Binding of mercurials to membrane suspensions and undenatured proteins.

Binding capacities of membrane suspensions and dissolved compounds for mercurials were titrated by a new potentiometric method. Critical steps included a silver electrode of new design, the use of L-cysteine as a thiol buffer, a nitrogen atmosphere, and pretreatment of samples with equimolar mercurial and cysteine. Titrations had a sharp endpoint, accurate +/- 26 nmole methylmercury or +/- 8 nmole mercuric salt. Measurements of binding capacity of bovine serum albumin averaged 93% of the titer predicted for one SH group per molecule; those of human hemoglobin yielded 86-91% of the titer predicted for two SH groups per molecule. Yields dropped with exposure of protein solutions or membrane suspensions to atmospheric oxygen. Brain microsomes had significantly higher binding capacities (per milligram of protein) than red blood cell ghosts. The ratio of endpoint titers of CH3HgCl to HgCl2 averaged 2:1 in assays of cysteine, proteins, and membranes, showing that the assay was free of denaturation artifacts and protein-protein interference. Solutions of EDTA showed measurable binding of Hg2+ but not of CH3Hg+. Satisfactory titrations were also obtained with N-ethylmaleimide.     

Addendum

A number of solid complexes of theophylline with CH₃Hg¹¹, Hg¹¹, as well as both CH₃Hg¹¹ and Hg¹¹, have been isolated from aqueous solution as the result of the reactions of theophylline and mercury-containing species at the appropriate pH and mole ratios of reactants. The complexes have been characterized by elemental analysis, ¹H and ¹³C NMR and infrared spectroscopy, and X-ray crystal structure analysis. The results obtained indicate that the initial site of mercury binding is strongly pH dependent. No evidence could be found for CH₃Hg¹¹ substitution at the C₈ position in theophylline, contrasting with the situation in xanthosine and inosine.     

Interactions between selenium and methylmercury in rat brain

The interaction of selenium with methylmercury was investigated in brain of animals labeled with ⁷⁵SeO₃^2? and CH₃²⁰³Hg⁺. Brains were fractionated into subcellular components and the cytosol was further fractionated by chromatography on Sephadex G-150 and G-200. The main result of these studies was evidence suggesting a shift of ⁷⁵Se from the cytosol to the mitochondrial fraction in brain when CH₃Hg⁺ was given. Concurrent equimolar (10 μmoles/kg) selenite injections increased the uptake of Hg but did not alter ²⁰³Hg distribution in brain. Changing the dose of CH₃Hg⁺ from 1 to 38 μmoles/kg had little effect on Hg uptake (% of dose per g). Gel filtrations on Sephadex G-150 and G-200 revealed that ²⁰³Hg in cytosol followed a pattern more closely related to protein (A₂₈₀) than to ⁷⁵Se, although a considerable portion of both isotopes eluted with proteins in the void volume. Assays of whole brain homogenates revealed a slight reduction in glutathione peroxidase activity in CH₃Hg⁺-treated rats which was not seen when equimolar selenite was injected with the CH₃Hg⁺.     

Dissolved and suspended mercury. species in the Wabigoon River (Ontario,Canada): seasonal and regional variations

Seasonal and regional variations in the speciation, sediment-water partitioning, and dynamics of mercury (Hg) were studied at selected sites along the Hg-polluted Wabigoon River, and at unpolluted headwater and tributary sites, during April–September, 1979. ‘Dissolved’ and ‘particulate’ forms of Hg in the water were separated by continuous-flow centrifugation in the field. The Hg and other pollutants such as wood chips and salt had been discharged from a chlor-alkali plant and paper mill at Dryden, Ontario. Concentrations and loadings of particulate methyl mercury (CH₃Hg⁺) and total particulate Hg (and loadings of total ‘dissolved’ Hg) were greatest during the spring flood (April-May) owing to accelerated resuspension and transport of sediments. Concentrations of ‘dissolved’ CH₃Hg⁺, however, were highest in the summer (July–September), probably reflecting stimulation of microbial methylating activity by elevated temperatures, together with factors such as reduced levels of metal-scavenging particulates and minimal dilution by runoff. Total dissolved Hg concentrations were relatively high in September at polluted sites only, possibly because of desorption from sediments due to elevated concentrations of Cl⁻ ions. Loadings of dissolved CH₃Hg⁺ tended to be high in the summer but were generally depressed (suggesting sorption by suspended particles) during the major spring-flood episode in May. During July–August dissolved CH₃Hg⁺ was a function of total dissolved Hg, suggesting rapid biomethylation of desorbed inorganic Hg; but in general dissolved and suspended CH₃Hg⁺ levels depended on environmental variables and were unrelated to total Hg concentrations. In the summer only, total dissolved Hg was a function of dissolved Cl⁻. Hg species in particulates were associated with sulfides, hydrated Fe and Mn oxides, organic matter (notably high molecular weight humic and humic-Fe components), and selenium (Se); but CH₃Hg⁺ and total Hg differed in their specific preferences for binding agents, implying that binding sites discriminate between CH₃Hg⁺ and Hg²⁺ ions. CH₃Hg⁺ was associated with sulfide and (in the spring only) with Fe oxides, whereas total Hg was associated with organic matter and Se and with DTPA- and NaOH-extractable Fe in the spring but with Mn oxide and NaOH-extractable organics in the summer. Sulfides were most abundant in May, indicating that they were eroded from bottom sediments, but Fe and Mn oxides were most abundant in the summer, probably owing to activities of filamentous iron bacteria and other micro-organisms. Particulate Hg was 98–100% nonextractable by mild solvents such as Ca acetate, CaCl₂, dilute acetic acid, and (at polluted sites only) DTPA solutions, suggesting that the particulate Hg mobilized in the spring may not be readily available to organisms; association with Se and high molecular weight humic matter also supports this hypothesis. Hg probably becomes more bio-available in the summer, as suggested by the upsurge in dissolved CH₃Hg⁺ and total dissolved Hg levels, and by increases in the solubility of particulate Hg in acetic acid, DTPA, H₂O₂, and NaOH solutions, as well as an increase in the relative importance of lower molecular weight fractions of NaOH-extractable Hg (in September). Regional variations in Hg speciation and partitioning reflected a gradient in sediment composition from wood chips near Dryden to silt-clay mud further downstream. Hg in silt-clay mud relatively far (> 35 km) downstream from the source of pollution or in unpolluted areas appeared to be more readily solubilized by Cl⁻ ions or chelators such as DTPA, more readily methylated (as indicated by downstream increases in dissolved CH₃Hg⁺ levels and CH₃Hg⁺/total Hg ratios), and was to a greater degree organically bound (H₂O₂-extractable), and thus was probably more bio-available, than Hg in wood-chip deposits. Possible explanations include weaker binding of Hg by the mud, the more finely divided state of the mud, and improved microbial growth at lower concentrations of toxic pollutants. Owing to enrichment in sulfides and Fe oxides, resuspended wood-chip sediments were especially efficient scavengers of CH₃Hg⁺. The results indicate that in any pollution abatement plan aimed at lowering the Hg levels in the biota of lakes fed by the Wabigoon River, immobilization, removal, or detoxification of dissolved as well as particulate forms of Hg in the river would probably have to be considered. Possibly, Hg species could be ‘scrubbed’ from the river water by increasing the suspended load and by sedimentation and treatment with Hg-binding agents in special receiving basins.     

The chemistry and transport of mercury in a small wetland in the Adirondack region of New York, USA

The biogeochemistry of Hg was evaluated in a small wetland in the Adirondack region of New York. Concentrations of total Hg (Hg_T) in streamwater draining the wetland showed little temporal variation. The annual areal watershed flux of Hg_T (2.2 µg/m²-yr) was considerably smaller than regional inputs of atmospheric deposition of Hg_T, indicating that the terrestrial environment is a net sink for atmospheric deposition of Hg_T. Drainage inputs of Hg_T were conservatively transported through the beaver impoundment. The annual flux of total methyl mercury (CH₃Hg⁺ _T was greater than literature values of atmospheric deposition suggesting that the watershed is a net source of CH₃Hg⁺ _T . Stream concentrations of CH₃Hg⁺ _T increased during low-flow summer conditions in a riparian wetland, and particularly at the outlet of the beaver impoundment. Net production of CH₃Hg⁺ _T occurred in the beaver impoundment (0.45 µg/m²-yr). Rates of net methylation for the beaver impoundment were comparable to values reported in the literature for wetlands.     

The human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 (NAT1) is irreversibly inhibited by inorganic (Hg) and organic mercury (CH3Hg): Mechanism and kinetics

Human arylamine N-acetyltransferase 1 (NAT1) is a xenobiotic-metabolizing enzyme that biotransforms aromatic amine chemicals. We show here that biologically-relevant concentrations of inorganic (Hg²⁺) and organic (CH₃Hg⁺) mercury inhibit the biotransformation functions of NAT1. Both compounds react irreversibly with the active-site cysteine of NAT1 (half-maximal inhibitory concentration (IC₅₀) = 250 nM and k_inact = 1.4 × 10⁴ M⁻¹ s⁻¹ for Hg²⁺ and IC₅₀ = 1.4 μM and k_inact = 2 × 10² M⁻¹ s⁻¹ for CH₃Hg⁺). Exposure of lung epithelial cells led to the inhibition of cellular NAT1 (IC₅₀ = 3 and 20 μM for Hg²⁺ and CH₃Hg⁺, respectively). Our data suggest that exposure to mercury may affect the biotransformation of aromatic amines by NAT1.     

Biosorption of inorganic and methyl mercury by a biosorbent from Aspergillus niger

A biosorbent prepared by alkaline extraction of Aspergillus niger biomass was evaluated for its potential to remove mercury species – inorganic (Hg²⁺) and methyl mercury (CH₃Hg⁺) – from aqueous solutions. Batch experiments were carried out to determine the pH and time profile of sorption for both species in the pH range 2–7. The Hg²⁺ exhibited more rapid sorption and higher capacity than the CH₃Hg⁺. Further, removal of both mercury species from spiked ground water samples was efficient and not influenced by other ions. Sorption studies with esterified biosorbent indicated loss of binding of both mercury species (>80%), which was regained when the ester groups were removed by alkaline hydrolysis, suggesting the involvement of carboxyl groups in binding. Further, no interconversion of sorbed species occurred on the biomass. The biosorbent was reusable up to six cycles without serious loss of binding capacity. Our results suggest that the biosorbent from Aspergillus niger can be used for removal of mercury and methyl mercury ions from polluted aqueous effluents.     

Skeletal muscle glycogen phosphorylase is irreversibly inhibited by mercury: Molecular,cellular and kinetic aspects

Muscle glycogen phosphorylase (GP) plays an important role in muscle functions. Mercury has toxic effects in skeletal muscle leading to muscle weakness or cramps. However, the mechanisms underlying these toxic effects are poorly understood. We report that GP is irreversibly inhibited by inorganic (Hg²⁺) and organic (CH₃Hg⁺) mercury (IC₅₀ = 380 nM and k_inact = 600 M⁻¹ s⁻¹ for Hg²⁺ and IC₅₀ = 43 μM and k_inact = 13 M⁻¹ s⁻¹ for CH₃Hg⁺) through reaction of these compounds with cysteine residues of the enzyme. Our data suggest that the irreversible inhibition of GP could represent one of the mechanisms that contribute to mercury-dependent muscle toxicity.     

Membrane lipids as intracellular binders of chlorpromazine and related drugs

Equilibrium dialysis studies with chlorpromazine (CPZ) showed affinity and binding capacity values which were not significantly different with the following binders: rat liver microsomes, mitochondria, mitochondrial membranes, brain synaptosomes, myelin vesicles, and red blood cell membranes. There was no binding to cytosol or mitochondrial matrix. The same binding values as above were obtained with protein-free liposomes of lipids extracted from microsomes, mitochondrial and red cell membranes and of pure egg lecithin. The binding values of the two classes of binding sites of all these preparations were K₁ = 2.7 ± 1.0 · 10⁴ M^?1, K₂ = 3.8 ± 1.7 · 10³ M^?1, C₁ = 580 ± 230 and C₁₊₂ = 1410 ± 500 nmole/mg phospholipid. These values were not altered by elimination of the polar head groups of phospholipids with phospholipase C. The results were confirmed by a UV spectroscopic method whereby the strongest binding signals were obtained with CPZ in the presence of fatty acids such as oleate. It is concluded that the major intracellular binders for CPZ and related drugs are the nonpolar moieties of membrane phospholipids, whereby hydrophobic interactions are mainly involved.     

Mercury's neurotoxicity is characterized by its disruption of selenium biochemistry

Background

Methylmercury (CH₃Hg⁺) toxicity is characterized by challenging conundrums: 1) “selenium (Se)-protective” effects, 2) undefined biochemical mechanism/s of toxicity, 3) brain-specific oxidative damage, 4) fetal vulnerability, and 5) its latency effect. The “protective effects of Se” against CH₃Hg⁺ toxicity were first recognized >50?years ago, but awareness of Se's vital functions in the brain has transformed understanding of CH₃Hg⁺ biochemical mechanisms. Mercury's affinity for Se is ~1 million times greater than its affinity for sulfur, revealing it as the primary target of CH₃Hg⁺ toxicity.

Mechanism of inhibition of rat brain (Na +-K +)-stimulated adenosine triphosphatase reaction by cadmium and methyl mercury

The mechanisms of inhibition of rat brain Na ⁺-K ⁺- ATPase by cadmium chloride (CdCl₂) and methylmercuric chloride (CH₃HgCl) were studied in vitro by assessing the effects of these heavy metals on this enzyme and associated component parameters. Both the heavy metals significantly inhibited the overall Na ⁺-K ⁺ -ATPase in a concentration-dependent manner with an estimated median inhibitory concentration (IC-50) of 3.2 × 10^?5M for CdCl₂ and 6 × 10^?6M for CH₃HgCl. Protection of enzyme against heavy metal inhibition by 5 × 10^?5M to 1 × 10^?4 M dithiothreitol (DTT) and glutathione (GSH) or cysteine (CST) indicates that both monothiols and dithiols have the same ability in regenerating sulfhydryl (–SH) groups or chelating the metals. Inhibition of K⁺-p-nitrophenyl phosphatase (K⁺-PNPPase), the component enzyme catalyzing the K⁺-dependent dephosphorylation in the overall Na ⁺-K ⁺ATPase reaction by these heavy metals, indicates that the mechanism of inhibition involves binding to this phosphatase. Reversal of K⁺-PNPPase inhibition by DTT, GSH, and CST suggests sulfhydryl groups as binding sites. Binding of ³H-oubain, a cardiac glycocide and inhibitor of both phosphorylation and dephosphorylation, to brain fraction was significantly decreased by CH₃HgCl, and this inhibition was reversed by the three thiol compounds, suggesting presence of –SH group(s) in the ouabain receptor site. Cadmium chloride failed to inhibit the binding of this receptor, indicating that the mechanics of inhibition of ATPase by CH₃HgCl and CdCl₂ are different from each other. The results suggest that the critical conformational property of enzyme common to both kinase (E₁) and phosphatase (E₂) is susceptible to CH₃HgCl whereas only phosphatase is sensitive to CdCl₂.     

Sequestration of organometallic compounds by synthetic and naturally occurring polycarboxylate ligands. Binding of monomethylmercury(II) by polyacrylic and alginic acids

Abstract

The sequestering capacity of synthetic and naturally occurring polycarboxylate ligands towards mono-methylmercury(II) was evaluated by stability quantitative data on the interaction of CH₃Hg⁺ with different molecular weight synthetic polyacrylates (2 and 20kDa average M.wt) and alginate (70–100 kDa) extracted from brown algae Macrocystis pyrifera. The influence of ionic medium was evaluated by measurements on the CH₃Hg⁺-polyacrylate systems in NaNO₃ medium at different ionic strengths (0.10, 0.25, 0.50 and 0.75 mol L^?1), and a Debye-HiJckel type equation was used for the dependence of complex formation constants on ionic strength. Measurements on the CH3Hg⁺ - alginate system were carried out at l = 0.10 mol L^?1 in NaNO₃ medium. By using the stability data, the sequestering capacity of both ligands towards monomethylmercury(II) was determined at different pH values. Results obtained show that the binding ability of polyacrylic ligands (PAA) is stronger than the alginate (AA), following the trend PAA (20 kDa)> PAA (2kDa)>AA.     

Development of a transgenic tobacco plant for phytoremediation of methylmercury pollution

To develop the potential of plant for phytoremediation of methylmercury pollution, a genetically engineered tobacco plant that coexpresses organomercurial lyase (MerB) with the ppk-specified polyphosphate (polyP) and merT-encoding mercury transporter was constructed by integrating a bacterial merB gene into ppk/merT-transgenic tobacco. A large number of independent transgenic tobaccos was obtained, in some of which the merB gene was stably integrated in the plant genome and substantially translated to the expected MerB enzyme in the transgenic tobacco. The ppk/merT/merB-transgenic tobacco callus showed more resistance to methylmercury (CH₃Hg⁺) and accumulated more mercury from CH₃Hg⁺-containing medium than the ppk/merT-transgenic and wild-type progenitors. These results suggest that the MerB enzyme encoded by merB degraded the incorporated CH₃Hg⁺ to Hg²⁺, which then accumulated as a less toxic Hg-polyP complex in the tobacco cells. Phytoremediation of CH₃Hg⁺ and Hg²⁺ in the environment with this engineered ppk/merT/merB-transgenic plant, which prevents the release mercury vapor (Hg⁰) into the atmosphere in addition to generating potentially recyclable mercury-rich plant residues, is believed to be more acceptable to the public than other competing technologies, including phytovolatilization.     

Hg2+-induced leakage of electrolytes and inhibition of NO3 − utilization inNostoc calcicola

Summary The effect of mercury (Hg²⁺) in the absence and presence of methylmercury (CH₃Hg⁺), cadmium (Cd²⁺), copper (Cu²⁺), nickel (Ni²⁺) and calcium (Ca²⁺) on Nostoc calcicola Bréb. has been studied in terms of electrolyte leakage, NO₃ ^– uptake and in vivo nitrate reductase (NR) activity to discover any possible correlation among such parameters under Hg²⁺ stress. Leakage of electrolytes from Hg²⁺-treated cyanobacterial cells was directly proportional to Hg²⁺ concentrations and exposure time. In comparison to NO₃ ^– uptake, an about 60-fold slower rate of NR activity was observed in the untreated cultures, the former being five times more Hg²⁺-sensitive. A non-competitive synergistic interaction of Hg²⁺ with CH₃Hg⁺ or Cd²⁺ and antagonistic with that of Ni²⁺ or Ca²⁺ has been observed for both the processes of NO₃ ^– utilization. The antagonistic interaction of Cu²⁺ with Hg²⁺ in terms of NO₃ ^– uptake and synergistic with respect to NR activity, has been attributed to the dual bonding preference of Cu²⁺ for cellular ligands. These findings suggest that (a) a statistically significant correlation exists among such parameters; (b) Hg²⁺ predominantly attacks the cyanobacterial cell membrane; (c) Hg²⁺ inhibits NO₃ ^– utilization; (d) the presence of other cations increases or decreases the inhibitory actions of Hg²⁺.     

Hg2+-stimulated NADH-oxidase activity of Ascaris muscle microsomal lipoamide dehydrogenase.

In the presence of Hg²⁺$\text{Ascaris}$ lipoamide dehydrogenase stimulated the reduction of oxygen, ferricyanide, and 2,6-dichlorophenolindophenol with NADH, which was inhibited by lipoic acid. On the other hand, Cu²⁺ stimulated the reduction of the artificial dyes, but only a little the reduction of oxygen. Hg²⁺ changed the visible absorption spectrum of the lipoamide dehydrogenase, but did not change the fluorescence curve. Lipoic acid decreased the fluorescence, but did not change the visible absorption spectrum. The $\text{Ascaris}$ lipoamide dehydrogenase have two SH groups per one subunit and 5–6 moles of HgCl₂ and 3–4 moles of CuSO₄ per one subunit were required for the maximal activity.     

Beta-adrenergic receptor characteristics of postnatal rat myocardial cell preparations

Summary Primary mycolardial cell cultures and freshly isolated cardiac cells in suspension resprensent two isolated, whole cell models for investigating cellular transsarcolemmal⁴⁵Ca⁺⁺ exchange in response to a receptor-coupled stimulus. Studies were performed to characterize beta-adrenergic receptor binding, beta-adrenergic receptor mediated cellular calcium (⁴⁵Ca⁺⁺) exchange, and viability in purified primary myocardial cell cultures and freshly isolated cardiac cells in suspension obtained from 3-to 3-d-old Sprague-Dawley rats. In addition, beta-adrenergic receptor binding was characterized in whole-heart crude membrane preparations. All three preparations had saturable beta-adrenergic binding sites with the antagonist [¹²⁵I]iodopindolol ([¹²⁵I]IPIN). The suspensions had a significantly lower B _max (42±6 fmol/mg protein) than the membranes and cultures (77±8 and 95±10 fmol/mg protein, respectively). The K _D of the cultures (218±2.0 pM) was significantly higher than that for the suspensions (107 ±1.3 pM) and membranes (93±1.3 pM). Viability was significantly lower in the suspensions (57%) when compared to 94% viability in myocardial cell cultures after 3 h of incubation in Kreb's Henseleit buffer. Incubation of the cultures with 5.0×10⁻⁷ M isoproterenol resulted in a significant increase in⁴⁵Ca⁺⁺ exchange as early as 15 s. In contrast,⁴⁵Ca⁺⁺ exchange into the suspensions was not increased. Although both primary cell cultures and cardiac cells in suspension possess saturable beta-adrenergic receptors, only the monolayer cultures exhibited functional beta-adrenergic receptor-mediated⁴⁵Ca⁺⁺ exchange. Of the two intact cell models investigated, these data suggest that primary myocardial cell cultures are more suitable than cell suspensions for investigating beta-adrenergic receptor binding and functions in the postnatal rat heart. This research was supported by The University of Texas Research Institute, a grant from the Texas Advanced Research Technology Program awarded to S. W. Leslie and R. E. Wilcox, and contract 223-86-2109 from the Food and Drug Administration.     

Spectrin as a stabilizer of the phospholipid asymmetry in the human erythrocyte membrane

After treatment of intact human erythrocytes with SH-oxidizing agents (e.g. tetrathionate and diamide) phospholipase A₂ cleaves approx. 30% of the phosphatidylserine and 50% of the phosphatidylethanolamine without causing hemolysis (Haest, C.W.M. and Deuticke, B. (1976) Biochim. Biophys. Acta 436, 353–365). These phospholipids are scarcely hydrolysed in fresh erythrocytes and are assumed to be located in the inner lipid layer of the membrane (Verkleij, A.J., Zwaal, R.F.A., Roelofsen, B., Comfurius, P., Kastelijn, D. and van Deenen, L.L.M. (1973) Biochim. Biophys. Acta 323, 178–193). The enhancement of the phospholipid cleavage is now shown to be accompanied by a 50% decrease of the membrane SH-groups and a cross-linking of spectrin, located at the inner surface of the membrane, to oligomers of < 10⁶ dalton.Blocking approx. 10% of the membrane SH groups with N-ethylmaleimide suppresses both the polymerization of spectrin and the enhancement of the phospholipid cleavage. N-Ethylmaleimide, under these conditions, reacts with three SH groups per molecule of spectrin, 0.7 SH groups per major intrinsic 100 000 dalton protein (band 3) and 1.1 SH groups per molecule of an extrinsic protein of 72 000 daltons (band 4.2). Blocking studies with iodoacetamide demonstrate that the SH groups of the 100 000-dalton protein are not involved in the effects of the SH-oxidizing agents.It is suggested that a release of constraints imposed by spectrin enables phosphatidylserine and phosphatidylethanolamine to move from the inner to the outer lipid layer of the erythrocyte membrane and that spectrin, in the native erythrocyte, stabilizes the orientation of these phospholipids to the inner surface of the membrane.     

Distribution of mercury, methyl mercury and organic sulphur species in soil, soil solution and stream of a boreal forest catchment

Concentrations of methyl mercury, CH₃Hg (II), total mercury, Hg_tot = CH₃Hg (II) + Hg (II), and organic sulphur species were determined in soils, soil solutions and streams of a small (50 ha) boreal forest catchment in northern Sweden. The CH₃Hg (II)/Hg_tot ratio decreased from 1.2–17.2% in the peaty stream bank soils to 0.4–0.8% in mineral and peat soils 20 m away from the streams, indicating that conditions for net methylation of Hg (II) are most favourable in the riparian zone close to streams. Concentrations of CH₃Hg (II) bound in soil and in soil solution were significantly, positively correlated to the concentration of Hg_tot in soil solution. This, and the fact that the CH₃Hg (II)/Hg_tot ratio was higher in soil solution than in soil may indicate that Hg (II) in soil solution is more available for methylation processes than soil bound Hg (II). Reduced organic S functional groups (Org-S_RED) in soil, soil extract and in samples of organic substances from streams were quantified using S K-edge X-ray absorption near-edge structure (XANES) spectroscopy. Org-S_RED, likely representing RSH, RSSH, RSR and RSSR functionalities, made up 50 to 78% of total S in all samples examined. Inorganic sulphide [e.g. FeS₂ (s)] was only detected in one soil sample out of 10, and in none of the stream samples. Model calculations showed that under oxic conditions nearly 100% of Hg (II) and CH₃Hg (II) were complexed by thiol groups (RSH) in the soil, soil solution and in the stream water. Concentrations of free CH₃Hg⁺ and Hg²⁺ ions in soil solution and stream were on the order of 10^–18 and 10^–32M, respectively, at pH 5. For CH₃Hg (II), inorganic bi-sulphide complexes may contribute to an overall solubility at concentrations of inorganic sulphides higher than 10^–9M, whereas considerably higher concentrations of inorganic sulphides (lower redox-potential) are required to increase the solubility of Hg (II).     

High affinity binding sites on plasma membrane obtained from the lymphoblastoid cultured 1301 cell line for highly radioactive serum thymic factor

The interaction of the synthetic serum thymic factor (FTS, facteur thymique sérique) with a plasma membrane preparation of human T lymphocytes from the lymphoblastoid T cell line 1301 was studied using ³H-labelled FTS (specific activity 120 Ci/mmol). The binding is temperature dependent and function of the concentration of both ³H-labelled FTS and membrane proteins. At 37°C, using 1 nM of ³H-labelled FTS a steady state is observed within 80 min. The binding is reversible, specific and saturable. Scatchard analysis reveals the existence of at least two binding sites with respective K_d of the order of 0.516±0.2 nM and 110±27.8 nM with concentrations of 0.186±0.045 pmol and 2.026±0.367 pmol per mg of membrane protein.     

Specificity of Collybistin-Phosphoinositide Interactions: IMPACT OF THE INDIVIDUAL PROTEIN DOMAINS*

The regulatory protein collybistin (CB) recruits the receptor-scaffolding protein gephyrin to mammalian inhibitory glycinergic and GABAergic postsynaptic membranes in nerve cells. CB is tethered to the membrane via phosphoinositides. We developed an in vitro assay based on solid-supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes doped with different phosphoinositides on silicon/silicon dioxide substrates to quantify the binding of various CB2 constructs using reflectometric interference spectroscopy. Based on adsorption isotherms, we obtained dissociation constants and binding capacities of the membranes. Our results show that full-length CB2 harboring the N-terminal Src homology 3 (SH3) domain (CB2_SH3+) adopts a closed and autoinhibited conformation that largely prevents membrane binding. This autoinhibition is relieved upon introduction of the W24A/E262A mutation, which conformationally “opens” CB2_SH3+ and allows the pleckstrin homology domain to properly bind lipids depending on the phosphoinositide species with a preference for phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate. This type of membrane tethering under the control of the release of the SH3 domain of CB is essential for regulating gephyrin clustering.