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PubMed2004

Microfibril-associated glycoprotein-1 binding to tropoelastin: multiple binding sites and the role of divalent cations.

Microfibrils and elastin are major constituents of elastic fibers, the assembly of which is dictated by multimolecular interactions. Microfibril-associated glycoprotein-1 (MAGP-1) is a microfibrillar component that interacts with the soluble elastin precursor, tropoelastin. We describe here the adaptation of a solid-phase binding assay that defines the effect of divalent cations on the interactions between MAGP-1 and tropoelastin. Using this assay, a strong calcium-dependent interaction was demonstrated, with a dissociation constant of 2.8 +/- 0.3 nm, which fits a single-site binding model. Manganese and magnesium bestowed a weaker association, and copper did not facilitate the protein interactions. Three constructs spanning tropoelastin were used to quantify their relative contributions to calcium-dependent MAGP-1 binding. Binding to a construct spanning a region from the N-terminus to domain 18 followed a single-site binding model with a dissociation constant of 12.0 +/- 2.2 nm, which contrasted with the complex binding behavior observed for fragments spanning domains 17-27 and domain 27 to the C-terminus. To further elucidate binding sites around the kallikrein cleavage site of domains 25/26, MAGP-1 was presented with constructs containing C-terminal deletions within the region. Construct M1659, which spans a region from the N-terminus of tropoelastin to domain 26, inclusive, bound MAGP-1 with a dissociation constant of 9.7 +/- 2.0 nm, which decreased to 4.9 +/- 1.0 nm following the removal of domain 26 (M155n), thus displaying only half the total capacity to bind MAGP-1. These results demonstrate that MAGP-1 is capable of cumulative binding to distinct regions on tropoelastin, with different apparent dissociation constants and different amounts of bound protein.

Cations, Divalent↗
PubMed1984

Lymphocyte bacterial rosette test: methodological details and effect of metabolic inhibitors and divalent cations.

Lymphocytes bind certain bacteria. This property has been utilized in the lymphocyte bacterial rosette assay to identify T and B cell subsets. Here we determined the optimum conditions for the assay, studied the effect of metabolic inhibitors and divalent cations and compared bacterial rosette-forming lymphocyte subpopulations with those defined with monoclonal antibodies. The strains used were Brucella melitensis, Bacillus subtilis, Staphylococcus aureus and Escherichia coli. Optimum attachment was obtained at 4 degrees C in 6% BSA with ultrasonicated bacteria. Pretreatment of lymphocytes with the microfilament-disruptive drug cytochalasin B suppressed the binding of bacteria, whereas colchicine (inhibitor of microtubules), puromycin (inhibitor of translation), sodium azide (inhibitor of oxidative phosphorylation) and 2-deoxyglucose (inhibitor of glycolysis) had no effect. Divalent cations were required for the attachment of bacteria. B. melitensis bound to DR-positive cells, whereas the other bacterial strains rosetted OKT4- OKT8- and DR-positive cells without exhibiting helper or suppressor T cell or B lymphocyte specificity.

Antibodies, Monoclonal↗
PubMed1988

Divalent cations induce protofibril gelation.

Soluble fibrin oligomers (protofibrils) undergo phase change merely by adding 1-2 mM Ca2+ or 25-100 microM Zn2+. The cation-induced "protofibrin" clots appear similar to normally formed fibrin gels. Maximal clot turbidity of protofibrin gels increases with cations in a concentration-dependent manner. Magnesium (less than 0.5 mM) is ineffective in inducing protofibril gelation. Turbidity and degree of polymerization (DP) [indirectly expressed as AT (activation time)] appear to be positively correlated, regardless of whether the divalent cation is Ca2+ or Zn2+. Cross sections of Ca2+-induced protofibrin fibers are approximately 6-18-fibrin-monomers-thick. With both Ca2+ and 40 microM Zn2+, fiber cross section increases to 30-50 monomers thick. Negatively stained Zn2+-and Ca2+-induced protofibrin gels exhibit banding periodicity of approximately 240 A, similar to that of normally generated fibrin gels. Regions of lateral merging of individual segments of the protofibrin leads to increased cross section of the fiber and forms a branch required for gelation. These findings indicate that the rate of coagulation and the ultimate thickness of the fibers both relate to lateral associative processes of protofibrils, which are augmented by physiologic concentrations of 2+ and Zn2+.

Biopolymers↗
PubMed1981

Further observations on calcium and other divalent cations metabolism in intact Ehrlich ascites tumour cells.

The metabolism of calcium has been investigated in the Ehrlich Ascites Tumour Cells (ATC). ATC extrude Ca2+ actively by an energy-dependent mechanism, supported by both respiration and glycolysis. Extrusion takes place even against a very steep concentration gradient (10 mM Ca2+). Cell calcium content is decreased by monovalent cations (Na+,K+ and Li+), which act independently from their metabolic effects. La3+ inhibits ATC Ca2+ extrusion whereas Ruthenium Red slightly decreases cell calcium content. The antibiotic ionophore A 23187 strongly increases ATC Ca2+ level. the metabolism of other divalent cations (Mg2+, Sr2+ and Mn2+) has been studied. Mg2+ does not show appreciable changes in the various metabolic conditions tested, while Mn2+ and Sr2+ behave quite differently from Ca2+, suggesting a different distribution of these cations in ATC. The experimental findings indicate that Ehrlich Ascites Tumour Cells regulate their calcium content by mechanisms related to plasma membranes while the size and activity of mitochondrial compartment is of minor importance.

Animals↗
PubMed1988

Regulation of norepinephrine uptake by adenine nucleotides and divalent cations: role for extracellular protein phosphorylation.

This study examined the hypothesis that ATP, released together with norepinephrine (NE) from brain noradrenergic nerve terminals, may serve as a cosubstrate for an extracellular protein phosphorylation system that regulates the reuptake of the transmitter, NE. The possible regulation of high-affinity uptake (uptake 1) of [3H]NE by divalent cations and ATP, both of which are involved in protein phosphorylation, was examined in rat cerebral cortical synaptosomes. A marked inhibition of uptake 1 by 5'-adenylylimidodiphosphate [App(NH)p], a nonhydrolyzable, competitive antagonist of ATP, was observed. A similar inhibition of uptake was observed when Ca2+ and Mg2+ were both omitted from the incubation medium. App(NH)p distinguished the actions of Ca2+ from those of Mg2+: Ca2+-stimulated uptake 1 was blocked by App(NH)p; Mg2+-stimulated uptake was not. In parallel experiments, the patterns of protein phosphorylation in crude and purified preparations of synaptosomes were examined under conditions similar to those used in uptake assays. A striking correlation was found between the inhibition of uptake 1, by either App(NH)p or Ca-omission, and inhibition of the phosphorylation of one specific, 39,000-dalton, Ca2+-dependent, protein component in synaptosomes. This 39K protein was distinct from the alpha subunit of pyruvate dehydrogenase, a mitochondrial protein of similar electrophoretic mobility. These findings are consistent with the possibility that an ectokinase on synaptosomes utilizes extracellular ATP and Ca2+ in phosphorylating a protein(s) associated with the regulation of NE uptake.

Adenosine Triphosphate↗
PubMed2000

Interaction of porin from Yersinia pseudotuberculosis with lipopolysaccharides. Effect of ionic strength, pH, and divalent cations on the binding parameters.

Interaction of the pore-forming protein (porin) from Yersinia pseudotuberculosis with S- and R-forms of the endogenous lipopolysaccharide (LPS) was studied at various ionic strengths (20-600 mM NaCl), concentrations of divalent cations (5-100 mM CaCl2, MgCl2), and pH values from 3.0 to 9.0. The interaction of the R-LPS with porin has been shown in all experimental conditions to be in consensus with the model suggesting binding at independent sites of two types. S-LPS binds to interacting sites of relatively high affinity and to independent sites of low affinity at all pH values examined and at low NaCl concentration. The cooperative interaction of the S-LPS and porin is not observed at high ionic strength and in divalent cation-free medium. The number of binding sites of porin and association constants (Ka) for both LPS forms decrease significantly on increasing the solution ionic strength. The Ka values for the R- and S-LPS change oppositely on changing the pH: the Ka value for the R-LPS is maximal (Ka = 6.7 x 10(5) M-1), but that for S-LPS is minimal (Ka = 0.4 x 10(5) M(-1) at pH 5.0-5.5. The number of high-affinity and low-affinity binding sites for both LPS forms is maximal at pH 5.0-5.5. In this case, the numbers of high- and low-affinity sites for R-LPS are 3 and 10, respectively, and those for the S-LPS are 7 and 20, respectively. These data suggest an important role of electrostatic interactions on binding of LPS to porin. The contribution of conformational changes of the ligand and protein and hydrophobic interactions are discussed.

Binding Sites↗
PubMed1988

Increased outer membrane ornithine-containing lipid and lysozyme penetrability of Paracoccus denitrificans grown in a complex medium deficient in divalent cations.

Paracoccus denitrificans grown in a complex medium was highly susceptible to lysozyme, in contrast to cells grown in a complex medium supplemented with Mg2+ and Ca2+ or in a succinate-salts medium. The complex medium was deficient in divalent cations needed for optimum outer membrane stability. The major change in molecular compositions of outer membranes isolated from cells grown under the different conditions was a higher ratio of ornithine-containing lipid to phospholipid in complex-medium-grown cells (0.63) than in cells grown in complex medium with Mg2+ and Ca2+ (0.22) or in succinate-salts medium (0.14). We suggest that the dipolarionic ornithine-containing lipid is less dependent than acidic phospholipids on divalent cations for its incorporation into the outer membrane.

Calcium↗
PubMed1979

Effects of some divalent cations on synaptic transmission in frog spinal neurones.

1. Synaptic transmission between dorsal root afferents and motoneurones was studied in the isolated and hemisected spinal cord of frogs, using intracellular and extracellular recording techniques, and ionic substitutions of divalent cations in the bathing fluid. 2. Delayed components of excitatory post-synaptic potentials (e.p.s.p.s) evoked in motoneurones by dorsal root supramaximal stimuli, as well as the Ca2+-dependent slow after-hyperpolarization which follows antidromic spikes, were reversibly blocked by superfusing the cords with 'Ca2+-free' media containing Co2+ (4 mM) or Mg2+ (6-10 mM). However, short latency e.p.s.p.s persisted in these media for more than 8 hr. 3. The minimum synaptic delay of the Co2+ and Mg2+, resistant e.p.s.p.s, measured from the peak negativity of the extracellularly recorded presynaptic spike to the onset of the e.p.s.p., was 0.3 msec at 10 +/- 1 degrees C. 4. The Co2+, Mg2+-resistant e.p.s.p.s were graded, and could be elicited by stimulation of segmental or adjacent roots. Those evoked by each of two adjacent roots showed linear summation when the roots were stimulated simultaneously. 5. The Co2+, Mg2+-resistant e.p.s.p.s decreased in amplitude at stimulating frequencies between 10 and 100 Hz, and with paired stimuli at intervals shorter than 20-40 msec. These reductions in amplitude were paralleled by decreases in amplitude of the presynaptic population spike. 6. Solutions free of divalent ions, containing EGTA (2 mM) abolished the Co2+, Mg2+-resistant e.p.s.p.s. They remained blocked for a variable time after returning to Ca2+-free Ringer containing Mg2+ (8 mM). Their continued abolition at this stage is probably not due to changes in electrical properties of motoneuronal membranes. Eventually, the Mg2+-resistant e.p.s.p.s started recovering in the Ca2+-free Ringer containing Mg2+. The time of onset of this recovery depended on the duration of exposure to EGTA. 7. Sr2+ (2-11 mM), although less effective than Ca2+, restored the composite e.p.s.p.s evoked by dorsal root supramaximal stimuli, as well as the Ca2+-dependent slow after-hyperpolarization of the motoneurone. The composite e.p.s.p.s could not be restored with Ba2+ (2-10 mM). 8. The results suggest that the Co2+, Mg2+-resistant e.p.s.p is generated by electrical coupling between some afferent fibres (probably primary afferents) and motoneurones. The after-effects of EGTA treatments probably reflect uncoupling of electrotonic junctions. In contrast, the delayed components of the composite e.p.s.p.s are generated through chemical synapses whose divalent cation requirement is similar to that of the neuromuscular junction.

Action Potentials↗
PubMed1993

Multiple sites for subtilisin cleavage of tubulin: effects of divalent cations.

Limited digestion of pig brain GDP-tubulin by subtilisin was carried out in the presence of Mg2+, Mn2+, Ca2+, Zn2+, or Be2+. Isoelectric focusing, followed by SDS-PAGE, revealed characteristic divalent cation-dependent changes in the alpha- and beta-tubulin cleavage patterns. Previous studies revealed that the beta-cleavage pattern is different for heterodimers and microtubules [Lobert and Correia, 1992: Arch. Biochem. Biophys. 296: 152-160]. Divalent cation effects on subtilisin digestion of tubulin indicate different classes of divalent cation binding sites. Western blot analysis locates the proteolytic zone at residue 430 or higher in both subunits for all conditions. Turbidity and electron microscopy reveal that GDP-tubulin cleaved by subtilisin in the presence of Mg2+, Ca2+, or Mn2+ forms sheets of rings. Mn2+ induces ring formation in uncleaved GDP-tubulin. Isotype-depleted tubulin was generated by the removal of class III beta-tubulin using immunoaffinity chromatography. Subtilisin digestion of the depleted fraction and the purified class III beta-tubulin demonstrates that cleavage occurs at three to four distinct sites. Thus, subtilisin-digested tubulin is more heterogeneous than was previously reported and the cleavage sites depend on solution conditions, divalent cations, and the state of assembly. This has important implications for experiments that utilize subtilisin-digested tubulin for studying microtubule-associated protein binding.

Animals↗
PubMed1981

Divalent cations and transmitter release at low concentration of tetrodotoxin.

Transmitter release from frog motor terminals was studied in the presence of very low concentrations of tetrodotoxin (TTX, 4.10(-10)--6.10(-9) g/ml). TTX reversibly reduced the amplitude of the end-plate potential (epp), while leaving the amplitude distribution to follow Poisson's law. The effects of a number of divalent cations were studied in the presence of TTX. It was found that after the addition of TTX there was an increase in the constant of dissociation of calcium and strontium from a hypothetical membrane "release site," while the dissociation constants of magnesium and manganese remained unaltered. It is concluded that the release site is probably intracellular and that a reduced presynaptic spike amplitude, as well as magnesium and manganese ions, decrease the access of calcium and strontium to the site.

Animals↗
PubMed1999

Divalent cations differentially regulate integrin alphaIIb cytoplasmic tail binding to beta3 and to calcium- and integrin-binding protein.

We have used recombinant or synthetic alphaIIb and beta3 integrin cytoplasmic peptides to study their in vitro complexation and ligand binding capacity by surface plasmon resonance. alpha.beta heterodimerization occurred in a 1:1 stoichiometry with a weak KD in the micromolar range. Divalent cations were not required for this association but stabilized the alpha.beta complex by decreasing the dissociation rate. alpha.beta complexation was impaired by the R995A substitution or the KVGFFKR deletion in alphaIIb but not by the beta3 S752P mutation. Recombinant calcium- and integrin-binding protein (CIB), an alphaIIb-specific ligand, bound to the alphaIIb cytoplasmic peptide in a Ca2+- or Mn2+-independent, one-to-one reaction with a KD value of 12 microM. In contrast, in vitro liquid phase binding of CIB to intact alphaIIbbeta3 occurred preferentially with Mn2+-activated alphaIIbbeta3 conformers, as demonstrated by enhanced coimmunoprecipitation of CIB with PAC-1-captured Mn2+-activated alphaIIbbeta3, suggesting that Mn2+ activation of intact alphaIIbbeta3 induces the exposure of a CIB-binding site, spontaneously exposed by the free alphaIIb peptide. Since CIB did not stimulate PAC-1 binding to inactive alphaIIbbeta3 nor prevented activated alphaIIbbeta3 occupancy by PAC-1, we conclude that CIB does not regulate alphaIIbbeta3 inside-out signaling, but rather is involved in an alphaIIbbeta3 post-receptor occupancy event.

Antigens, CD↗
PubMed2000

Impermeability of the GIRK2 weaver channel to divalent cations.

A single amino acid mutation (G156S) in the putative pore-forming region of the G protein-sensitive, inwardly rectifying K(+) channel subunit, GIRK2, renders the conductance constitutively active and nonselective for monovalent cations. The mutant channel subunit (GIRK2wv) causes the pleiotropic weaver disease in mice, which is characterized by the selective vulnerability of cerebellar granule cells and Purkinje cells, as well as dopaminergic neurons in the mesencephalon, to cell death. It has been proposed that divalent cation permeability through constitutively active GIRK2wv channels contributes to a rise in internal calcium in the GIRK2wv-expressing neurons, eventually leading to cell death. We carried out comparative studies of recombinant GIRK2wv channels expressed in Xenopus oocytes and COS-7 cells to determine the magnitude and relative permeability of the GIRK2wv conductance to Ca(2+). Data from these studies demonstrate that the properties of the expressed current differ in the two systems and that when recombinant GIRK2wv is expressed in mammalian cells it is impermeable to Ca(2+).

Animals↗
PubMed1978

Loss of resealing ability in erythrocyte membranes. Effect of divalent cations and spectrin release.

Washed human erythrocyte membranes can recover impermeability to macromolecules upon warming in solutions of sufficient ionic strength. This ability is rapidly lost from most ghost preparations in dilute salt solution at temperatures of 15 degrees C of higher. Divalent cations both reseal ghosts in the absence of high ionic strength and prevent loss of resealing ability. The effective concentrations are 40 micron for Ca2+ and 200 micron for Mg2+. The loss of resealing ability is associated with the release of spectrin polypeptides from the inner surface of the membrane. In ghost preparations that have not become irreversibly leaky, or in the presence of Ca2+, loss of spectrin does not occur. These results suggest that an intact spectrin netwoek is required for resealing to macromolecules, and divalent cations stabilize this network. In light of this information, the effect of temperature on resealing kinetics is described.

Calcium↗
PubMed1983

Norepinephrine-induced loss of phosphatidylinositol from isolated rat liver plasma membrane. Effects of divalent cations.

Norepinephrine at 5 microM induces a rapid (60 s) and specific loss of phosphatidylinositol (PtdIns) when added to isolated rat liver plasma membranes. The hormone action is inhibited by the alpha-adrenergic antagonist phentolamine (20 microM). Depletion of Mg2+ and Ca2+ singly or in combination from the incubation buffer mimicks the hormone effect on PtdIns breakdown. No further effect on PtdIns degradation could be measured when norepinephrine was added to the cation-depleted buffers. Addition of the Ca2+ ionophore A23187 to the isolated membranes has no effect. It is concluded that PtdIns degradation can be provoked in isolated rat liver plasma membrane through alpha-adrenergic receptor activation and that this effect is dependent on divalent cations in the sense that loss of cations from the membrane allows degradation to commence.

Animals↗
PubMed1986

Regulation of membrane histamine H1-receptor binding sites by guanine nucleotides, mono- and divalent cations.

Agonist interaction with histamine H1-receptor in [3H] mepyramine bovine aortic membranes labeled with [3H] mepyramine is selectively regulated by cations and guanine nucleotides. GTP and his nonhydrolisable analog Gpp(NH)p' markedly decrease histamine affinity for [3H] mepyramine binding sites. The effect of GTP is reversed in the presence of divalent cation, magnesium. Calcium and sodium ions have little effect on histamine binding whereas magnesium ions decrease the affinity of histamine for the radioantagonist binding sites about tenfold. GTP has little effect on [3H] mepyramine binding and the interaction of H1-antagonist triprolidine with histamine H1-receptors. The above results indicate that the effect of guanine nucleotides, mono and divalent cations involves the effect on membrane signal transducing mechanism probably GTP-binding protein(s) cation regulatory site(s) rather than receptor binding site directly.

Animals↗
PubMed1991

Substrate analogues and divalent cations as inhibitors of glutamate decarboxylase from Escherichia coli.

To examine the idea that glutamate decarboxylase from E. coli can be a convenient source for the study of the effects of compounds on GABA synthesis in the nervous system, a series of substrate analogues and divalent cations were tested as potential inhibitors of the bacterial enzyme. Those analogues exhibiting inhibitor activity did so in a competitive manner. The most effective inhibitors were 3-mercaptopropionic acid, 4-bromoisophthalic acid and isophthalic acid which exhibited Ki values of 0.13 mM, 0.22 mM and 0.31 mM, respectively. Eight other analogues produced lesser degrees of inhibition. In addition, seven divalent metal cations were tested as inhibitors of the enzyme. However, only Hg2+, Cd2+, Cu2+ and Zn2+ were effective at a concentration of 0.1mM. When these results were compared to the patterns of inhibition of glutamate decarboxylase from mouse brain, certain differences in the manner in which the enzymes responded to the inhibitors, emerged. Consequently, the bacterial decarboxylase may not be a good model for the study of drug action on brain GABA synthesis.

3-Mercaptopropionic Acid↗
PubMed2003

Structural reorganization of proteins revealed by radiolysis and mass spectrometry: G-actin solution structure is divalent cation dependent.

The solution structures of isolated monomeric actins in their Mg(2+)-ATP and Ca(2+)-ATP bound forms and in complexes with gelsolin segment-1 have been probed using hydroxyl radicals (*OH) generated by synchrotron X-ray radiolysis. Proteolysis and mass spectrometry analysis of 28 peptides containing 58 distinct reactive probe sites within actin were used to monitor conformational variations linked to divalent cation and gelsolin segment-1 binding. The solvent accessibilities of the probe sites, as measured by footprinting in solution for the Ca(2+)-G-actin and Mg(2+)-G-actin complexes with gelsolin segment-1, were consistent with available crystallographic data. This included a specific protection at the contact interface between the partners, as revealed by reduced reactivity of peptide 337-359 in the complex. Aside from the specific protection indicated previously, the oxidation rates for the reactive residues of the isolated Ca(2+)-G-actin were similar to those of the actin gelsolin segment-1 complexes; however, the reactivity of numerous residues in the isolated Mg(2+)-G-actin form was significantly reduced. Specifically, Mg(2+)-G-actin has a set of protected sites relative to Ca(2+)-G-actin that suggest a structural reorganization in subdomains 4 and 2 and a C-terminus more closely packed onto subdomain 1. These conformational variations for isolated Mg(2+)-G-actin provide a structural basis for its greater tendency to polymerize into filaments as compared to Ca(2+)-G-actin.

Actins↗
PubMed1978

Divalent cation stimulation of in vitro fibre assembly from epidermal keratin protein.

Keratin was extracted from purified cornified cells of newborn rats in Tris-HCl-buffered 8 M urea containing beta-mercaptoethanol. Microfilaments were assembled in vitro by reducing the ionic strength of buffer and the urea concentration. One millimolar concentration of KCl and NaCl did not affect filament formation, but the same concentration of divalent cations greatly altered this process. CaCl2 and MgCl2 induced gelation of keratin by formation of bundles of birefringent macrofilaments. ZnCl2, CuSO4 and HgCl2 formed greater numbers of macrofilaments and the protein aggregated.

Amino Acids↗