Toxic effects of mercury(II), cadmium(II) and lead(II) porphyrins on Trypanosoma brucei brucei growth

The effects of free mercury(II), cadmium(II) and lead(II) ions and their metalloporphyrin-derivatives on Trypanosoma brucei brucei growth in culture were studied. All experiments were conducted in the dark. IC(50) values on growth obtained in 24-h time-course experiments were 1.5 x 10(-7), 2.4 x 10(-6), 4.4 x 10(-6) and 2.6 x 10(-5) M for mercury(II) porphyrin, cadmium(II) porphyrin, lead(II) porphyrin and free base porphyrin, respectively. While the IC50 values for Hg2+, Cd2+ and Pb2+ were 3.6 x 10(-6), 1.5 x 10(-5) and 1.6 x 10(-5) M, respectively. These results clearly indicate that the toxicity of the metalloporphyrin complexes of mercury(II), cadmium(II) and lead(II) to T. b. brucei parasites was much higher compared to their free metal ions and free base porphyrin at low concentrations. It was also observed after 8 h incubation that the metalloporphyrins were effective in inhibiting the division of the parasites at concentrations >1.25 x 10(-7) M for mercury(II) porphyrin, concentrations >1.2 x 10(-6) M for cadmium(II) and lead(II) porphyrins and at concentrations >3.6 x 10(-6) M for Hg2+ ion. These observations were not detected in samples treated with the free metal ions and the free base porphyrin at the same concentrations. Interestingly, trypanosomes treated with metalloporphyrin complexes displayed different morphological features from those cells treated with free base porphyrin or metal ions. The chemotherapeutic potential of the metalloporphyrins of H2TMPyP for treatment of African trypanosomiasis is discussed.     

Oxidative cleavage of DNA by tridentate copper (II) complex

Copper (II) complex 1 having planar tridentate ligand, bzimpy, where bzimpy is 2,6-bis(benzimidazo-2-yl) pyridine was synthesized and characterized by UV-visible, FAB (fast atom bombardment) mass and infrared spectroscopy. From absorption titration data, the binding constant of Cu(II) with DNA was calculated to be (1.8+/-0.02)x10(4) M(-1). Thermal denaturation study of DNA with 1 revealed deltaT(m) of 5+/-0.5 degrees C. Viscosity measurement showed that complex binds with DNA through intercalative mode. Copper (II) complex induces cleavage in plasmid DNA in the presence of coreductants such as ascorbic acid or glutathione.     

Kinetic data for redox reactions of cytochrome c with Fe(CN)5X complexes and the question of association prior to electron transfer

Use of rigorous equilibration kinetics to evaluate rate constants for the Fe(CN)6 4- reduction of horse-heart cytochrome c in the oxidized form, cyt c (III), has shown that limiting kinetics do not apply with concentrations of Fe(CN)6 4- (the reactant in excess) in the range 2-10 x 10(-4) M, I = 0.10 M (NaCl). The reaction conforms to a first-order rate law in each reactant, and at 25 degrees C, pH 7.2 (Tris), it is concluded that K for association prior to electron transfer is less than 200 M-1. From previous studies at 25 degrees C, ph 7.0 (10(-1) M phosphate), I = 0.242 M (NaCl), a value K = 2.4 x 10(3) M-1 has been reported. Had such a value applied, some or all of the redox inactive complexes Mo(CN)8 4-, Co(CN)6 3-, Cr(CN)6 3-, Zr(C2O4)4 4- present in amounts 5-20 x 10(-4) M would have been expected to associate at the same site and partially block the redox process. No effect on rats was observed. With the reductants Fe(CN)5(4-NH2-py)3- and Fe(CN)5(imid)3-, reactions proceeded to greater than 90% completion and rate laws were again first order in each reactant. Rate constants (M-1 sec-1) at 25 degrees C, pH 7.2 (Tris), I = 0.10 M (NaCl), are Fe(CN)6 4- (3.5 x 10(4)), Fe(CN)5(4-NH2py)3- (6.7 x 10(5), and Fe(CN)5(imid)3- (4.2 x 10(5). Related reactions in which cyt c(II) is oxidized are also first order in each reactant, Fe(CN)6 3- (9.1 x 10(6)), Fe(CN)5(NCS)3- (1.3 x 10(6)), Fe(CN)5(4-NH2py)2- (3.8 x 10(6) at pH 9.4), and Fe(CN)5(NH3)2- (2.75 x 10(6) at ph 8). Redox inactive Co(CN)6 3- (1.0 x 10(-3) M) has no effect on the reaction of Fe(CN)6 3- which suggests that a recent interpretation for the Fe(CN)6 3- oxidation of cyt c(II), I = 0.07 M, may also require reappraisal.     

Characterization of an inhibitory metal binding site in carboxypeptidase A

The specificity of metal ion inhibition of bovine carboxypeptidase A ([(CPD)Zn]) catalysis is examined under stopped-flow conditions with use of the fluorescent peptide substrate Dns-Gly-Ala-Phe. The enzyme is inhibited competitively by Zn(II), Pb(II), and Cd(II) with apparent KI values of 2.4 x 10(-5), 4.8 x 10(-5), and 1.1 x 10(-2) M in 0.5 M NaCl at pH 7.5 and 25 degrees C. The kcat/Km value, 7.3 x 10(6) M-1 s-1, is affected less than 10% at 1 x 10(-4) M Mn(II) or Cu(II) and at 1 x 10(-2) M Co(II), Ni(II), Hg(II), or Pt(IV). Zn(II) and Pb(II) are mutually exclusive inhibitors. Previous studies of the pH dependence of Zn(II) inhibition [Larsen, K. S., & Auld, D. S. (1989) Biochemistry 28, 9620] indicated that [(CPD)Zn] is selectively inhibited by a zinc monohydroxide complex, ZnOH+, and that ionization of a ligand, LH, in the enzyme's inhibitory site (pKLH 5.8) is obligatory for its binding. The present study allows further definition of this inhibitory zinc site. The ionizable ligand (LH) is assigned to Glu-270, since specific chemical modification of this residue decreases the binding affinity of [(CPD)Zn] for Zn(II) and Pb(II) by more than 60- and 200-fold, respectively. A bridging interaction between the Glu-270-coordinated metal hydroxide and the catalytic metal ion is implicated from the ability of Zn(II) and Pb(II) to induce a perturbation in the electronic absorption spectrum of cobalt carboxypeptidase A ([(CPD)Co]).(ABSTRACT TRUNCATED AT 250 WORDS)     

Chiral and achiral macrocyclic copper(II) complexes: Synthesis, characterization, and comparative binding studies with calf-thymus DNA

The new chiral macrocyclic complexes [1,2-bis(1H-benzimidazol-2-yl)-1-(1,8-dihydro-1,3,5,8,10,12-hexaazacyclotetradecane)-2-hydroxyethanolate] copper(II) and -nickel(II) perchlorate, 3 and 4, respectively, were synthesized by the reaction of 1,2-bis(1H-benzimidazol-2-yl)ethane-1,2-diol (L) and (1,8-dihydro-1,3,5,8,10,12-hexaazacyclotetradecane)copper(II) and -nickel(II) diperchlorate complexes, 1 and 2, respectively. All complexes were characterized by various spectroscopic techniques. Molar-conductance measurements showed that all of the complexes are ionic in nature. In complexes 3 and 4, the metal center is encapsulated by the ligand L in a pentacoordinated environment. The optical-rotation values ([alpha](D)) of 3 and 4 at 25 degrees indicate that the complexes are chiral. Absorption- and fluorescence-spectral studies, cyclic voltammetry, and viscosity measurements have been carried out to assess the comparative binding of complexes 1 and 3 with calf thymus (CT)-DNA. Analysis of the results suggests that the new chiral complex 3 binds to CT-DNA through a partial intercalation mode that is different from the binding mode of parent achiral complex 1. The complexes 1 and 3 bind to CT-DNA with binding constants K(b) of 2.7 x 10(4) and 6.6 x 10(4) M(-1), respectively. Circular-dichroism (CD) studies have been further employed to ascertain the binding mode of complex 3, which is consistent with the other spectral studies.     

Light-scattering and scanning transmission electron microscopic investigation of the hemocyanin of the bivalve, Yoldia limatula (Say)

1. The hemocyanin of the bivalve, Yoldia limatula (Say) was found by light-scattering to have a mol. wt of 8.0 +/- 0.6 x 10(6). Mass measurements by scanning transmission electron microscopy (STEM) gave a particle mass of 8.25 +/- 0.42 x 10(6) for the native particle and 4.09 +/- 0.20 x 10(6) for the half-molecule. 2. The hemocyanin subunits fully dissociated in 8.0 M urea and 6.0 M GdmCl at pH 8.0, and at pH 11.0, 0.01 M EDTA have mol. wts of 4.38 x 10(5), 4.22 x 10(5) and 4.71 x 10(5), close to one-twentieth of the parent molecular weight of Y. limatula hemocyanin and most gastropod hemocyanins. 3. Analyses of the urea dissociation transitions studied at pH 8.0, 1 x 10(-2) M Mg2+, 1 x 10(-2) M Ca2+ and pH 8.0, 3 x 10(-3) M Ca2+ suggest few hydrophobic amino acid groups, of the order of 10 to 15 at the contact areas of each half-molecule or decamer. 4. The further dissociation of the decamers to dimers and the dimers to monomers indicates the presence of a larger number of amino acid groups of ca 35-40/dimer and 100-120/monomer. 5. This suggests hydrophobic stabilization of the dimer to dimer and monomer to monomer contacts within the decamers, as observed with other molluscan hemocyanins.     

In vitro toxicity of palladium(II) and gold(III) porphyrins and their aqueous metal ion counterparts on Trypanosoma brucei brucei growth

The trypanocidal effects of aqueous gold(III) and palladium(II) and their metalloporphyrin derivatives on Trypanosoma brucei brucei growth in culture have been studied using an Alamar Blue indicator assay. All the experiments were conducted in the dark. As previously described for mercury(II), cadmium(II) and lead(II) porphyrins [Chem.-Biol. Interact. 139 (2002) 177], the toxicity of the metalloporphyrin complex of palladium(II) to T. b. brucei parasites was much higher compared to the aqueous free palladium(II) and free base porphyrin. Palladium(II) porphyrin, free palladium(II), and the free base porphyrin were trypanocidal to T. b. brucei at concentrations >1.5 x 10(-6), >6.1 x 10(-6) and >1.9 x 10(-5) M, respectively. While gold(III) porphyrin was effective against the parasites at concentrations >4.8 x 10(-6) M, its aqueous gold(III) was toxic at concentrations as low as 2.0 x 10(-7) M due to the generation of free radicals in the presence of this metal ion which enhanced its toxicity to the T. b. brucei parasites. Although some cell division was observed in some of the cells treated with palladium(II) porphyrin, some dividing cells had no nucleus due to unequal division and delivery of the nuclei into the daughter cells. As a result, the rate of cell division decreased with time and cell death occurred within 24 h. Interestingly, trypanosomes treated with metalloporphyrin complexes displayed different morphological features from those cells treated with free base porphyrin or metal ions. Of all the porphyrins and free metal ions tested, only mercury(II) porphyrin and aqueous gold(III) ion were toxic to the trypanosomes in the 10(-7) M range. The chemotherapeutic potential of these observations is discussed.     

Dissociation of cAMP accumulation and phosphate uptake in opossum kidney (OK) cells with parathyroid hormone (PTH) and parathyroid hormone related protein (PTHrP)

Bovine parathyroid hormone (PTH) 1-34 [bPTH(1-34)] and human PTH related protein [hPTHrP(1-34)] stimulated cAMP accumulation in opossum kidney (OK) cells with Km of 5 x 10(-9) M, but inhibition of phosphate uptake was obtained with 17-fold lower Km of 3 x 10(-10) M. Phosphate uptake was partially inhibited with [Nle8.18Tyr34]bPTH(3-34)NH2 without concomitant cAMP stimulation. With hPTHrP(7-34)NH2, cAMP accumulation was increased in parallel to inhibition of phosphate uptake. [D-Trp12Tyr34]bPTH(7-34)NH2 and [Tyr34]hPTH(7-34)NH2 had no agonist activity on cellular cAMP and inhibition of phosphate uptake. bPTH(1-34)-stimulated cAMP accumulation was antagonized by [Nle8.18Tyr34]bPTH(3-34)NH2, [D-Trp12Tyr34]bPTH(7-34)NH2, hPTHrP(7-34)NH2 and [Tyr34]hPTH(7-34)NH2 with Ki of 1.4 x 10(-7), 2 x 10(-7), 4.7 x 10(-7) and 3.7 x 10(-6) M, respectively. But [Nle8.18Tyr34]bPTH(3-34)NH2 and [D-Trp12Tyr34]bPTH(7-34)NH2 reversed the inhibition of phosphate uptake only marginally, and hPTHrP(7-34)NH2 and [Tyr34]hPTH(7-34)NH2 were inactive. With hPTHrP(1-34) the Ki for cAMP accumulation of [Nle8,18Tyr34]bPTH(3-34)NH2 and hPTHrP(7-34)NH2 were 1.9 x 10(-7) and 7.2 x 10(-7) M, and inhibition of phosphate uptake was partially reversed with [Nle8,18Tyr34]bPTH(3-34)NH2, but not with hPTHrP(7-34)NH2. The present results indicate that truncated hPTHrP(7-34)NH2, unlike [Tyr34]hPTH(7-34)NH2 and [D-Trp12Tyr34]bPTH(7-34)NH2, elevates cellular cAMP and inhibits phosphate uptake. bPTH(1-34)- and hPTHrP(1-34)-evoked cAMP accumulation is suppressed by PTH and PTHrP fragments while inhibition of phosphate uptake remains largely unaltered.     

Synthesis, crystal structure, cytotoxic activity and DNA-binding properties of the copper (II) and zinc (II) complexes with 1-[3-(2-pyridyl)pyrazol-1-ylmethyl]naphthalene

A new ligand L, 1-[3-(2-pyridyl)pyrazol-1-ylmethyl]naphthalene, and its two metal complexes, [Cu(L)3](ClO4)2 (1) and [Zn(L)3](ClO4)2(H2O)2 (2), have been synthesized and characterized. The crystal structure of complex 1 was determined by single crystal X-ray diffraction, which crystallized in monoclinic, space group P2(1)/n with unit cell parameters, a = 12.710(4) angstroms, b = 12.135(3) angstroms, c = 33.450(9) angstroms, beta = 93.281(5) degrees and Z = 4. The Cu atom was six-coordinated to N(1), N(2), N(4), N(5), N(7) and N(8) from three L ligands and formed a slightly distorted octahedral geometry. Complexes 1 and 2, and ligand L were subjected to biological tests in vitro using three different cancer cell lines (HL-60, BGC-823 and MDA-MB-435). Complex 1 showed significant cytotoxic activity against three cancer cell lines. The interactions of complexes 1 and 2, and ligand L with calf thymus DNA were then investigated by thermal denaturation, viscosity measurements and spectrophotometric methods. The experimental results indicated that complexes 1 and 2 bound to DNA by intercalative mode via the ligand L. The intrinsic binding constants of complexes 1 and 2, and ligand L with DNA were 1.8 x 10(4), 5.4 x 10(3) and 2.76 x 10(3) M(-1), respectively.     

Electron paramagnetic resonance, kinetics of formation and decomposition studies of (bis(hydroxyethyl)amino-tris(hydroxymethyl)-methane)oxochromate(V): a model chromium(V) complex for DNA damage studies

A new chromium complex, (bis(hydroxyethyl)amino-tris(hydroxymethyl)methane)oxochromate(V), has been characterized by epr spectroscopy. The chromium(V) complex was formed by the ligand displacement reaction of bis(2-ethyl-2-hydroxybutanato) oxochromate(V) with bis(hydroxyethyl)amino-tris(hydroxy-methyl)methane (BT). Both epr and kinetic data indicate that the reaction proceeds through a chromium(V) intermediate. Kinetics of formation of the intermediate exhibit a rate saturation at higher [BT] (> 30 mM) indicating a rate law constituting an equilibrium between the parent Cr(V) complex and the bis-tris ligand followed by a pure first order process. The g-value of the intermediate is consistent with a Cr(V) complex in which the BT is coordinated in a bidentate fashion replacing a coordinated hydroxy butanoic acid ligand, affording a mixed ligand complex. The equilibrium step (K = 36 M-1) consists of monodentate coordination by the BT ligand and the limiting first order rate constant (1.9 x 10(-2) s-1) manifests the rate of chelation by the polydentate ligand. The intermediate is converted to the product upon further chelation through the complete displacement of the remaining 2-ethyl-2-hydroxy butanoic acid by a first order process (k = 0.023 s-1). The epr data support a pair of products that are in rapid equilibrium. In these products, BT functions either as a tetra or a penta-dentate ligand coordinating through four or five alkoxy sites. The enthalpy and entropy of activations related to the two chelation steps were found to be 32 +/- 2 kJ/mol and -(1.7 +/- 0.2) x 10(2) J/mol K for the intermediate, and 36 +/- 1 kJ/mol and -(1.5 +/- 0.2) x 10(2) J/mol K for the product. Our data support an associative mechanism for the chelation steps. The Cr(V)-BT product is more stable than the parent complex. The second order disproportionation rate constant for the Cr(V)-BT complex was evaluated to be 0.1 M-1 s-1 compared to 8.0 M-1 s-1 for the parent complex. This is the first example of a chromium(V) complex with a non-macrocyclic ligand coordinating through oxygen donor atoms which is stable in aqueous solution at neutral pH over a long period of time.     

Synthesis, characterization, and DNA-binding properties of the Ln(III) complexes with 6-hydroxy chromone-3-carbaldehyde-(2'-hydroxy) benzoyl hydrazone

A new ligand, 6-hydroxy chromone-3-carbaldehyde-(2'-hydroxy) benzoyl hydrazone (L), was prepared by condensation of 6-hydroxy-3-carbaldehyde chromone (CDC) with 2-hydroxy benzoyl hydrazine. Its four rare earth complexes have been synthesized and characterized on the basis of elemental analyses, molar conductivities, mass spectra, 1H NMR, thermogravimetry/differential thermal analysis (TG-DTA), UV-vis spectra, fluorescence spectra, and IR spectra. The general formula of the complexes is [LnL2.(NO3)2].NO3 [Ln=La(1), Sm(2), Dy(3), Eu(4)]. Spectrometric titration, ethidium bromide displacement experiments, and viscosity measurements indicate that Eu(III) complex and ligand, especially the Eu(III) complex, strongly bind with calf-thymus DNA, presumably via an intercalation mechanism. The intrinsic binding constants of Eu(III) complex and ligand with DNA were 3.55 x 10(6) and 1.33 x 10(6)M(-1) through fluorescence titration data, respectively. In addition, the suppression ratio for O2-* and OH* of the ligand and its complexes was studied by spectrophotometric methods. The experimental results show that La (1), Sm (2), and Eu (4) complexes are better effective inhibitor for OH* than that of mannitol. It indicates that the complexes have the activity to suppress O2-* and OH* and exhibit more effective antioxidants than ligand alone.     

DNA damage and 2'-deoxyguanosine oxidation induced by S(IV) autoxidation catalyzed by copper(II) tetraglycine complexes: synergistic effect of a second metal ion

S(IV) (SO(2),HSO(3)(-)andSO(3)(2-)) autoxidation catalyzed by Cu(II)/tetraglycine complexes in the presence of DNA or 2'-deoxyguanosine (dGuo) resulted in DNA strand breaks and formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), respectively. Ni(II), Co(II) or Mn(II) (1.0x10(-4)M) complexes had much smaller effects. Cu(II)/tetraglycine (1.0x10(-4)M) in the presence of Ni(II) or Mn(II) (10(-7)-10(-6)M) and S(IV) showed remarkable synergistic effect with these metal ions producing a higher yield of 8-oxodGuo. Oxidation of dGuo and DNA damage were attributed to oxysulfur radicals formed as intermediates in S(IV) autoxidation catalyzed by transition metal ions. SO*(3)(-) and HO* radicals were detected by EPR-spin trapping experiments with DMPO (5,5-dimethyl-1-pyrroline-N-oxide).     

Spectroscopic properties of the metalloregulatory Cd(II) and Pb(II) sites of S. aureus pI258 CadC

Staphylococcus aureus pI258 CadC is an extrachromosomally encoded metalloregulatory repressor protein from the ArsR superfamily which negatively regulates the expression of the cad operon in a metal-dependent fashion. The metalloregulatory hypothesis holds that direct binding of thiophilic divalent cations including Cd(II), Pb(II), and Zn(II) by CadC allosterically regulates the DNA binding activity of CadC to the cad operator/promoter (O/P). This report presents a detailed characterization of the metal binding and DNA binding properties of wild-type CadC. The results of analytical ultracentrifugation experiments suggest that both apo- and Cd(1)-CadC are stable or weakly dissociable homodimers characterized by a K(dimer) = 3.0 x 10(6) M(-1) (pH 7.0, 0.20 M NaCl, 25.0 degrees C) with little detectable effect of Cd(II) on the dimerization equilibrium. As determined by optical spectroscopy, the stoichiometry of Cd(II) and Pb(II) binding is approximately 0.7-0.8 mol/mol of wild-type CadC monomer. Chelator (EDTA) competition binding isotherms reveal that Cd(II) binds very tightly, with K(Cd) = 4.3 (+/-1.8) x 10(12) M(-1). The results of UV-Vis and X-ray absorption spectroscopy of the Cd(1) complex are consistent with a tetrathiolate (S(4)) complex formed by four cysteine ligands. The (113)Cd NMR spectrum reveals a single resonance of delta = 622 ppm, consistent with an S(3)(N,O) or unusual upfield-shifted S(4) complex. The Pb(II) complex reveals two prominent absorption bands at 350 nm (epsilon = 4000 M(-1) cm(-1)) and 250 nm (epsilon = 41 000 M(-1) cm(-1)), spectral properties consistent with three or four thiolate ligands to the Pb(II) ion. The change in the anisotropy of a fluorescein-labeled oligonucleotide containing the cad O/P upon binding CadC and analyzed using a dissociable CadC dimer binding model reveals that apo-CadC forms a high-affinity complex [K(a) = (1.1 +/- 0.3) x 10(9) M(-1); pH 7.0, 0.40 M NaCl, 25 degrees C], the affinity of which is reduced approximately 300-fold upon the binding of a single molar equivalent of Cd(II) or Pb(II). The implications of these findings on the mechanism of metalloregulation are discussed.     

Comparison of angiotensin II (Ang II) effects in the internal anal sphincter (IAS) and lower esophageal sphincter smooth muscles

Studies were performed to compare the actions of Ang II in the internal anal sphincter (IAS) vs. lower esophageal sphincter (LES) smooth muscles in vitro, in opossum and rabbit. Studies also were carried out in isolated smooth muscle cells. In opossum, Ang II produced no discernible effects in the IAS, but did produce a concentration-dependent contraction in the LES. Conversely, in the rabbit, while Ang II caused a modest response in the LES, it caused a significant contraction in the IAS. The contractile responses of Ang II in the opossum LES were mostly resistant to different neurohumoral antagonists but were antagonized by AT1 antagonist losartan. AT2 antagonist PD 123,319, rather than inhibiting, prolonged the contractile action of Ang II. The contractile actions of Ang II in the opossum LES were not modified by the tyrosine kinase inhibitors (genistein and tyrphostin 1 x 10(-6) M) but were partially attenuated by the PKC inhibitor H-7 (1 x 10(-6) M), Ca2+ channel blocker nicardipine (1 x 10(-5) M), Rho kinase inhibitor HA-1077 (1 x 10(-7) M) or p(44/42) MAP kinase inhibitor PD 98059 (5 x 10(-5) M). The combination of HA-1077 and H-7 did not cause an additive attenuation of Ang II responses. Western blot analyses revealed the presence of both AT1 and AT2 receptors. We conclude that Ang lI-induced contraction of sphincteric smooth muscle occurs primarily by the activation of AT1 receptors at the smooth muscle cells and involves multiple pathways, influx of Ca2+, and PKC, Rho kinase and p(44/42) MAP kinase.     

The hemoglobin of the aquatic snail, Planorbella duryi (Wetherby)

1. The hemoglobin of the pond snail, Planorbella duryi has a molecular weight of 1.64 x 10(6) to 1.77 x 10(6) as determined by light-scattering at 630 nm and a sedimentation coefficient of 36 S. 2. The analysis of the circular dichroism spectrum obtained in the 190-250 nm region suggests a high degree of helical folding of the polypeptide chains of P. duryi hemoglobin analogous to human hemoglobin and myoglobin, with estimates of alpha-helical folding of about 60-65%, 0-5% beta-structure, and the remaining portion of the chains in unordered form. 3. The dissociated subunits in 6.0 M GdmCl, in the absence and in the presence of reducing reagent (0.1 M dithiothreitol), have a molecular weight of 3.73 +/- 0.23 x 10(5) and 1.93 +/- 0.04 x 10(5), suggesting a di-decameric assembly of the parent hemoglobin organized in the form of five dimers held together by disulfide-linkages. 4. The native hemoglobin is strongly resistant to both pH dissociation and dissociation by urea and such salts as NaCl and NaClO4. Dissociation and denaturation could only be effected in concentrated GdmCl solutions. 5. The influence of the various dissociating agents on the quaternary structure suggest ionic stabilization of the decameric assembly, which is stabilized by salt bridges between the subunits.     

Cl- uptake mechanism in freshwater-adapted tilapia (Oreochromis mossambicus)

In this study, the correlation between Cl(-) influx in freshwater tilapia and various transporters or enzymes, the Cl(-)/HCO(3)(-) exchanger, Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase were examined. The inhibitors 2x10(-4) M ouabain (a Na(+),K(+)-ATPase inhibitor), 10(-5) M NEM (a V-type H(+)-ATPase inhibitor), 10(-2) M ACTZ (acetazolamide, a carbonic anhydrase inhibitor), and 6x10(-4) M DIDS (a Cl(-)/HCO(3)(-) exchanger inhibitor) caused 40%, 60%-80%, 40%-60%, and 40%-60% reduction in Cl(-) influx of freshwater tilapia, respectively. The inhibitor 2x10(-4) M ouabain also caused 50%-65% inhibition in gill Na(+),K(+)-ATPase activity. Western blot results showed that protein levels of gill Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase in tilapia acclimated in low-Cl(-) freshwater were significantly higher than those acclimated to high-Cl(-) freshwater. Based on these data, we conclude that Na(+),K(+)-ATPase, V-H(+)-ATPase, the Cl(-)/HCO(3)(-) exchanger, and carbonic anhydrase may be involved in the active Cl(-) uptake mechanism in gills of freshwater-adapted tilapia.     

Acetyl-CoA carboxylase inhibitors from avocado (Persea americana Mill) fruits

A methanol extract of avocado fruits showed potent inhibitory activity against acetyl-CoA carboxylase, a key enzyme in fatty acid biosynthesis. The active principles were isolated and identified as (5E,12Z,15Z)-2-hydroxy-4-oxoheneicosa-5,12,15-trienyl (1), (2R,12Z,15Z)-2-hydroxy-4-oxoheneicosa-12,15-dienyl (2), (2R*,4R*)-2,4-dihydroxyheptadec-16-enyl (3) and (2R*,4R*)-2,4-dihydroxyheptadec-16-ynyl (4) acetates by instrumental analyses. The IC50 of the compounds were 4.0 x 10(-6), 4.9 x 10(-6), 9.4 x 10(-6), and 5.1 x 10(-6) M, respectively.     

Linear free-energy analysis of mercury(II) and cadmium(II) binding to three-stranded coiled coils

Investigators have studied how proteins enforce nonstandard geometries on metal centers to assess the question of how protein structures can define the coordination geometry and binding affinity of an active-site metal cofactor. We have shown that cysteine-substituted versions of the TRI peptide series [AcG-(LKALEEK)(4)G-NH(2)] bind Hg(II) and Cd(II) in geometries that are different from what is normally found with thiol ligands in aqueous solution. A fundamental question has been whether this structural perturbation is due to protein influence or a change in the metal geometry preference. To address this question, we have completed linear free-energy analyses that correlate the association of three-stranded coiled coils in the absence of a metal with the binding affinity of the peptides to the heavy metals, Hg(II) and Cd(II). In this paper, six new members of this family have been synthesized, replacing core leucine residues with smaller and less hydrophobic residues, consequently leading to varying degrees of self-association affinities. At the same time, studies with some smaller and longer sequenced peptides have also been examined. All of these peptides are seen to sequester Hg(II) and Cd(II) in an uncommon trigonal environment. For both metals, the binding is strong with micromolar dissociation constants. For binding of Hg(II) to the peptides, the dissociation constants range from 2.4 x 10(-)(5) M for Baby L12C to 2.5 x 10(-)(9) M for Grand L9C for binding of the third thiolate to a linear Hg(II)(pep)(2) species. The binding of Hg(II) to the peptide Grand L9C is similar in energetics for metal binding in the metalloregulatory protein, mercury responsive (merR), displaying approximately 50% trigonal Hg(II) formation at nanomolar metal concentrations. Approximately, 11 kcal/mol of the Hg(II)(Grand L9C)(3)(-) stability is due to peptide interactions, whereas only 1-4 kcal/mol stabilization results from Hg(II)(RS)(2) binding the third thiolate ligand. This further validates the hypothesis that the favorable tertiary interactions in protein systems such as merR go a long way in stabilizing nonnatural coordination environments in biological systems. Similarly, for the binding of Cd(II) to the TRI family, the dissociation constants range from 1.3 x 10(-)(6) M for Baby L9C to 8.3 x 10(-)(9) M for TRI L9C, showing a similar nature of stable aggregate formation.     

Effects of amiridin and tacrine, drugs effective in Alzheimer's disease, on synaptosomal uptake of neuromediators]

The study of the drugs effective in the treatment of cognitive deficits and memory loss associated with senile dementia of the Alzheimer's type--tacrine and amiridin, acetylcholinesterase inhibitor physostigmine and nootrop piracetam on uptake of 3H-serotonin (3H-5-HT), 3H-adrenaline (3H-AD), 3H-noradrenaline (3H-HA), 2H-dopamine (3H-DA), 3H-gamma-aminobutyric acid (3H-GABA), 3H-glutamic acid (3H-GLU), 3H-aspartic acid (3H-ASP) and 3H-glycine (3H-GLI) showed that tacrine and amiridin (5 x 10(-5) M) statistically significantly (P less than 0.05) inhibited the uptake of 3H-DA and 3H-5-HT. Physostigmine at concentration 5 x 10(-4) M statistically significantly (P less than 0.05) inhibited uptake of 3H-5-HT only. Piracetam at concentration range 1-5 x 10(-3) M had no effect on uptake of all investigated neurotransmitters. The above finding suggest that the uptake of neurotransmitter in nerve terminals is not the main target of amiridin and tacrine.