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At least 613 records · Page 34Linked to original sources
PubMed2006

Mercury release from deforested soils triggered by base cation enrichment.

The Brazilian Amazon has experienced considerable colonization in the last few decades. Family agriculture based on slash-and-burn enables millions of people to live in that region. However, the poor nutrient content of most Amazonian soils requires cation-rich ashes from the burning of the vegetation biomass for cultivation to be successful, which leads to forest ecosystem degradation, soil erosion and mercury contamination. While recent studies have suggested that mercury present in soils was transferred towards rivers upon deforestation, little is known about the dynamics between agricultural land-use and mercury leaching. In this context, the present study proposes an explanation that illustrates how agricultural land-use triggers mercury loss from soils. This explanation lies in the competition between base cations and mercury in soils which are characterized by a low adsorption capacity. Since these soils are naturally very poor in base cations, the burning of the forest biomass suddenly brings high quantities of base cations to soils, destabilizing the previous equilibrium amongst cations. Base cation enrichment triggers mobility in soil cations, rapidly dislocating mercury atoms. This conclusion comes from principal component analyses illustrating that agricultural land-use was associated with base cation enrichment and mercury depletion. The overall conclusions highlight a pernicious cycle: while soil nutrient enrichment actually occurs through biomass burning, although on a temporary basis, there is a loss in Hg content, which is leached to rivers, entering the aquatic chain, and posing a potential health threat to local populations. Data presented here reflects three decades of deforestation activities, but little is known about the long-term impact of such a disequilibrium. These findings may have repercussions on our understanding of the complex dynamics of deforestation and agriculture worldwide.

Agriculture↗
PubMed1997

Biophysical characterization of cationic lipid: DNA complexes.

To better understand the structures formed by the interaction of cationic lipids with DNA, we undertook a systematic analysis to determine the biophysical characteristics of cationic lipid:DNA complexes. Four model cationic lipids with different net cationic charge were found to interact in similar ways with DNA when that interaction was compared in terms of the apparent molar charge ratio of lipid to DNA. When DNA was present in charge excess over the cationic lipid, the complex carried a net negative charge as determined by zeta potential measurements. Under these conditions, some DNA was accessible to ethidium bromide, and free DNA was observed in agarose gels and in dextran density gradients. Between a lipid:DNA charge ratio of 1.25 and 1.5:1, all the DNA became complexed to cationic lipid, as evidenced by its inaccessibility to EtBr and its complete association with lipid upon agarose gel electrophoresis and density gradient separations. These complexes carried a net positive charge. The transition between negatively and positively charged complexes a occurred over a very small range of lipid to DNA ratios. Employing a fluorescent lipid probe, the addition of DNA was shown to induce lipid mixing between cationic lipid-containing vesicles. The extent of DNA-induced lipid mixing reached a maximum at a charge ratio of about 1.5:1, the point at which all the DNA was involved in a complex and the complex became positively charged. Together with freeze-fracture electron micrographs of the complexes, these biophysical data have been interpreted in light of the existing models of cationic lipid:DNA complexes.

Cations↗
PubMed2000

Tunable pH-sensitive liposomes composed of mixtures of cationic and anionic lipids.

The pH-dependent fusion properties of large unilamellar vesicles (LUVs) composed of binary mixtures of anionic and cationic lipids have been investigated. It is shown that stable LUVs can be prepared from the ionizable anionic lipid cholesteryl hemisuccinate (CHEMS) and the permanently charged cationic lipid N,N-dioleoyl-N, N-dimethylammonium chloride (DODAC) at neutral pH values and that these LUVs undergo fusion as the pH is reduced. The critical pH at which fusion was observed (pH(f)) was dependent on the cationic lipid-to-anionic lipid ratio. LUVs prepared from DODAC/CHEMS mixtures at molar ratios of 0 to 0.85 resulted in vesicles with pH(f) values that ranged from pH 4.0 to 6.7, respectively. This behavior is consistent with a model in which fusion occurs at pH values such that the DODAC/CHEMS LUV surface charge is zero. Related behavior was observed for LUVs composed of the ionizable cationic lipid 3alpha-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Chol) and the acidic lipid dioleoylphosphatidic acid (DOPA). Freeze-fracture and (31)P NMR evidence is presented which indicates that pH-dependent fusion results from a preference of mixtures of cationic and anionic lipid for "inverted" nonbilayer lipid phases under conditions where the surface charge is zero. It is concluded that tunable pH-sensitive LUVs composed of cationic and anionic lipids may be of utility for drug delivery applications. It is also suggested that the ability of cationic lipids to adopt inverted nonbilayer structures in combination with anionic lipids may be related to the ability of cationic lipids to facilitate the intracellular delivery of macromolecules.

Anions↗
PubMed1981

Fluorescence study of the divalent cation-transport mechanism of ionophore A23187 in phospholipid membranes.

The mechanism for transport of divalent cations across phospholipid bilayers by the ionophore A23187 was investigated. The intrinsic fluorescence of the ionophore was used in equilibrium and rapid-mixing experiments as an indicator of ionophore environment and complexation with divalent cations. The neutral (protonated) form of the ionophore binds strongly to the membrane, with a high quantum yield relative to that in the aqueous phase. The negatively charged form of the ionophore binds somewhat less strongly, with a lower quantum yield, and does not move across the membrane. Complexation of the negatively charged form with divalent cations was measured by the decrease in fluorescence. An apparent rate constant (kapp) for transport of the ionophore across the membrane was determined from the rate of fluorescence changes observed in stopped-flow rapid kinetic experiments. The variation of kapp was studied as a function of pH, temperature, ionophore concentration, membrane lipid composition, and divalent cation concentration and type. Analysis and comparison with equilibrium constants for protonation and complexation show that A23187 and its metal:ionophore complexes bind near the membrane-water interface in the lipid polar-head region. The interfacial reactions occur rapidly, compared with the transmembrane reactions, and are thus in equilibrium during transport. The transport cycle can be described as follows: a 1:1 complex is formed between the membrane bound A23187-(Am-) and the aqueous divalent cation with dissociation constant K1 approximately 4.6 x 10(-4) M. This is in equilibrium with a 1:2 (metal:ionophore) complex (K2 approximately 3.0 x 10(-4) [ionophore/lipid]) that is responsible for transporting the divalent cations across the membrane. The rate constant for translocation of the 1:2 complex is 0.1-0.3 s-1. Dissociation of the complex of the trans side and protonation occur rapidly. The rate constant for translocation of H+ . A23187- is 28 s-1. A theory is presented that is capable of reproducing the kinetic data at any calcium concentration. The cation specificity for ionophore complex transport (kapp), determined at low ionophore concentration for a series of divalent cations, was found to be proportional to the equilibrium constant for 1:1 complexation. The order of ion specificity for these processes was found to be Ca2+ greater than Mg2+ greater Sr2+ greater than Ba2+. Interactions with Na+ were not observed. Maximal values of kapp were observed for vesicles prepared from pure dimyristoyl phosphatidylcholine. Inclusion of phosphatidyl ethanolamine, phosphatidic acid, or dipalmatoyl phosphatidylcholine resulted in lower values of kapp. Calcium transport by A23187 is compared with that of X537A, and it is shown that the former is 67-fold faster. The difference in rates is due to differences in the ability of each ionophore to form a 1:2 complex from a 1:1 complex.

Anti-Bacterial Agents↗
PubMed1983

Large divalent cations and electrostatic potentials adjacent to membranes. A theoretical calculation.

We have extended the Gouy-Chapman theory of the electrostatic diffuse double layer by considering the finite size of divalent cations in the aqueous phase adjacent to a charged surface. The divalent cations are modeled as either two point charges connected by an infinitely thin, rigid "rod" or two noninteracting point charges connected by an infinitely thin, flexible "string." We use the extended theory to predict the effects of a cation of length 10 A (1 nm) on the zeta and surface potentials of phospholipid bilayer membranes. The predictions of the rod and string models are similar to one another but differ markedly from the predictions of the Gouy-Chapman theory. Specifically, the extended model predicts that a large divalent cation will have a smaller effect on the potential adjacent to a negatively charged bilayer membrane than a point divalent cation, that the magnitude of this discrepancy will decrease as the Debye length increases, and that a large divalent cation will produce a negative zeta potential on a membrane formed from zwitterionic lipids. These predictions agree qualitatively with the experimental results obtained with the large divalent cation hexamethonium. We discuss the biological relevance of our calculations in the context of the interaction of cationic drugs with receptor sites on cell membranes.

Cations, Divalent↗
PubMed1999

Location of a cation-binding site in the loop between helices F and G of bacteriorhodopsin as studied by 13C NMR.

The high-affinity cation-binding sites of bacteriorhodopsin (bR) were examined by solid-state 13C NMR of samples labeled with [3-13C]Ala and [1-13C]Val. We found that the 13C NMR spectra of two kinds of blue membranes, deionized (pH 4) and acid blue at pH 1.2, were very similar and different from that of the native purple membrane. This suggested that when the surface pH is lowered, either by removal of cations or by lowering the bulk pH, substantial change is induced in the secondary structure of the protein. Partial replacement of the bound cations with Na+, Ca2+, or Mn2+ produced additional spectral changes in the 13C NMR spectra. The following conclusions were made. First, there are high-affinity cation-binding sites in both the extracellular and the cytoplasmic regions, presumably near the surface, and one of the preferred cation-binding sites is located at the loop between the helix F and G (F-G loop) near Ala196, consistent with the 3D structure of bR from x-ray diffraction and cryoelectron microscopy. Second, the bound cations undergo rather rapid exchange (with a lifetime shorter than 3 ms) among various types of cation-binding sites. As expected from the location of one of the binding sites, cation binding induced conformational alteration of the F-G interhelical loop.

Bacteriorhodopsins↗
PubMed1998

Membrane fusion plays an important role in gene transfection mediated by cationic liposomes.

By confocal laser scanning microscopy (CLSM) we have studied the membrane fusion between cationic liposomes and the endosome membranes involved in gene transfection mediated by cationic liposomes. Antisense oligonucleotides were transferred by cationic liposomes with a cationic cholesterol derivative, cholesteryl-3beta-carboxyamidoethylenedimethylamine (I). Cationic liposomes were made by a mixture of the derivative I and DOPE. The intracellular distribution of fluorescein-conjugated antisense oligonucleotides (phosphorothioate) was studied by CLSM. The images showed that the antisense oligonucleotides were preferentially transferred into the nucleus of target cells (NIH3T3, COS-7 and HeLa cells) by the liposomes with derivative I. However, their transfection was completely blocked by nigericin which was able to dissipate the pH gradient across the endosome membranes, although the liposome/DNA complex was found in the cytoplasm of the target cells. This was quite in contrast with the fluorescence images of the target cells treated with wortmannin, an inhibitor of endocytosis. The results suggest that at least two steps are effective for gene transfection mediated by the cationic liposomes with cationic cholesterol derivatives. One is the endocytosis of the liposome/DNA complex into the target cells and the other is the removal of antisense oligonucleotides (plasmid DNAs) from the complex in the endosomes. The latter step was preferentially preceded by the membrane fusion between the cationic liposomes and the endosome membranes at around pH 5.0.

3T3 Cells↗
PubMed2000

Adsorption of polyvalent cations to bilayer membranes from negatively charged lipid: estimating the lipid accessibility in the case of complete binding.

The effect of trivalent (Gd(3+) and Yb(3+)) and divalent (Be(2+) and Ca(2+)) cations on suspensions of multilamellar liposomes formed from brain PS and DMPS has been studied using microelectrophoresis and DSC techniques, respectively. The zeta potential values have been shown to strongly depend on the total lipid concentration in the suspension. At moderate concentrations of the polyvalent cations, the total cation concentration exceeds the bulk one several times due to adsorption of cations to the liposomes. A modification of the Gouy-Chapman-Stern theory in the case of unknown bulk concentration of the polyvalent cation is presented. An intrinsic association constant for Be(2+) ions was evaluated to be about K(2) approximately 50 M(-1). The algorithm for estimating the concentrations of the accessible (to exogenously added polyvalent cations) lipid-binding sites is described. These values are consistent with the subsurface concentrations of the polyvalent cations, which monotonously increase with the total concentration of the polyvalent cations. The calculated lipid accessibilities are shown to be in accordance with the DSC data.

Adsorption↗
PubMed2003

Influence of cation-pi interactions in different folding types of membrane proteins.

Cation-pi interactions play an important role to the stability of protein structures. In this work, we analyze the influence of cation-pi interactions in three-dimensional structures of membrane proteins. We found that transmembrane strand (TMS) proteins have more number of cation-pi interactions than transmembrane helical (TMH) proteins. In TMH proteins, both the positively charged residues Lys and Arg equally experience favorable cation-pi interactions whereas in TMS proteins, Arg is more likely than Lys to be in such interactions. There is no relationship between number of cation-pi interactions and number of residues in TMH proteins whereas a good correlation was observed in TMS proteins. The average cation-pi interaction energy for TMH proteins is -16 kcal/mol and that for TMS proteins is -27 kcal/mol. The pair-wise cation-pi interaction energy between aromatic and positively charged residues showed that Lys-Trp energy is stronger in TMS proteins than TMH proteins; Arg-Phe, Arg-Tyr and Lys-Phe have higher energy in TMH proteins than TMS proteins. The decomposition of energies into electrostatic and van der Waals revealed that the contribution from electrostatic energy is twice as that from van der Waals energy in both TMH and TMS proteins. The results obtained in the present study would be helpful to understand the contribution of cation-pi interactions to the stability of membrane proteins.

Amino Acids, Aromatic↗
PubMed1999

Properties of a hyperpolarization-activated cation current in interneurons in the rat lateral geniculate nucleus.

A hyperpolarization-activated cation conductance contributes to the membrane properties of a variety of cell types. In the thalamus, a prominent hyperpolarization-activated cation conductance exists in thalamocortical cells, and this current is implicated in the neuromodulation of complex firing behaviors. In contrast, the GABAergic cells in the reticular nucleus in the thalamus appear to lack this conductance. The presence and role of this cation conductance in the other type of thalamic GABAergic cells, local interneurons, is still unclear. To resolve this issue, we studied 54 physiologically and morphologically identified local interneurons in the rat dorsal lateral geniculate nucleus using an in vitro whole-cell patch recording technique. We found that hyperpolarizing current injections induced depolarizing voltage sags in these geniculate interneurons. The I-V relationship revealed an inward rectification. Voltage-clamp study indicated that a slow, hyperpolarization-activated cation conductance was responsible for the inward rectification. We then confirmed that this slow conductance had properties of the hyperpolarization-activated cation conductance described in other cell types. The slow conductance was insensitive to 10 mM tetraethylammonium and 0.5 mM 4-aminopyridine, but was largely blocked by 1-1.5 mM Cs+. It was permeable to both K+ and Na+ ions and had a reversal potential of -44 mV. The voltage dependence of the hyperpolarization-activated cation conductance in interneurons was also studied: the activation threshold was about -55 mV, half-activation potential was about -80 mV and maximal conductance was about 1 nS. The activation and deactivation time constants of the conductance ranged from 100 to 1000 ms, depending on membrane potential. The depolarizing voltage sags and I-V relationship were further simulated in a model interneuron, using the parameters of the hyperpolarization-activated cation conductance obtained from the voltage-clamp study. The time-course and voltage dependence of the depolarizing voltage sags and I-V relationship in the model cell were very similar to those found in geniculate interneurons in current clamp. Taken together, the results of the present study suggest that thalamic local interneurons possess a prominent hyperpolarization-activated cation conductance, which may play important roles in determining basic membrane properties and in modulating firing patterns.

Animals↗
PubMed2001

Vibrational spectroscopy of gas-phase neutral and cationic phenanthrene in their electronic groundstates.

Various experimental methods are applied to retrieve the vibrational structure of phenanthrene in its neutral and cationic groundstates. The linear infrared (IR) absorption spectra in the 400-1650 cm(-1) range of jet-cooled phenanthrene and its cation, both clustered with either an argon or a neon atom, are obtained via photo-induced cluster dissociation spectroscopy. The spectra observed are in good agreement with calculated spectra of the bare species. However, the observed spectrum of cationic phenanthrene shows more lines and lines with different intensities in the 900-1400 cm(-1) range than expected from calculations. Additional spectra of the perdeuterated phenanthrene Ar cation, and the warm (T approximately > room temperature) bare phenanthrene cation are recorded. Also the mass-analyzed threshold ionization spectra of bare phenanthrene and phenanthrene-Ar are recorded and compared with each other. Comparison of the spectral data recorded to calculated spectra of bare neutral, cationic and cationic perdeuterated phenanthrene, as well as to IR spectra recorded in matrix-isolation experiments, explicitly demonstrates that cluster dissociation spectroscopy is a valid and powerful method to obtain IR spectroscopic information of bare neutral and cationic jet-cooled poly-aromatic hydrocarbons.

Cations↗
PubMed1995

Multiple cationic residues of anthopleurin B that determine high affinity and channel isoform discrimination.

Site 3 sea anemone toxins modify inactivation of mammalian voltage-gated Na channels. One variant, anthopleurin A (ApA), effectively selects for cardiac over neuronal mammalian isoforms while another, anthopleurin B (ApB), which differs in 7 of 49 amino acids, modifies both cardiac and neuronal channels with high and approximately equal affinity. Previous investigations have suggested an important role for cationic residues in determination of toxin activity, and our single-site mutagenesis studies have indicated that isoform discrimination can be partially explained by the unique cationic residues Arg-12 and Lys-49 of anthopleurin B (ApB). Here, we have further investigated the role of cationic residues by characterizing toxin mutants in which two such residues are replaced simultaneously. The ApB double mutants R14Q-K48A (cationic residues identical in both ApA and ApB), R12S-K49Q (cationic residues unique to ApB), and R12S-R14Q (cationic residues located in the unstructured loop shared among anemone toxins) were constructed by site-directed mutagenesis and their biological activities characterized by sodium uptake assays in cell lines expressing the neuronal (N1E-115) or cardiac (RT4-B) isoform of the Na channel. Each double mutant displayed reduced activity compared with wild type, but none were completely inactive. Neutralization of the proximal cationic residues (R12 and R14) was the most effective, reducing affinity 72-fold (neuronal) and 56-fold (cardiac). Substitution of cationic residues that differed between ApB and ApA (R12S-K49Q) reduced affinity of the toxin for neuronal channels to a much greater extent than for cardiac channels, producing affinities only slightly lower than for ApA in each case.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2001

Monovalent cation-induced conformational change in glucose oxidase leading to stabilization of the enzyme.

Glucose oxidase (GOD) from Aspergillus niger is an acidic dimeric enzyme having a high degree of localization of negative charges on the enzyme surface and dimer interface. We have studied the effect of monovalent cations on the structure and stability of GOD using various optical spectroscopic techniques, limited proteolysis, size exclusion chromatography, differential scanning calorimetry, and enzymic activity measurements. The monovalent cations were found to influence the enzymic activity and tertiary structure of GOD, but no effect on the secondary structure of the enzyme was observed. The monovalent cation-stabilized GOD was found to have a more compact dimeric structure but lower enzymic activity than the native enzyme. The enzyme's K(m) for D-glucose was found to be slightly enhanced for the monovalent cation-stabilized enzyme (maximum enhancement of about 35% for LiCl) as compared to native GOD. Comparative denaturation studies on the native and monovalent cation-stabilized enzyme demonstrated a significant resistance of cation-stabilized GOD to urea (about 50% residual activity at 6.5 M urea) and thermal denaturation (Delta T(m) maximum of 10 degrees C compared to native enzyme). However, pH-induced denaturation showed a destabilization of monovalent cation-stabilized GOD as compared to the native enzyme. The effectiveness of monovalent cations in stabilizing GOD structure against urea and thermal denaturation was found to follow the Hofmeister series: K(+) > Na(+) > Li(+).

Aspergillus niger↗
PubMed2001

Site-specific cation binding mediates TATA binding protein-DNA interaction from a hyperthermophilic archaeon.

Pyrococcus woesei (Pw) is a hyperthermophilic archaeal organism that exists under conditions of high salt and elevated temperature. In a previous study [O'Brien, R., DeDecker, B., Fleming, K., Sigler, P. B., and Ladbury, J. E., (1998) J. Mol. Biol. 279, 117-125], we showed that, despite the similarity of primary and secondary structure, the TATA box binding protein (TBP) from Pw binds thermodynamically in a fundamentally different way to its mesophilic counterparts. The affinity of the interaction increases as the salt concentration is increased. The formation of the protein-DNA complex involves the release of water and the uptake of ions, which were hypothesized to be cations. Here we test this hypothesis by selecting potential cation binding sites at negatively charged, acidic residues in the complex interface. These were substituted using site-directed mutagenesis of specific residues. Changes in the thermodynamic parameters on formation of the mutant protein-DNA complex were determined using isothermal titration calorimetry and compared to the wild type interaction. Removal of a glutamate residue from the binding site resulted in the uptake of one less cation on formation of the complex. This glutamate (E12) is directly involved in the binding of cations in the complex interface. Substitution of another acidic residue proximal to the DNA binding site (D101) had no effect on cation uptake, suggesting that the location of the amino acid on the protein surface is important in dictating the potential to coordinate cations. Removal of the cation binding site provided a more favorable entropy of binding; however, this effect is significantly reduced at higher salt concentrations. The removal of the cation binding site led to an increase in affinity with respect to the wild-type TBP at low salt concentrations.

Alanine↗
PubMed2005

Interactions between cytochrome c2 and the photosynthetic reaction center from Rhodobacter sphaeroides: the cation-pi interaction.

The cation-pi interaction between positively charged and aromatic groups is a common feature of many proteins and protein complexes. The structure of the complex between cytochrome c(2) (cyt c(2)) and the photosynthetic reaction center (RC) from Rhodobacter sphaeroides exhibits a cation-pi complex formed between Arg-C32 on cyt c(2) and Tyr-M295 on the RC [Axelrod, H. L., et al. (2002) J. Mol. Biol. 319, 501-515]. The importance of the cation-pi interaction for binding and electron transfer was studied by mutating Tyr-M295 and Arg-C32. The first- and second-order rates for electron transfer were not affected by mutating Tyr-M295 to Ala, indicating that the cation-pi complex does not greatly affect the association process or structure of the state active in electron transfer. The dissociation constant K(D) showed a greater increase when Try-M295 was replaced with nonaromatic Ala (3-fold) as opposed to aromatic Phe (1.2-fold), which is characteristic of a cation-pi interaction. Replacement of Arg-C32 with Ala increased K(D) (80-fold) largely due to removal of electrostatic interactions with negatively charged residues on the RC. Replacement with Lys increased K(D) (6-fold), indicating that Lys does not form a cation-pi complex. This specificity for Arg may be due to a solvation effect. Double mutant analysis indicates an interaction energy between Tyr-M295 and Arg-C32 of approximately -24 meV (-0.6 kcal/mol). This energy is surprisingly small considering the widespread occurrence of cation-pi complexes and may be due to the tradeoff between the favorable cation-pi binding energy and the unfavorable desolvation energy needed to bury Arg-C32 in the short-range contact region between the two proteins.

Amino Acids, Aromatic↗
PubMed2006

Contribution of cation-pi interactions to protein stability.

Calculations predict that cation- interactions make an important contribution to protein stability. While there have been some attempts to experimentally measure strengths of cation-pi interactions using peptide model systems, much less experimental data are available for globular proteins. We have attempted to determine the magnitude of cation-pi interactions of Lys with aromatic amino acids in four different proteins (LIVBP, MBP, RBP, and Trx). In each case, Lys was replaced with Gln and Met. In a separate series of experiments, the aromatic amino acid in each cation-pi pair was replaced by Leu. Stabilities of wild-type (WT) and mutant proteins were characterized by both thermal and chemical denaturation. Gln and aromatic --> Leu mutants were consistently less stable than corresponding Met mutants, reflecting the nonisosteric nature of these substitutions. The strength of the cation-pi interaction was assessed by the value of the change in the free energy of unfolding [DeltaDeltaG(degrees) = DeltaG(degrees)(Met) - DeltaG(degrees)(WT)]. This ranged from +1.1 to -1.9 kcal/mol (average value -0.4 kcal/mol) at 298 K and +0.7 to -2.6 kcal/mol (average value -1.1 kcal/mol) at the Tm of each WT. It therefore appears that the strength of cation-pi interactions increases with temperature. In addition, the experimentally measured values are appreciably smaller in magnitude than calculated values with an average difference /DeltaG(degrees)expt - DeltaG(degrees)calc/av of 2.9 kcal/mol. At room temperature, the data indicate that cation-pi interactions are at best weakly stabilizing and in some cases are clearly destabilizing. However, at elevated temperatures, close to typical Tm's, cation-pi interactions are generally stabilizing.

Amino Acid Substitution↗
PubMed1997

Identification and characterization of functional cation coordination sites in platelet endothelial cell adhesion molecule-1.

We have employed 45CaCl2 binding studies, terbium (Tb3+) luminescence spectroscopy, and electrospray mass spectroscopy (ESI-MS) to identify divalent metal binding properties of soluble recombinant human PECAM-1 (srPECAM-1), and to define unique cation binding domains using short, linear peptide sequences from the protein. PECAM-1 was found to directly interact with 45CaCl2, binding 2.3 nmol of Ca2+/nmol of srPECAM-1 with a Kd of 1.17 nM. PECAM-1 was found to contain high-affinity cation binding sites involving amino acids Asp443, Asp444, and Glu446 of Ig-domain 5 and residues Glu487, Glu490, Asp491, Glu538, Glu540, and Glu542 of Ig-domain 6. The PECAM cation binding sites demonstrated broad specificity for all divalent cations, with Mn2+ having a higher affinity than Ca2+ or Mg2+. Direct binding of Tb3+ to these PECAM peptides was confirmed by ESI-MS. Modeling studies predict that the six cation binding residues within Ig-domain 6 are proximal to each other in three-dimensional space, and may form a single cation coordination site. The identification of cation binding sites in PECAM-1 will direct further work in examining its cation-dependent roles in cellular signaling.

Amino Acid Sequence↗
PubMed1997

Catalytic role of monovalent cations in the mechanism of proton transfer which gates an interprotein electron transfer reaction.

Within the methylamine dehydrogenase (MADH)-amicyanin protein complex, long range intermolecular electron transfer (ET) occurs between tryptophan tryptophylquinone (TTQ) of MADH and the type I copper of amicyanin. The reoxidations of two chemically distinct reduced forms of TTQ were studied, a quinol (O-quinol) generated by reduction by dithionite and the physiologically relevant aminoquinol (N-quinol) generated by reduction by methylamine. The latter contains a substrate-derived amino group which displaces the C6 carbonyl oxygen on TTQ. ET from N-quinol MADH to amicyanin is gated by the transfer of a solvent exchangeable proton [Bishop, G. R., & Davidson, V. L. (1995) Biochemistry 34, 12082-12086]. The factors which influence this proton transfer (PT) reaction have been examined. The rate of PT increases with increasing pH and with increasing salt concentration. The salt effect is due to specific monovalent cations and is not a general ionic strength effect. The rate enhancements by pH and cations do not reflect an elimination of the PT step that gates ET. Over the range of pH from 5.5 to 9.0 and with cation concentrations from 0 to 200 mM, the observed rate of the redox reaction is still that of PT. This is proven by kinetic solvent isotope effect studies which show that a primary isotope effect persists even at the highest values of pH and cation concentration. A model is presented to explain how specific cations contribute to catalysis and influence the rate of PT in this reaction. The pH dependence is attributed to an ionizable group that is involved in cation binding. The effect of the cation is stabilization of a negatively charged reaction intermediate that is formed during the deprotonation of the N-quinol, and from which rapid ET to the copper of amicyanin occurs. The relevance of these findings to other enzymes which exhibit reaction rates that are influenced by monovalent cations is also discussed.

Bacterial Proteins↗