of the solutes that affect the integrity of the water oxidizing protein complex by stabilizing or disrupting critical hydrophobic domains.     

Gelation of amylose

A range of physical techniques, including light-scattering, turbidity measurements, viscometry, dilatometry, rheological measurements, and X-ray diffraction, has been used to study the gelation of amylose. Gels form on cooling entangled amylose solutions and occur as a result of a phase separation which produces a three-dimensional polymer network. Crystallisation, as detected by X-ray diffraction, was observed to be a slower process originating in the polymer-rich phase.     

Effects of Anions on Swelling, Respiration, and Phosphorylation of Isolated Corn Mitochondria

The effects of a series of anions on swelling, respiration, and oxidative phosphorylation of corn mitochondria were studied. Active mitochondrial swelling similar to that found with HPO₄⁻² was demonstrated in the presence of IO₃⁻, NO₂⁻, MoO₄⁻², SO₄⁻², HAsO₄⁻², acetate, S₂O₃⁻², SeO₄⁻², CrO₄⁻², and WoO₄⁻². In general, those anions which caused active swelling also released respiration and reduced the efficiency of oxidative phosphorylation with exogenous NADH as substrate. The degree of passive swelling in the presence of certain of the monovalent anions was found to approximate the order of the lyotropic series (SCN⁻ > CIO₄⁻ > I⁻ > NO₃⁻ > CI⁻).     

Thiol-dependent metallo-endopeptidase characteristics of Pz-peptidase in rat and rabbit.

Pz-peptidase was purified from rat testis and rabbit muscle. Zinc was detectable in the rat enzyme. The activity of the enzyme from both species was slowly but completely abolished by EDTA and restored by Zn2+. Free thiol groups were also important for the catalytic activity of rat Pz-peptidase, as previously reported for the rabbit enzyme. We conclude that in both species Pz-peptidase has the characteristics of a thiol-dependent metallo-endopeptidase.     

Thiol-dependent generation of superoxide radical induced by menadione and vicasol

It was demonstrated that in the presence of blood serum menadione and vicasol reduce cytochrome c in two ways, i. e., via O2-. -dependent and O2-. -independent routes. T ability of the serum to generate O2-. in the presence of menadione is retained after dialysis. Albumin can also act as an electron donor during O2-. generation mediated by menadione; however, this ability of the serum does not correlate with the presence of albumin in it. N-Ethylmaleimide and Cu2+ inhibit the ability of albumin and blood serum to induce menadione-mediated generation of O2-. It is concluded that in the presence of thiol-containing proteins and O2-. vitamin K3 forms an oxidative system generating O2-.     

Effects of Lysozyme and Inorganic Anions on the Morphology of Streptococcus mutans BHT: Electron Microscopic Examination

The effects of hen egg white lysozyme and the inorganic salt sodium thiocyanate on the integrity of Streptococcus mutans BHT were studied by transmission electron microscopy. Both control cells and cells exposed to NaSCN possessed thick outer cell walls and densely staining inner cell walls juxtaposed to the plasma membranes. In the presence of NaSCN, however, the S. mutans BHT nucleoid was coagulated into thick electron-dense filaments. Exposure of S. mutans BHT to 150 μg of hen egg white lysozyme per ml resulted in the progressive destruction of both the cell walls and the plasma membranes. The enzyme appeared to affect the region of the cell wall septum, and exposure to 150 μg of hen egg white lysozyme per ml for as short a time as 10 min resulted in visible morphological cell wall alterations. At 30 min, ultrastructural observations revealed that the majority of the cells were in the process of expelling a portion of their cytoplasmic contents from the septal and other regions of the cells at the time of fixation. After 3 h of incubation in the presence of this high lysozyme concentration, gelled protoplasmic masses, which were free from the cells, were evident. In addition, extensive damage to the outer and inner cell walls and to the plasma membranes was apparent, although the cells maintained their shape. On some areas of the cell surface, the outer cell wall and plasma membrane were completely absent, whereas at other locations the outer cell wall was either split away from the inner cell wall and plasma membrane or distended from an area free of inner cell wall and plasma membrane. Upon addition of NaSCN to the hen egg white lysozyme-treated cells, both the gelled protoplasmic masses and the damaged cells exhibited an exploded appearance and existed as membrane ghosts, cell wall fragments, or dense aggregates of cytoplasmic components. The effects of a low lysozyme concentration (22.5 μg/ml) on S. mutans morphology were less pronounced at short incubation times (i.e., 10 and 30 min) than those that were observed with a high enzyme concentration; however, breaks in the cell walls and dissolution of the plasma membranes with resulting cell lysis were visible after a prolonged (3-h) incubation and after subsequent addition of NaSCN.     

不同阴离子对二色补血草盐腺Na~+分泌速率的影响

本文以二色补血草(Limonium bicolor)为实验材料,用Hoagland营养液和200mmol·L-1NaCl、NaBr、NaNO3溶液分别处理12h,测定二色补血草盐腺的Na+分泌速率、叶片Na+含量和MDA(丙二醛)含量以及质膜透性,并利用非损伤微测技术探索可能与盐腺相关的转运蛋白,以探讨不同阴离子对二色补血草盐腺分泌Na+的作用及其可能原因。结果表明:在NaCl处理12h时二色补血草叶片Na+分泌速率达到最大,然后逐渐下降;不同钠盐处理下叶片Na+分泌速率为NaCl>NaBr=NaNO3>Hoagland,而叶片Na+含量为NaBr>NaCl>NaNO3>Hoagland;不同盐处理下叶片质膜透性和MDA含量无显著性差异;利用Na-K-Cl共转运体专一性抑制剂bumetanide处理发现Na+分泌速率显著降低。这些结果表明Na-K-Cl共转运体可能参与盐腺分泌Na+。     

Gelation mechanism of agarose

The non-Newtonian behavior and dynamic viscoelasticity of a series of aqueous solutions of agarose were measured with a rheogoniometer. The flow curve, at 25°, of agarose solution approximated to plastic behavior at 0.1, 0.13, and 0.15% concentrations. Gelation occurred at concentration of 0.13% at low temperature (0°). The dynamic modulus of agarose showed a very high value at low temperature, and increased with an increase in temperature, showing a maximum value at 30°, then it decreased. In the presence of NaCl, KCl, CaCl₂, and MgCl₂ for a solution of agarose at 0.08% concentration, the transition temperature, at which dynamic modulus decreased rapidly, was observed at 60°. Gelation was also observed at low temperature (0°) in acid and alkaline range after reaching pH values of 2.3 and 9.5, respectively, by addition of 100m HCl, H₂SO₄, NaOH, and Ca(OH)₂ to a 0.08% agarose solution. A possible mode of intra- and inter-molecular hydrogen bonding within and between the agarose molecules in aqueous solution is proposed.     

Protective Effect of Some Anions on the Heat-Denaturation of Ovotransferrin

An alkaline serine-proteinase from Bacillus sp. PN51 isolated from bat feces collected in Phang Nga, Thailand, was purified and characterized. The molecular mass was estimated to be 35.0 kDa. The N-terminal 25 amino acid sequence was about 70% identical with that of Natrialba magadii halolysin-like extracellular serine protease. The enzyme showed the highest proteinase activity at 60 °C at pH 10.0. The activity was strongly inhibited by PMSF and chymostatin. The proteinase activity was not affected by the presence of 2% urea, 2% H₂O₂, 12% SDS, 15% triton X-100, or 15% tween 80. The proteinase preferred Met, Leu, Phe, and Tyr residues at the P₁ position, in descending order. The k _cat, K _m and k _cat/K _m values for Z-Val-Lys-Met-MCA were 16.8±0.14 min^?1, 5.1±0.28 μM, and 3.3±0.28 μM^?1 min^?1 respectively. This is the first report of an alkaline serine-proteinase with extremely high stability against detergents such as SDS.     

Effects of pH,Ionic Strength,Temperature, and Complexing Anions on the Sorption Behavior of Cobalt on Hydrous Silica

Sorption of Co(II) on SiO₂.xH₂O (silica gel) has been investigated as a function of time, amount of silica gel (0.10–1.00g), cobalt concentration (5.00 × 10^?5–1.20 × 10^?3 M), ionic strength (0.20–1.40 M NaClO₄), pH (～6.80–10.80), and temperature (273–318 K). Using the sorption kinetics data, the diffusion coefficient of Co(II) was calculated to be 6.86(±0.44) × 10^?12 m²sec^?1 under particle diffusion-controlled conditions. The sorption rate was determined as 2.61(±0.19) × 10^?3 sec^?1 at 298 K, pH 6.70(±0.05) and 0.20 M NaClO₄. The sorption data followed the Freundlich, Langmuir, and Dubinin-Radushkevich (D-R) isotherms. Cobalt sorption decreased with increased ionic strength. A gradual decrease in pH with increased ionic strength supported the sorption of Co(II) by an ion exchange mechanism. The effects of different ligands such as , F^?, and on the sorption of Co(II) were studied in the pH range 6.50 to 8.50. The sorption of cobalt on silica gel increased with increased temperature and had an endothermic enthalpy change (ΔH = 23.60(±0.57) kJ/mol).     

Effects of Organic Acid Anions on the Growth and Metabolism of Syntrophomonas wolfei in Pure Culture and in Defined Consortia

The effects of organic acid anions on the growth of Syntrophomonas wolfei was determined by varying the initial concentration of the acid anion in the medium. The addition of 15 mM acetate decreased the growth rate of a butyrate-catabolizing coculture containing Methanospirillum hungatei from 0.0085 to 0.0029 per hour. Higher initial acetate concentrations decreased the butyrate degradation rate and the yield of cells of S. wolfei per butyrate degraded. Inhibition was not due to the counter ion or the effect of acetate on the methanogen. Initial acetate concentrations above 25 mM inhibited crotonate-using pure cultures and cocultures of S. wolfei. Benzoate and lactate inhibited the growth of S. wolfei on crotonate in pure culture and coculture. Lactate was an effective inhibitor of S. wolfei cultures at concentrations greater than 10 mM. High concentrations of acetate and lactate altered the electron flow in crotonate-catabolizing cocultures, resulting in the formation of less methane and more butyrate and caproate. The inclusion of the acetate-using methanogen, Methanosarcina barkeri, in a methanogenic butyrate-catabolizing coculture increased both the yield of S. wolfei cells per butyrate degraded and the efficacy of butyrate degradation. Butyrate degradation by acetate-inhibited cocultures occurred only after the addition of Methanosarcina barkeri. These results showed that the metabolism of S. wolfei was inhibited by high levels of organic acid anions. The activity of acetate-using methanogens is important for the syntrophic degradation of fatty acids when high levels of acetate are present.