The properties of slightly soluble Zn(II)-protein complexes in the form of a suspension

The properties of slightly soluble Zn(II)-protein complexes in the form of a suspension. Slightly soluble Zn(II) complexes with selected hormones were obtained in the form of a suspension. Zn(II)-protein complexes were obtained at different ligand concentrations. The amount of Zn(II) in microM bound to a protein increases along with an increase in the added ligand concentration. This amount is statistically significant as far as its binding to INS is concerned. Proteins bind to Zn(II) through two classes of binding sites (n). Most Zn(II) binds to albumin and least to LH and FSH. The protein molecular weight and number of its amino acid residues have an influence on binding Zn(II) to a selected protein. Zn(II) has the highest affinity for PRL and INS, and the lowest for albumin and FSH. The kinetics analysis of Zn(II) binding to proteins has shown that Zn(II) creates the most stable complexes with PRL, INS, and LH. Relatively, weak complexes were obtained with albumin and FSH. The protein molecular weight and the number of its amino acid residues have a significant influence on the stability of Zn(II)-protein complex. The lower protein molecular weight and number of its amino acid residues, the more stable Zn(II)-protein complex is. The stability of complexes is also associated with the amount of Zn(II) bound to a protein and its affinity for a protein.     

Binding of Cu(II) or Zn(II) in a de novo designed triple‐stranded α‐helical coiled‐coil toward a prototype for a metalloenzyme

Abstract: We previously reported the IZ‐3adH peptide, which formed a triple‐stranded coiled‐coil after binding Ni(II), Cu(II), or Zn(II). In this paper, we report the peptide, IZ‐3aH, having a new metal binding specificity. The IZ‐3aH peptide was found to bind Cu(II) and Zn(II) and form a triple‐stranded coiled‐coil. However, it did not bind Ni(II). Metal ion titrations monitored by circular dichroism revealed that the dissociation constants, K_d were 9 μm for Zn(II) and 10 μm for Cu(II). The bound Cu(II) ion has a planar tetragonal geometry, where the coordination positions are three nitrogens of the His residues and one H₂O.     

Effects of cations and temperature on the binding of [3H]spiperone to sheep caudate nucleus

The specific [3H]spiperone binding by sheep caudate nucleus homogenate is increased by divalent cations. The effect of Ca2+ or Mn2+ (5 mM) is temperature-dependent, and it is optimal at about 37 degrees, but is relatively low below 15 degrees and above 50 degrees. In the absence of added Ca2+ or Mn2+, the maximal specific [3H]spiperone binding is observed at about 25 degrees, and the cations shift the optimum to about 37 degrees. Under the experimental conditions used, the KD is about 0.6 nM and is not influenced by Ca2+ or Mn2+, or by temperature (25 and 37 degrees). In addition to Ca2+ and Mn2+, Mg2+ and Zn2+ also increase the specific [3H]spiperone binding, but to a smaller extent. At the concentrations of Ca2+, Mn2+, Mg2+ and Zn2+ which produce a maximal increase in the [3H]spiperone binding, the membranes are nearly saturated with the cations which bind about 100 nmoles of Ca2+ or Mg2+/mg of protein, 170 nmoles Zn2+/mg of protein and at least 300 nmoles Mn2+/mg of protein. It is suggested that the cations increase the [3H]spiperone binding by either exposing more binding sites, by preventing denaturation or by increasing the solubility of [3H]spiperone in the membrane phase, or by a combination of these processes.     

Specific binding of a novel compound, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) to the active site of cAMP-dependent protein kinase

The interaction of the catalytic subunit of bovine cardiac muscle cAMP-dependent protein kinase with N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), the most potent and selective inhibitor toward cyclic nucleotide-dependent protein kinases in the series of isoquinolinesulfonamide derivatives, was studied. The addition of H-8 protected the catalytic subunit of the enzyme in a dose-dependent manner from irreversible inactivation by the ATP analogue p-fluorosulfonylbenzoyl-5'-adenosine (FSBA). The inactivation followed pseudo-first order kinetics and H-8 reduced the steady state constant of inactivation (Ki) without any effect on the first order rate constant (K3). The quantitative binding of H-8 to the enzyme was measured under conditions of thermodynamic equilibrium using a gel filtration method. The catalytic subunit bound approximately 1 mol of drug/mol of protein with apparent half-maximal binding at 1.0 microM drug, whereas the enzyme irreversibly modified by FSBA did not bind the drug, confirming that the enzyme has no site for H-8 in the catalytic subunit other than the active site. The binding studies also showed that H-8 does not require divalent cations such as Mg2+ to bind to the catalytic subunit of the protein kinase. The binding of H-8 to the active site was characterized using FSBA and other affinity labeling reagents which have been postulated to modify residues at or near the active site of the catalytic subunit. H-8 protected the enzyme against inactivation by FSBA and Cibacron Blue F3GA but did not afford any protection against the covalent modification of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl), suggesting that the binding site of H-8 does not involve the gamma-subsite of the ATP binding site in the catalytic subunit, since DTNB and NBD-Cl are thought to modify the residues complementary to gamma-phosphate of the ATP molecules.     

Allosteric Binding of Nickel(II) to Calmodulin

The binding of Ni(II) to calmodulin (CAM) in the presence andin the absence of Ca(II) was investigated by equilibrium dialysisin order to test the physicochemistry of direct Ni(II)-CAM interactionsthat might be responsible for the effects of this metal on CAMobserved in vivo. Samples containing 5 µm CAM, 5 mM Tris/HClbuffer (pH 7.4), and NaCl to maintain the ionic strength I =3600 µm, with or without 200 µm CaCl₂, were dialyzedat 37?C against 1–300 µm ⁶³NiCl₂. In the presenceof Ca(II), the CAM molecule has two binding sites for Ni(II)(K₁, = 7.25 ? 10⁵m^–1; = 3.79 ? 10³ M^–1) with markedcoopera-tivity (Hill coefficient = 1.20 ? 0.03 SE). In the absenceof Ca(II), a complicated Ni(II)-binding curve is obtained indicatingformation of many mutually interacting complex species. Bindingof Ni(II) to CAM in the presence of Ca(II) is inhibited slightlyby added MnCl₂ (50 µM) and very strongly by CuCl₂ andZnCl₂ (10 µm). To elucidate the mechanism of this inhibition,binding of Zn(II) (0.5–50 µm ⁶⁵ZnCl₂) to CAM inthe presence of Ca(II) (200 µM) was also studied. Themaximum molecular ratio of Zn(II) to CAM in the Zn(II)/Ca(II)/CAMcomplex approached 0.5. Thus, the observed inhibition by Zn(II)of the Ni(II) binding to Ca(II)/CAM does not involve competitionfor the same binding sites but is rather caused by a conformationalarrangement of CAM in its Ca(II)/Zn(II) complex that is differentthan the Ca(II) complex. This fact, as well as the observeddifference in binding of Ni(II) in the presence and absenceof Ca(II), stress the importance of conformation of the CAMmolecule to Ni(II) binding.     

Influence of zinc (II) binding on the structure of bovine α-lactalbumin

The effect of Zn(II) binding on the structure of bovine α-lactalbumin (LA) was investigated. α-Lactalbumin, a regulatory subunit of lactose synthase, binds Ca(II) and Zn(II) at different sites in a mutually non-exclusive manner. The structures of the metal-depleted form of LA (apo-LA) and Ca(II)-bound LA (holo-LA) have been well characterized. Here, the effect of Zn(II) binding on the structure of holo-LA has been investigated by comparison with the structure of holo-LA and apo-LA using CD and NMR spectroscopy. The CD spectrum of Zn(II)-holo-LA was similar to that of holo-LA, but the intensity of the negative peak in near-UV region was decreased. Zn(II) binding to holo-LA produced only small changes in NMR chemical shifts, but the integral volumes of the cross-peaks of NOESY signals in cluster II, which is in the vicinity of Zn(II) binding site, were affected. Zn(II) binding induces a local structural change on the holo-LA, but it does not induce a large backbone conformational change, © Munksgaard 1996.     

The metal binding properties of the CCCH motif of the 50S ribosomal protein L36 from Thermus thermophilus

Abstract: The TthL36 protein of the 50S ribosomal proteins from Thermus thermophilus has been found to contain the rare C(Xaa)₂C(Xaa)₁₂C(Xaa)₄H (CCCH) sequence motif, a zinc finger binding motif, which for other zinc finger proteins is known to cleave RNA hairpins. In order to investigate the metal‐binding properties of this T. thermophilus TthL36 protein, the core 26‐mer polypeptide containing this CCCH motif was prepared by solid‐phase peptide synthesis methods using Fmoc chemistry, purified by preparative RP‐HPLC and characterized by circular dichroism, high‐performance capillary zone electrophoresis and electrospray ionization mass spectrometry. Reaction of the acetamidomethyl (Acm)‐protected polypeptide with iodine under acidic conditions resulted in the formation of the fully de‐protected polypeptide. Of interest, the results demonstrate that the standard Acm‐deprotection method with the synthetic TthL36 polypeptide using mercuric acetate in the presence of a large excess of 2‐mercaptoethanol resulted in preferential formation of a very stable mercuro‐polypeptide complex. The properties of the Acm‐deprotected polypeptide in the presence of different metal ions were also investigated by spectroscopic methods. The results confirm that this TthL36 polypeptide containing the CCCH motif binds metal ions with different affinities, namely in the order Co(II) > Hg(II) > Zn(II).     

Zn(2+) enhancement of the recombinant 5-HT(3) receptor is modulated by divalent cations

The modulation by Zn(2+) of recombinant murine 5-hydroxytryptamine(3A) (5-HT(3A)) receptor responses and its modification by Ca(2+) or Mg(2+) were studied using whole-cell voltage clamp and radioligand binding techniques. In the absence of other added divalent cations Zn(2+) enhanced the response to 5-HT by increasing maximum peak current (I(max)) to a maximum of 122.5%, decreasing the rate of desensitization (maximum t(1/2)=210%), and decreasing the EC(50) by approximately two fold. In the presence of Ca(2+) or Mg(2+), the effects of Zn(2+) on I(max) and t(1/2) were still manifest, although higher Zn(2+) concentrations were required; however, the effect on EC(50) was abolished. Zn(2+) also enhanced [3H]agonist but not [3H]antagonist binding. We propose there is more than one Zn(2+) binding site on the 5-HT(3) receptor molecule, and that one or more of these sites may also bind Ca(2+) and Mg(2+).     

Binding of the polyflavane P13 on various pig myocardial membranes--effects on protein release and on Ca2+ and Mg2+ movements.

P13, a polyflavane compound, bound to myocardial membranes with various degrees of efficiency: sarcolemma > mitochondria > sarcoplasmic reticulum. The binding was enhanced by added cations. Partial release of bound P13 was obtained by washing and the same cations which enhanced the binding, inhibited the release. Washing-induced protein release from membranes was prevented by bound P13. The binding of P13 did not modify respiratory activity and energy-linked Ca²⁺ accumulation in intact mitochondria, but retained a better coupling activity in aged mitochondria by preventing cation efflux which occurs during ageing. P13 behaved like a membrane stabilizer since its binding diminished membrane leakage and therefore prevented protein and cation passive efflux through aged membranes.     

Co(II) and Cd(II) substitute for Zn(II) in the zinc finger derived from the DNA repair protein XPA, demonstrating a variety of potential mechanisms of toxicity

XPA is one of the key members of the protein complex of the nucleotide excision repair (NER) pathway of DNA repair. The CCCC zinc finger domain of XPA is involved in the interactions with other NER proteins. To study the possible molecular mechanisms of XPA inhibition, we previously investigated Zn(II) and Ni(II) interactions with the synthetic 37 amino acid peptide (XPAzf), AcDYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHKam, representing the XPA zinc finger sequence (Bal, W., Schwerdtle, T., and Hartwig, A. (2003) Mechanism of nickel assault on the zinc finger of DNA repair protein XPA. Chem. Res. Toxicol. 16, 242-248). In this work, we extended these studies on other carcinogenic metal ions, Co(II) and Cd(II). The binding constants and complex geometries were determined using UV-vis and CD spectroscopies, and oxidative damage to XPAzf was studied with HPLC. The conditional binding constants determined for Co(II) and Cd(II) in 50 mM phosphate buffer, pH 7.4, are 10(7.4)+/-(0.4) and 10(12.8)+/-(0.5), respectively, yielding binding constant ratios Zn(II)/Co(II) of 100 and Zn(II)/ Cd(II) of 0.001, which are the lowest values reported for zinc fingers so far. The Co(II) ion forms a tetrahedral complex with the sulfurs of XPAzf, which is isostructural with the native zinc finger. The Cd(II) complex is somewhat less structured. The oxidation of Zn(II)-saturated XPAzf by H2O2 is accelerated in the presence of Co(II), but the concentration profile of this effect indicates the formation of an active Co(II) complex external to the metal-sulfur center. The Cd(II)-saturated XPAzf is very resistant to oxidation by H2O2. Overall, our results indicate that XPAzf can undergo Co(II) and Cd(II) assault under specific conditions.     

Causes and consequences of zinc dyshomeostasis in rats with chronic aldosteronism

Iterations in Ca2+ and Mg2+ balance accompany aldosteronism (inappropriate for dietary Na+ intake). Increased Zn excretion and Zn translocation to injured tissues, including the heart, also occurs. Several causes and consequences of Zn dyshomeostasis in rats receiving aldosterone/salt treatment (ALDOST) were examined. (1) To study the role of urinary acidification in promoting hyperzincuria, acetazolamide (75 mg/kg), a carbonic anhydrase inhibitor, was used as cotreatment to raise urinary HCO3 excretion. (2) To assess Zn levels in the heart, including cardiomyocyte cytosolic free [Zn2+]i and mitochondrial Zn, the expression of metallothionein (MT-I), a Zn binding protein, and biomarkers of oxidative stress were examined. (3) Oxidative stress and cardiac pathology in response to ZnSO4 supplement (40 mg/d) were also studied. Comparison of controls and rats receiving 4 weeks ALDOST revealed the following: (1) an acidification of urine and metabolic alkalosis associated with increased urinary Zn excretion and hypozincemia, each of which were prevented by acetazolamide; (2) a rise in cardiac Zn, including increased [Zn2+]i and mitochondrial Zn, associated with increased tissue MT-I, 8-isoprostane, malondialdehyde, and gp91(phox), coupled with oxidative stress in plasma and urine; (3) ZnSO4 prevented hypozincemia, but not ionized hypocalcemia, and attenuated oxidative stress and microscopic scarring without preventing the vasculitis and perivascular fibrosis of intramural coronary arteries. Thus, the hyperzincuria seen with ALDOST is due to urinary acidification. The oxidative stress that appears in the heart is accompanied by increased tissue Zn serving as an antioxidant. Cotreatment with ZnSO4 attenuated cardiomyocyte necrosis; however, polynutrient supplement may be required to counteract the dyshomeostasis of all 3 cations that accompanies aldosteronism and contributes to cardiac pathology.     

Effect of transition or heavy metals on [3H]haloperidol binding in rat striatal membranes in vitro

Our previous experiments have shown that several metal cations affect dopaminergic uptake and release processes in synaptosomes in vitro. It is thus possible that other membrane-related steps of neurotransmission, such as receptor binding, are affected as well. We studied the effect of Mn2+, Cu2+, Cd2+, Zn2+, Hg2+, Pb2+ and of two organometals, methyl mercury and triethyl lead, on [3H]haloperidol binding in the striatal P2 fraction assuming that such a study would reveal direct effects of the ions on dopaminergic D2 receptor binding. According to non-linear curve fitting and Scatchard analysis, [3H]haloperidol bound to two sites in striatal tissue. The Kd of the higher affinity site was 0.14 +/- 0.05 nM and the Bmax 226.3 +/- 50.3 fmol/mg protein. The respective values for the lower affinity site were 2.49 +/- 0.56 nM and 678.3 +/- 111.4 fmol/mg protein. Among the divalent cations, Hg2+ (IC50 0.7 microM) and Cu2+ (IC50 2.9 microM) inhibited the high affinity [3H]haloperidol binding most potently. The inhibition by Cu2+ was due to a decrease in the binding affinity (increase in the Kd) while the number of binding sites remained unchanged. Zn2+ inhibited the binding by 41.8% and Cd2+ by 38.7% at 10 microM concentration while Pb2+ and Mn2+ did not affect binding significantly at this or lower concentrations. Methyl mercury (IC50 0.9 microM) and triethyl lead (IC50 2.6 microM) inhibited binding as well. Both these organometallic cations decreased the binding affinity but did not change significantly the number of binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of nickel assault on the zinc finger of DNA repair protein XPA

Xeroderma pigmentosum group A complementing protein (XPA) is a member of the protein complex of the nucleotide excision repair (NER) pathway of DNA repair, participating in the assembly of the incision complex. The 4S zinc finger domain of XPA is involved the interactions with other NER proteins. As demonstrated previously, the activity of XPA is compromised by several metal ions implicated in DNA repair inhibition, including Ni(II), Cd(II), and Co(II) (Asmuss, M., Mullenders, L. H. F., Elker, A., and Hartwig, A. (2000) Carcinogenesis 21, 2097-2104). To study the possible molecular mechanisms of XPA inhibition, we investigated Zn(II) and Ni(II) interactions with the synthetic 37 peptide (XPAzf), representing the XPA zinc finger sequence AcDYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHKam. The binding constants were determined using fluorescence and UV-vis spectroscopies, structural insights were provided by CD, and oxidative damage to XPAzf was studied with HPLC. The binding constants for Zn(II) and Ni(II) are (8.5 +/- 1.5) x 10(8) (log value 8.93(7)) and (1.05 +/- 0.07) x 10(6) M(-)(1) (6.02(3)), respectively, in 10 mM phosphate buffer, pH 7.4, and (6 +/- 4) x 10(9) (9.8(2)) and (2.9 +/- 0.5) x 10(6) M(-)(1) (6.46(8)) in 50 mM phosphate buffer, pH 7.4, yielding binding constant ratios Zn(II)/Ni(II) of 800 +/- 100 and 2300 +/- 500, respectively. The Ni(II) ion forms a square planar complex with the sulfurs of XPAzf, opposed to the tetrahedral structure of the native Zn(II) complex. Consequently, the overall zinc finger structure is lost in the Ni(II)-substituted peptide. Zn(II)-saturated XPAzf is remarkably resistant to air oxidation and is only slowly oxidized by 0.01 mM, 0.1 mM, and 1 mM H(2)O(2) in a concentration-dependent fashion. However, the presence of just 10-fold molar excess of Ni(II) is sufficient to accelerate this process for all three H(2)O(2) concentrations tested. Overall, our results indicate that XPAzf can undergo Ni(II) assault in specific conditions.     

Synthesis, characterization, antibiogram and DNA binding studies of novel Co(II), Ni(II), Cu(II), and Zn(II) complexes of Schiff base ligands with quinoline core

A series of Co(II), Ni(II), Cu(II), and Zn(II) complexes of Schiff base ligands L¹H₃ and L²H have been prepared. The ligands are synthesized by the condensation of 2-hydroxy-3-formylquinoline with salicyloylhydrazide and 2-hydrazinobenzothiazole in absolute ethanol. The prepared complexes were characterized by the analytical and spectral techniques. The stoichiometry of the complexes is found to be 1:1. The presence of coordinated and lattice water is confirmed by the TG and DTA studies. Subsequently all the prepared complexes were screened for antimicrobial activity against bacteria and fungi. The Cu(II) complexes have been found to be more active than the ligand. In addition the DNA binding/cleaving capacity of the compounds was analyzed by absorption spectroscopy, viscosity measurement, thermal denaturation, and gel electrophoresis methods.     

Engineering of a disulfide loop instead of a Zn binding loop restores the anti-proliferative,anti-angiogenic and anti-tumor activities of the N-terminal fragment of endostatin: Mechanistic and therapeutic insights

Although considerable effort has been devoted to understanding the molecular mechanism of endostatin's anti-cancer activity, the role of its Zn bound N-terminal loop has not been completely clarified. To investigate whether Zn binding or the N-terminal loop is involved in the anti-cancer properties of endostatin, we compared the structure and biological activity of a native Zn binding endostatin peptide (ES-Zn) with three variants: a Zn free variant (ES), a variant containing both a Zn binding site and a disulfide bond (ES-SSZn), and a variant including a disulfide loop but incapable of Zn binding (ES-SS). Spectroscopic studies indicated that ES-Zn and ES-SS consist of random coil and β structures, whereas ES-SSZn and ES fold into random coils. Theoretical analysis proposed that ES-Zn and ES-SS have a similar binding site to α_Vβ₃ integrin. The anti-proliferative activity of endostatin was retained by all peptides except ES, and the in vitro anti-angiogenic property was preserved in ES-Zn and ES-SS. Remarkably, breast tumor growth and CD31 activity were inhibited more effectively by ES-SS than by ES-Zn. Therefore, a correlation exists between the N-terminal loop and anti-cancer properties of endostatin fragment and a disulfide loop may be more promising than a Zn binding loop for inhibiting tumor growth.     

Effects of zinc and copper on cadmium uptake by brush border membrane vesicles

The effects of essential metals, zinc (Zn) and copper (Cu), on cadmium (Cd) uptake were investigated in brush border membrane vesicles (BBMV) isolated from the rat renal cortex and LLC-PK₁ cells. BBMV were incubated with Cd in the presence or absence of Zn or Cu, and then washed with a chelating agent, EGTA, to remove Cd bound to the outer surface of BBMV. Co-incubation with Zn or Cu decreased Cd accumulation in these BBMV in a concentration-dependent manner. Kinetic analysis of the initial accumulation of Cd suggested that Cd is taken up into rat BBMV via an unsaturable component and a saturable component (K_m = 13.8 μM, V_max = 1.44 nmol/mg protein/min), and co-incubation with Zn significantly increased the K_m of the saturable component without affecting the V_max, whereas Cu significantly increased the K_m-value and decreased the V_max-value. Increasing the osmolarity of the incubation medium slightly decreased Cd accumulation in the absence of Zn or Cu, whereas it did not decrease Cd accumulation in the presence of these metals. These results suggest the possibility that, in addition to passive diffusion, Cd is also taken up from the renal brush border membrane via carrier-mediated mechanisms that are inhibited by Zn competitively and by Cu non-competitively. Furthermore, these results suggest that: (1) Cd binds externally and internally to BBMV, (2) little Cd is transported into the intravesicular space, and (3) both Zn and Cu decrease the binding and transport of Cd.     

1H-NMR studies of calmodulin: the character of the calcium binding sites

The effects of various divalent cations on the Ca2+-binding sites of calmodulin were observed by 400 MHz 1H-NMR. The first and second Ca ions bound to sites III and IV (stage I), while the third and fourth bound to sites I and II (stage II). Zn2+, Hg2+ and Mn2+ bound to the first and third Ca2+-binding sites, but not to the second and fourth. Zn2+, Hg2+ or Mn2+ could bind to the first Ca2+-binding site by themselves and could bind to the third site only after the conformational change which occurs when two Ca2+ ions bind to first and second sites. Although Mg2+ did not bind to the first, second or fourth Ca2+-binding sites, it did bind to the third site. These results suggest that the order of Ca2+-binding and the order of affinity of the binding sites are not parallel; the first and third Ca2+-binding sites have high Ca2+-affinity, with the third being highest, whereas the second and fourth sites are of lower affinity. Also, we suggest in this study that the first and second sites are exposed on the surface of the protein, while the third and fourth ones are buried in the interior; the latter are exposed by the conformational change accompanying the binding of calcium to the first and second sites. Furthermore, the form of the interface by which calmodulin binds to target enzyme was altered slowly and continuously by the calcium-induced conformational change. The target enzyme was chosen and bound selectively to calmodulin among various enzymes by each interface form.     

Interactions of cations and anions with the binding of uptake blockers to the dopamine transporter

Uptake blockers and substrates are likely to recognise a common binding domain on the dopamine neuronal transporter (DAT). Among cations that form ionic gradients at the level of the cellular plasma membrane, Na+ is the only one that can stimulate their binding. The binding stimulation appears over Na+ concentrations ranging from 0 to 10-60 mM; at higher Na+ concentrations, binding reaches a plateau or decreases, according to the uptake blocker that is studied. The majority of the other cations, including K+, Ca2+, Mg2+ and Tris+, inhibit the binding of uptake blockers. Several metals impair binding to the DAT and/or the dopamine transport, but, under specific conditions, some of them, and chiefly Zn2+, stimulate binding. The complex relationships between cations, uptake blockers and the DAT suggest that cations recognise at least three different sites: the first one, site 1, is for cation-induced binding inhibition; the second one, site 2, is for Na+-induced binding stimulation; and the third one, site 3, is for Zn2+-induced binding stimulation. Modelling of the interactions between Na+, K+ and radioligands allows a better understanding of the effects of cations at sites 1 and 2, and of uptake blockers at site 1. Some anions also facilitate the binding of uptake blockers to the DAT, as far as they are associated with Na+. The dependence of the binding of dopamine on ions could be involved in its preferential inward transport and used by uptake blockers for their own binding to the DAT.     

Anti-inflammatory and selective COX-2 inhibitory activities of metal complexes of Schiff bases derived from aldoses

Mn(II)-, Fe(II)-, Co(II)-, Ni(II)-, and Zn(II)-Schiff base complexes containing anthranilic acid and aldoses as part of the base were prepared and characterized by microanalytical, thermogravimetric, and spectroscopic data. The complexes were found to be four-coordinate, anhydrous, and ML₂ type. The spectral and magnetic data indicate a tetrahedral geometry for Mn(II) and Fe(II) complexes, and a planar geometry for Co(II), Ni(II), and Zn(II) complexes. Mn(II) and Zn(II) complexes showed a significant anti-inflammatory activity against kaolin-paw-edema. All the complexes exhibited selective inhibition of COX-2 in two different cell models.     

Binding Affinity and Capacity for the Uremic Toxin Indoxyl Sulfate

Protein binding prevents uremic toxins from removal by conventional extracorporeal therapies leading to accumulation in maintenance dialysis patients. Weakening of the protein binding may enhance the dialytic elimination of these toxins. In ultrafiltration and equilibrium dialysis experiments, different measures to modify the plasma binding affinity and capacity were tested: (i), increasing the sodium chloride (NaCl) concentration to achieve a higher ionic strength; (ii), increasing the temperature; and (iii), dilution. The effects on the dissociation constant K_D and the protein bound fraction of the prototypical uremic toxin indoxyl sulfate (IS) in plasma of healthy and uremic individuals were studied. Binding of IS corresponded to one site binding in normal plasma. K_D increased linearly with the NaCl concentration between 0.15 (K_D = 13.2 ± 3.7 µM) and 0.75 M (K_D = 56.2 ± 2.0 µM). Plasma dilution further reduced the protein bound toxin fraction by lowering the protein binding capacity of the plasma. Higher temperatures also decreased the protein bound fraction of IS in human plasma. Increasing the NaCl concentration was effective to weaken the binding of IS also in uremic plasma: the protein bound fraction decreased from 89% ± 3% to 81% ± 3% at 0.15 and 0.75 M NaCl, respectively. Dilution and increasing the ionic strength and temperature enhance the free fraction of IS allowing better removal of the substance during dialysis. Applied during clinical dialysis, this may have beneficial effects on the long-term outcome of maintenance dialysis patients.