The monensin-mediated transport of Na+ and K+ through phospholipid bilayers studied by 23Na- and 39K-NMR

Addition of monesin to preparations of large unilamellar vesicles made from egg yolk phosphatidylcholine (EPC) in sodium or potassium chloride solution and from dioleoylphosphatidylcholine (DOPC) in sodium chloride solutions gives rise to dynamic 23Na- and 39K-NMR spectra. The dynamic spectra arise from the monensin-mediated transport of the metal ions through the membrane. The kinetics of the transport are followed as a function of monensin and metal ion concentrations and are compatible with a model in which one monensin molecule transports one metal ion. Rate constants for the association and dissociation of the monensin-metal complex in the membrane/water interface are extracted and the stability constants for complex formation are evaluated. The rate constants in DOPC are similar to those in EPC, confirming that diffusion is not rate-limiting in the transport process and that dissociation of the complex is the rate-limiting step. Although potassium on its own is transported more rapidly, sodium forms the more stable complex and is therefore transported preferentially in competition with potassium.     

Inactivation of thiostrepton by copper(II)

Summary Among the various bivalent metal ions tested, only copper(II) was found to bind to thiostrepton (M _rr 1650) in a stoichiometric ratio of 4:1. The binding of four copper ions to a thiostrepton molecule resulted in (a) irreversible loss in biological activity and (b) a change in the ultraviolet absorption spectrum of the antibiotic. Potentiometric titration of thiostrepton in the presence of copper(II) revealed dissociation of the antibiotic with a loss of 11 protons/molecule. Based on the preferential ability of copper(II) to bind to thiostrepton in the presence of some copper-complexing compounds containing similar ligand groups to the antibiotic, the possible co-ordinating atoms of the thiostrepton molecule involved in binding to the metal ion are discussed.     

Structure,function and selective inhibition of bacterial acetyl-coa carboxylase

Acetyl-CoA carboxylase (ACC) catalyses the first committed step in fatty acid biosynthesis: a metabolic pathway required for several important biological processes including the synthesis and maintenance of cellular membranes. ACC employs a covalently attached biotin moiety to bind a carboxyl anion and then transfer it to acetyl-CoA, yielding malonyl-CoA. These activities occur at two different subsites: the biotin carboxylase (BC) and carboxyltransferase (CT). Structural biology, together with small molecule inhibitor studies, has provided new insights into the molecular mechanisms that govern ACC catalysis, specifically the BC and CT subunits. Here, we review these recent findings and highlight key differences between the bacterial and eukaryotic isozymes with a view to establish those features that provide an opportunity for selective inhibition. Especially important are examples of highly selective small molecule inhibitors capable of differentiating between ACCs from different phyla. The implications for early stage antibiotic discovery projects, stemming from these studies, are discussed.     

The transport of Na+ and K+ ions through phospholipid bilayers mediated by the antibiotics salinomycin and narasin studied by 23Na- and 39K-NMR spectroscopy

Addition of the ionophoric antibiotics salinomycin or narasin to preparations of large unilamellar vesicles made from egg yolk phosphatidylcholine in sodium or potassium chloride solutions gives rise to dynamic effects in the 23Na- and 39K-NMR spectra. The dynamic spectra arise from the ionophore-mediated transport of the metal ions through the membrane. The kinetics of the transport are followed as a function of the concentrations of ionophore and the metal ion and are compatible in all cases with a model in which one ionophore molecule transports one metal ion. For both ionophores the transport of potassium ions is appreciably faster than that of sodium and in both cases the rate-limiting step for sodium transport is dissociation of the ionophore-metal complex. Assuming dissociation to be rate limiting in all four cases it is shown that the transport rate differences between the pairs of complexes of each metal arise solely from differences in the rates of formation. The stability constants for ionophore-metal complex formation in the membrane/water interface are evaluated.     

Structure and metal ion binding of the first transmembrane domain of DMT1

DMT1 is an integral membrane protein with 12 putative transmembrane domains. As a divalent metal ion transporter, it plays an important role in metal ion homeostasis from bacteria to human. Loss-function mutations at the conserved motif DPGN located within the first transmembrane domain (TMD1) of DMT1 indicate the significance of TMD1 in the biological function of the protein. In the present work, we study the structure, topology and metal ion binding of DMT1-TMD1 peptide by nuclear magnetic resonance using sodium dodecyl sulfate and dodecylphosphocholine micelles as membrane mimics. We find that the peptide forms an α-helix-extended segment-α-helix configuration in which the motif DPGN locates at the central flexible region. The N-terminal part of the peptide is deeply embedded in micelles, while the motif section and the C-terminal part are close to the surface of micelles. The peptide can bind to Mn2+ and Co2+ ions by the side chains of the negatively charged residues in the motif section and the C-terminal part of TMD1. The crucial role of the central flexible region and the C-terminal part of TMD1 in metal ion capture is confirmed by the binding of the N-terminal part truncated TMD1 to metal ions.     

Energy distribution and ionic selectivity of the bacterial potassium channel

An explanation for the ionic selectivity of the bacterial potassium channel K(CS)A is offered, which is based on a comparison of energy interactions of lithium, sodium, and potassium cations with the atoms of the selective filter of an protein pore. Using quantum-chemical calculations, the presence of a deeper potential hole for potassium ions was discovered, which explains the energy preferableness in their permeability. It has been shown that the traditional methods of force field AMBER, CHARMM, OPLS in reference parametrization and also at their partial reparametrization give incorrect ratings of energy distribution of ions in the channel.     

Metal binding characteristics of human salivary and porcine pancreatic amylase

With the exception of calcium very little is known about metal binding characteristics of either human salivary or porcine pancreatic amylase. In order to learn more about these protein-metal binding interactions, calcium-free human salivary and porcine pancreatic amylase [P(protein)] were obtained by carboxymethylcellulose chromatography of the partially purified proteins. Because these proteins acquired small amounts of calcium after further preparatory studies, they were dialyzed against 1 mM EDTA, pH 7.4, at 22 degrees C, which removed essentially all acquired calcium. The calcium-free amylases were then subjected to equilibrium dialysis against copper or zinc solutions with or without added glycine. The experimental data were fitted to appropriate mathematical equations, and binding constants of the metal complexes were calculated. Both human salivary and porcine pancreatic amylase were found to have two metal ion binding sites, only one of which was selective for calcium. Copper or zinc appeared to bind to the second site forming the species CuCaLP (or ZnCaP), where L, a ligand, is the glycine anion. Neither copper nor zinc displaced calcium from human salivary amylase, although copper bound to both binding sites in human salivary apoamylase to form the species Cu2L2P in which the amylase molecule appeared to form a bridge between the two copper atoms. In the case of the zinc-human salivary apoamylase system, the experimental data could not be analyzed quantitatively since the protein formed an insoluble complex species. Copper displaced calcium from porcine pancreatic amylase and formed a mixed ligand species similar to that formed with human salivary apoamylase. Zinc bound to both metal binding sites of porcine pancreatic apoamylase, forming species ZnP and Zn2P, although it did not displace calcium from the protein. While calcium in amylase is known to be critical for its amylolytic activity, little is known about the function of either zinc or copper in amylase albeit both of these metals are important in biological systems.     

A novel HDAC inhibitor with a hydroxy-pyrimidine scaffold

Histone deacetylases (HDACs) are enzymes involved in many important biological functions. They have been linked to a variety of cancers, psychiatric disorders, and other diseases. Since small molecules can serve as probes to study the relevant biological roles of HDACs, novel scaffolds are necessary to develop more efficient, selective drug candidates. Screening libraries of molecules may yield structurally diverse probes that bind these enzymes and modulate their functions in cells. Here we report a small molecule with a novel hydroxy-pyrimidine scaffold that inhibits multiple HDAC enzymes and modulates acetylation levels in cells. Analogs were synthesized in an effort to evaluate structure-activity relationships.     

Iodinated bovine prolactin. Characterization and binding to mammary gland cells.

Iodinated bovine prolactin (2.6 iodine atoms/molecule; labelled with a trace of 125I to give a specific activity of 0.041 muCi/mg) was prepared by the chloramine T method. It was active in two bioassays (pigeon crop sac and dispersed mouse mammary cell), though somewhat less active than the unmodified hormone. In an immunoassay, iodinated prolactin was more effective than the unmodified hormone at displacing 125I-prolactin from antibody. High specific activity 125I-prolactin (1 iodine atom/molecule; 70 muCi/microgram) was used for autoradiographic studies on the binding of prolactin to mouse mammary cells. In vivo the labelled hormone found in the mammary gland was associated with membranes of mammary epithelial cells and with alveolar lumen contents. In vitro 125I-prolactin was shown to bind to dispersed mouse mammary epithelial cells.     

Selective control of calcium levels by naloxone.

Acute treatment with morphine sulfate produces a selective loss of calcium from synaptosomal particulate fractions of rat brain. No changes in sodium, potassium or magnesium content were observed for myelin, synaptosomal particulate or mitochondrial fractions. Acute opiate treatment (90 min.) while causing calcium loss, produced no changes in regional brain content for sodium, potassium or magnesium. Naloxone, in the presence of morphine, reversed the calcium loss in both regional brain areas and synaptosomal particulate fractions. An hypothesis is offered that naloxone may bind to synaptosomal membranes protecting a morphine sensitive calcium pool, or may reverse the calcium loss seen after opiate agonist treatment.     

Affinities or Apparent Affinities of the Transport Adenosine Triphosphatase System

The interactions of potassium ions and ATP on transport ATPase activity are discussed, and the interpretation of these interactions is shown to be often ambiguous. Caldwell''s (1968) Physiological Review model is discussed with particular reference to the observed kinetics of sodium: sodium exchange in red cells. Recent experimental work on the properties of the ouabain-sensitive component of potassium efflux from red cells is described. This component of efflux occurs only if either sodium or potassium are present in the external medium, but the effects of external sodium and potassium are not additive. The relation between ouabain-sensitive potassium efflux and the external concentration of sodium (in a potassium-free medium) or of potassium (in low- and high-sodium media) are described. When starved sodium-poor red cells are poisoned with iodoacetamide, loaded with phosphate, and incubated in high-sodium potassium-free media, the ouabain-sensitive efflux of potassium appears to be accompanied by the reversal of the entire ATPase system. About two to three potassium ions leave by the ouabain-sensitive route for each molecule of ATP synthesized. If potassium is present in the external medium, no ouabain-sensitive synthesis of ATP occurs and the ouabain-sensitive efflux of potassium presumably involves the reversal of only the last part of the ATPase system.     

The effect of the rcation/ranion ratio on the structural and chemical properties of N-chloroarenesulfonamidates

A comparative structural study on sodium and potassium N-chloroarenesulfonamidates has been carried out using X-ray diffraction and IR spectroscopy as experimental techniques. As shown by crystallographic studies, the sodium ions tend to incorporate more water oxygen atoms in their coordination spheres than the potassium ions, while the potassium congeners prefer the interaction with sulfonamidate moieties. The marked dissimilarity between the compositions of the coordination spheres as well as the different tendency of the sodium and potassium salts to absorb moisture from the air have been attributed to the different sizes of the sodium and potassium ions. The opposite shifts of the ν_as(SO₂) and ν(SN) bands upon dehydration have been ascribed to an increased polarization of the sulfonyl group by the metal centers. Based on IR spectroscopic observations, a weakening of the N-Cl bonds is suspected in the water-free compounds, which may contribute to the known thermal instability of the dehydrated salts of N-chloroarenesulfonamides. Various sections of IR spectra as well as X-ray powder diffraction patterns have proved to be suitable to identify the water-free and hydrated samples.     

The adaptive function of melanin-based plumage coloration to trace metals

Trace metals produced by anthropogenic activities are of major importance in urban areas and might constitute a new evolutionary force selecting for the ability to cope with their deleterious effects. Interestingly, melanin pigments are known to bind metal ions, thereby potentially sequestering them in inert body parts such as coat and feathers, and facilitating body detoxification. Thus, a more melanic plumage or coat coloration could bring a selective advantage for animals living in polluted areas. We tested this hypothesis by investigating the link between melanin-based coloration and zinc and lead concentrations in feathers of urban feral pigeons, both at capture time and after one year of captivity in standardized conditions. Results show that differently coloured pigeons had similar metal concentrations at capture time. Metal concentrations strongly decreased after one year in standardized conditions, and more melanic pigeons had higher concentrations of zinc (but not lead) in their feathers. This suggests that more melanic pigeons have a higher ability to store some metals in their feathers compared with their paler counterparts, which could explain their higher success in urbanized areas. Overall, this work suggests that trace metal pollution may exert new selective forces favouring more melanic phenotypes in polluted environments.     

The interaction of analogues of the antimicrobial lipopeptide, iturin A2, with alkali metal ions

Electrospray mass spectrometry was employed as a tool in this first study on the molecular interaction between the alkali metal ions and antifungal lipopeptide iturin A, and some analogues. Cationisation by sodium and signal intensity of lipopeptide species depended on sodium concentration, but was independent of sample solvent, carrier solvent polarity and sample pH between 4 and 11. 8-Beta, a linear analogue of iturin A2 (8-Beta; beta-aminotetradecanoyl-NYNQPNS), and its shorter linear lipopeptide analogues, associated either one or two alkali metal cations, while the N-->C cyclic peptides associated with only one cation. The chirality of the beta-NC14 residue had a limited influence on the cationisation. It was observed that 8-Beta contained at least four interaction sites for a cation of which two, the C-terminal carboxylate and the side-chain of tyrosine, can take part in ionic interaction with a cation. It is proposed that the remaining two interaction centres of alkali metal ions are within the two type II beta-turns found in conformation of natural iturin A. This was corroborated by the diminished capacity of the shorter peptides, in which one of the beta-turns was eliminated to bind a second larger cation. All the lipopeptides showed the same order of alkali metal ion selectivity: Na+ > K+ > Rb+. These results indicated a size limitation in the interaction cavity or cavities. The absence of, or observation of only low abundance, di-cationised complexes of cyclic peptides the indicated association of the cation in the interior of the peptide ring. It is thus hypothesised that alkali metal ions can bind in one of the two beta-turns in the natural iturin A molecule.     

Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 1. Crystallography and site-directed mutagenesis of metal binding sites.

The structure and function of the xylose (glucose) isomerase from Actinoplanes missouriensis have been analyzed by X-ray crystallography and site-directed mutagenesis after cloning and overexpression in Escherichia coli. The crystal structure of wild-type enzyme has been refined to an R factor of 15.2% against diffraction data to 2.2-A resolution. The structures of a number of binary and ternary complexes involving wild-type and mutant enzymes, the divalent cations Mg2+, Co2+, or Mn2+, and either the substrate xylose or substrate analogs have also been determined and refined to comparable R factors. Two metal sites are identified. Metal site 1 is four-coordinated and tetrahedral in the absence of substrate and is six-coordinated and octahedral in its presence; the O2 and O4 atoms of linear inhibitors and substrate bind to metal 1. Metal site 2 is octahedral in all cases; its position changes by 0.7 A when it binds O1 of the substrate and by more than 1 A when it also binds O2; these bonds replace bonds to carboxylate ligands from the protein. Side chains involved in metal binding have been substituted by site-directed mutagenesis. The biochemical properties of the mutant enzymes are presented. Together with structural data, they demonstrate that the two metal ions play an essential part in binding substrates, in stabilizing their open form, and in catalyzing hydride transfer between the C1 and C2 positions.     

Ion-enhanced fluorescence staining of sodium dodecyl sulfate-polyacrylamide gels using bis(8-p-toluidino-1-naphthalenesulfonate)

A method for the sensitive fluorescent staining of sodium dodecyl sulfate (SDS) gels that extends the applicability and sensitivity of existing procedures has been developed. SDS-protein complexes are able to bind the noncovalent hydrophobic probe, bis(8-p-toluidino-1-naphthalenesulfonate) (bisANS) with an increase in quantum yield that is considerably larger than that observed with the commonly used monomeric form, 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS). The quantum yield of bisANS bound to the SDS-protein complex is greatly enhanced by incubation with one of a number of cations including potassium and barium. The use of bisANS with metal ion enhancements provides a method for staining SDS gels that can be more sensitive than commonly used methods based on the binding of Coomassie blue, and provides a simple and rapid method for the detection and quantitation of proteins. The use of metal ion enhancements also greatly increases the sensitivity of staining methods based on the use of 1,8-ANS. The present method is much more sensitive than previous noncovalent, flourescent, postelectrophoresis stains, but it retains their considerable advantages of speed, simplicity, and the ability to perform secondary procedures on the separated materials.     

Mechanisms of metal ion incorporation into metalloproteins

Although the structure and function of protein metallocenters have been extensively characterized, much less is known about their assembly. Here, I describe several general strategies for metallocenter biosynthesis and provide literature precedents for each mechanism. The simplest mechanism involves reversible metal ion binding to amino acid ligands of the apo-protein. In a variation of this mechanism, the apo-protein first must be phosphorylated, carboxylated or otherwise covalently modified in order to create the metal ion binding site. Alternatively, passive metal ion binding may require the presence of an associated compound, such as a nucleotide, carbonate or inorganic sulfide, which is co-incorporated into the protein along with the metal ion. In addition, reversible binding may occur using a pre-formed organometallic cofactor such as a metal-tetrapyrrole. Electron transfer reactions are coupled to biosynthesis of certain metallocenters, i.e. oxidation or reduction of the metallocenter or apo-protein may be required prior to binding, or once bound the metallocenter may be oxidatively trapped in the protein. An effector molecule may bind to apo-protein to open up or stabilize the metallocenter binding site, then after the metallocenter is incorporated the effector molecule dissociates. A transferase or insertase protein first may bind the metallocenter and then incorporate it into the appropriate apo-protein. Finally, metal cofactors may be covalently attached to proteins. Regardless of the metallocenter biosynthetic mechanism, intracellular metal ion concentrations must be sufficient; hence, metal ion transport systems also are briefly discussed.     

Identification of new batrachotoxin-sensing residues in segment IIIS6 of the sodium channel

Ion permeation through voltage-gated sodium channels is modulated by various drugs and toxins. The atomistic mechanisms of action of many toxins are poorly understood. A steroidal alkaloid batrachotoxin (BTX) causes persistent channel activation by inhibiting inactivation and shifting the voltage dependence of activation to more negative potentials. Traditionally, BTX is considered to bind at the channel-lipid interface and allosterically modulate the ion permeation. However, amino acid residues critical for BTX action are found in the inner helices of all four repeats, suggesting that BTX binds in the pore. In the octapeptide segment IFGSFFTL in IIIS6 of a cockroach sodium channel BgNa(V), besides Ser_3i15 and Leu_3i19, which correspond to known BTX-sensing residues of mammalian sodium channels, we found that Gly_3i14 and Phe_3i16 are critical for BTX action. Using these data along with published data as distance constraints, we docked BTX in the Kv1.2-based homology model of the open BgNa(V) channel. We arrived at a model in which BTX adopts a horseshoe conformation with the horseshoe plane normal to the pore axis. The BTX ammonium group is engaged in cation-π interactions with Phe_3i16 and BTX moieties interact with known BTX-sensing residues in all four repeats. Oxygen atoms at the horseshoe inner surface constitute a transient binding site for permeating cations, whereas the bulky BTX molecule would resist the pore closure, thus causing persistent channel activation. Our study reinforces the concept that steroidal sodium channel agonists bind in the inner pore of sodium channels and elaborates the atomistic mechanism of BTX action.     

A soluble fraction requirement in the transfer reaction of protein synthesis by rice embryo ribosomes

The requirements for the transfer of (14)C-phenylalanine from yeast soluble ribonucleic acid to protein in vitro by rice (Oryza sativa L. var. Bluebonnet) ribosomes have been investigated. An absolute requirement for polyuridylic acid, 2-mercaptoethanol, guanosine triphosphate, magnesium, and potassium or ammonium ions and ribosomes has been demonstrated. Ribosomes washed in 0.5% sodium deoxycholate also required the presence of rice supernatant. The optimum concentration of magnesium ion for the reaction was approximately 7 mm, while 60 mm of either ammonium or potassium ion gave maximum transfer of phenylalanine in this heterologous system. The optimum concentration of guanosine triphosphate required varied with the presence or absence of the phosphoenolpyruvate-pyruvate kinase generating system. Without the system, the optimum concentration was 1.5 mm, but in its presence the optimum was approximately 0.1 mm.     

Improved synthesis of unique estradiol-linked platinum(II) complexes showing potent cytocidal activity and affinity for the estrogen receptor alpha and beta

We have recently reported the synthesis of a platinum(II) complex, made of estradiol, the female sex hormone, and a cisplatin analog, an anticancer drug, linked together by an eleven carbon atoms chain. The novel estradiol-Pt(II) hybrid molecule was synthesized in nine chemical steps with 10% overall yield. This new compound has been tested in vitro on estrogen-dependent (MCF-7) and -independent (MDA-MD-231) (ER(+) and ER(-)) cell lines. Interestingly, the biological activity was quite significant, more potent than that of cisplatin, the compound currently used in chemotherapy. The estrogen receptor binding affinity (ERBA) of this compound was very similar to that of 17beta-estradiol (E(2)) on both estrogen receptors (ERs), alpha and beta. In order to further study this type of molecule, we have decided to synthesize several analogs with the same estrogenic scaffold but with various chain lengths separating the estradiol from the toxic part of the molecule. This was planned in order to study the effect of the length of the linking chain on the biological activity of the hybrids. Four E(2)-Pt(II) hybrid molecules having 6-14 carbon atoms linking chain have been synthesized using a new synthetic methodology. They are synthesized in only eight chemical steps with 21% overall yield. The 17beta-estradiol-linked platinum(II) complexes have been tested for their receptor binding affinity as well as for their cytocidal activity on several breast cancer cell lines. The synthesis and biological results are reported herein.