Regulation of hepatic adenylate cyclase by glucagon, GTP, divalent cations, and adenosine.
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Adenosine↗
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Results for “Cations, Divalent”
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Activity of PP2C beta is increased by divalent cations and lipophilic compounds depending on the substrate.
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Cations, Divalent↗
Letter: Divalent cations in predicting antidepressant response to lithium.
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Calcium↗
Divalent cation transport and susceptibility to infectious and autoimmune disease: continuation of the Ity/Lsh/Bcg/Nramp1/Slc11a1 gene story.
Solute carrier family 11 member a1 (Slc11a1), formerly known as Nramp1/Ity/Lsh/Bcg, is a proton/divalent cation antiporter that regulates susceptibility to infectious and autoimmune disease. Here we review recent studies on (1) the role of Slc11a1 in iron metabolism and iron recycling in macrophages; (2) the use of mouse breeding and introgression of knockouts onto Slc11a1 congenic backgrounds for genes encoding the multiple pleiotropic functions associated with Slc11a1; and (3) associations/linkages of SLC11A1 with human disease and how these relate to functional promoter region polymorphisms.
Animals↗
The twenty aminoacyl-tRNA synthetases from Escherichia coli. General separation procedure, and comparison of the influence of pH and divalent cations on their catalytic activities.
A general separation procedure of the twenty E. coli aminoacyl-tRNA synthetases including either a 105 000 g centrifugation or a poly-ethyleneglycol-dextran two-phases partition fractionation, and chromatographies on DEAE-cellulose, phosphocellulose and hydroxyapatite is described. The specific activities of the synthetases have been determined after each chromatographic step and compared to their respective activities in the 105 000 g supernatant. Some aminoacyl-tRNA synthetases were obtained at 80 per cent purity. The presence of phenylmethylsulfonyl fluoride does not significantly modify either the elution patterns of the synthetases during the various chromatographic steps or their specific activities. Thus, contrarily to enzymes from various eukaryotic organisms no significant inactivation of the E. coli aminoacyl-tRNA synthetases occurs via proteolytic processes during the purification procedure. The effects of various factors: pH, magnesium, and other bivalent cations including spermidine, were tested on the aminoacylation and the [32P] PPi-ATP isotope-exchange reactions, and the optimal aminoacylation and isotope-exchange conditions determined for 18 of the 20 E. coli aminoacyl-tRNA synthetases.
Amino Acyl-tRNA Synthetases↗
Inhibition of willardiine-induced currents through rat GluR6/KA-2 kainate receptor channels by Zinc and other divalent cations.
Heteromeric GluR6/KA-2 kainate receptor were expressed in HEK293 cells and an inhibition of willardiine-induced currents by cations was studied. Zinc was much more effective than Ca(2+), Ba(2+) and Mg(2+) at 235, 265 and 1382 fold increase in IC(50), respectively. The inhibition was not voltage-dependent. The present data showed that the binding site for the cations are different from that for willardiine and that the currents are inhibited by the cations via at least two distinct binding sites to Zn(2+) and Ca(2+). These data suggest that Zn(2+) play an important role in modulating glutamate receptors at the nervous system because of a presence of Zn(2+) and various effects of Zn(2+) on the receptors.
Alanine↗
Effect of divalent cations on KCl- and noradrenaline-induced contractile responses in rat aorta after nifedipine treatment.
Nifedipine (1 microM) relaxed the sustained contractile responses induced by 1 microM noradrenaline or 60 mM KCl in rat aortic strips. After washing, a second addition of the spasmogens gave smaller tonic contractions than the first one. Even more, a third addition of KCl also gave a smaller contraction than the first one, but a complete recovery of the contractile response to noradrenaline was obtained by a third addition of this agonist. Application of cumulative amounts of Ca2+ or Ba2+ (2.4-24 mM) on the residual contraction in response to these agents after nifedipine treatment, but in the absence of the blocker, restored the magnitude of the contractile responses. Addition of cumulative amounts of Mg2+ (2.4-24 mM) did not modify or even relax the contractile responses to KCl and noradrenaline, respectively.
Animals↗
Divalent cation changes in cerebellar Purkinje cells after climbing fiber deafferentation.
A secondary ion mass spectrometry (SIMS) microscope was used to detect intracellular stores of calcium, magnesium, sodium and potassium. Measurements were made in semithin sections of fixed tissues of normal and climbing fiber deafferented cerebellar cortex. Quantitative data were collected from 150 microns diameter image fields in the molecular and granule layers. The results indicate smaller quantities of both calcium and magnesium in the deafferented cerebellar cortex compared to the normals, the molecular as well as the granule layer being affected. The results are discussed in terms of the usefulness and limitations of the SIMS microscope for histological preparations.
Afferent Pathways↗
RNA aptamers to S-adenosylhomocysteine: kinetic properties, divalent cation dependency, and comparison with anti-S-adenosylhomocysteine antibody.
To explore the potential of RNA aptamers as small-molecule discriminating devices, we have characterized the properties of aptamers selected from a library of approximately 10(14) variants through their interaction with S-adenosylhomocysteine (SAH, AdoHcy). Competition studies with SAH and azaSAM analogues revealed that the Hoogsteen face of adenine is the main contributor to binding, whereas specificity for SAH is conferred by a secondary contact point at or near the sulfur/thioether of homocysteine (Hcy). Binding specificities were determined by both affinity chromatography and a novel method designed for the biosensor. The kinetic properties of individual aptamers, including the "classic" ATP aptamer that also emerged in our selection, were studied by biosensor analysis. Association rates were slow, but the complexes were stable, suggesting micro- to submicromolar affinities. A solution affinity of approximately 0.1 microM was found for the strongest binding variant under the conditions used for selection (5 mM Mg(2+)). Systematic studies of the effect of Mg(2+) and Mn(2+) on binding, however, revealed that the affinity of the aptamers could be substantially improved, and at optimized conditions of Mn(2+) the affinity of one of the aptamers approached that of an anti-SAH antibody with similar/identical binding specificity. Comparisons with the MAb suggest that the on rate is the limiting factor for high-affinity binding by these aptamers, and comparison with a truncated aptamer shows that shortening of RNA constructs may alter binding kinetics as well as sensitivity to ions.
Adenine↗
Divalent cation modulation of the ribonuclease functions of human immunodeficiency virus reverse transcriptase.
The stimulatory effect of Mg2+ and Mn2+ on the ribonuclease H (RNase H) functions of HIV-1 reverse transcriptase (RT) has been evaluated using a model 90-nt RNA template/36-nt DNA primer. Wild type enzyme exhibits similar endonuclease and directional processing activities in response to both cations, while RNase H activity (hydrolysis of double-stranded RNA) is only evident in the presence of Mn2+. Enzyme altered at the p66 residue Glu478 (Glu478-->Gln478), which participates in metal ion binding, is completely inactive in Mg2+. However, Mn2+ restores specifically its endoribonuclease activity. In the presence of Mn2+, mutant RT also catalyzes specific removal of the tRNA replication primer, eliminating the possibility of contaminating Escherichia coli RNase H in our recombinant enzyme. However, the efficiency with which mutant RT catalyzes transfer of nascent DNA between RNA templates (an event mandating RNase H activity) is severely reduced. These findings raise the possibility that directional processing activity is required to accelerate transfer of nascent DNA between templates during retroviral replication.
Base Sequence↗
Conformation and interaction of phenylalanine with the divalent cation at the active site of human recombinant tyrosine hydroxylase as determined by proton NMR.
Recombinant human tyrosine hydroxylase has been purified as a metal-free apoenzyme (apo-hTH1) which tightly binds one Fe2+, Co2+, or Zn2+ per subunit with activation only by Fe2+ and competitive inhibition by the other cations. L-tyrosine and L-phenylalanine are alternative substrates for this enzyme, giving similar Vmax values, although the KM value for phenylalanine is about 8-fold greater than for tyrosine. Apo-hTH1 enhances the paramagnetic effects of Co2+ on 1/T1 and 1/T2 values of the protons of enzyme-bound phenylalanine both in the presence and in the absence of the oxidized form of the cofactor L-erythro-7,8-dihydrobiopterin (BH2), which was used as an inactive analog of the natural cofactor (6R)-1-erythro-tetrahydrobiopterin (BH4). No effects of hTH1-Zn2+ on 1/T1 or 1/T2 are found. From paramagnetic effects of hTH1-Co2+ on 1/T1 of phenylalanine protons at 250 and 600 MHz, in the presence of BH2, a correlation time (tau c) of 1.8 +/- 0.1 ps was found. Using this tau c value, and assuming that only one proton of the pairs H3,H5, and H2,H6 is experiencing the total paramagnetic effect (asymmetric limiting case), distances from enzyme-bound Co2+ to phenylalanine (+/- 1.2 A) of 6.1 A (H3 or H5), 6.3 A (H2 or H6), 7.0 A (H4), 7.3 A (H alpha), > or = 7.4 A (H beta-pro-S), and > or = 7.6 A (H beta-pro-R) were calculated. The distances to H3 or H5 and to H2 or H6 are slightly increased to 6.8 and 7.0 A, respectively, if each proton of both degenerate pairs equally experiences the paramagnetic effect of Co2+ (symmetric limiting case). These distances place the aromatic ring of phenylalanine in the second coordination sphere of the metal, which would permit an Fe-bound oxy or peroxy species to approach molecular contact with C3/C4, suggesting a direct role of Fe2+ in the hydroxylation reaction. The same correlation time and similar distances were found in the absence of BH2 with H4 of phenylalanine slightly closer to the metal. In the ternary hTH1-Zn(2+).BH2.phenylalanine complex, eight interproton distances in the enzyme-bound phenylalanine were determined by NOESY spectra at 600 MHz at 35-, 50-, and 75-ms mixing times. The conformation of enzyme-bound phenylalanine, consistent with the six Co(2+)-proton distances and the eight interproton distances, is partially extended with torsional angles chi 1 = 97 degrees +/- 3 degrees and chi 2 = -78 degrees +/- 2 degrees.
Apoenzymes↗
Role of divalent cations in the 3',5'-exonuclease reaction of DNA polymerase I.
X-ray studies of the proofreading 3',5'-exonuclease site of the large (Klenow) fragment of DNA polymerase I have detected a binuclear metal complex consisting of a pentacoordinate metal (site A) which shares a ligand, Asp-355, with an octahedral metal (site B) [Freemont, P. S., Friedman, J. M., Beese, L. S., Sanderson, M. R., & Steitz, T. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8924-8928; Beese, L. S., & Steitz, T. A. (1991) EMBO J. 10, 25-33]. Kinetic studies of the activation of the 3',5'-exonuclease reaction by Co2+, Mn2+, or Mg2+, at low concentrations of DNA, reveal sigmoidal activation curves for the three metal ions with Hill coefficients of 2.3-2.4 and K0.5 values of 16.6 microM, 4.2 microM, and 343 microM, respectively. The binding of Co2+ to the enzyme results in the appearance of an intense visible absorption spectrum of the metal ion with maxima at 633, 570, and 524 nm and extinction coefficients of 190, 194, and 150 M-1 cm-1, respectively, suggesting the formation of a pentacoordinate Co2+ complex. Optical titration with Co2+ yields a sigmoidal titration curve which is best fit by assuming the cooperative binding of three Co2+ ions with a K0.5 of 39.9 microM, comparable to the value of 16.6 microM obtained kinetically. Displacement of Co2+ by 1 equiv of Zn2+, which binds tightly to the A site of the 3',5'-exonuclease, shifts the optical spectrum to 524 nm and lowers the extinction coefficient to 30 -1 cm-1, indicative of octahedral coordination.2+ the formation of the binuclear complex.
Binding Sites↗
Effects of divalent cations bound to the catalytic site on ATP-induced conformational changes in the sarcoplasmic reticulum Ca(2+)-ATPase: stopped-flow analysis of the fluorescence of N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine attached to cysteine-674.
Cys-674 of the sarcoplasmic reticulum Ca(2+)-ATPase was labeled with N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine without a loss of the catalytic activity. The steady-state fluorescence of the label decreased with increasing concentration of Mg.ATP, Mn.ATP, or Ca.ATP in the presence of Ca2+ and 0.1 M KCl at 0 degree C, pH 7.0. The maximum extent of the decrease was 23%. The Mg.ATP-, Mn.ATP-, or Ca.ATP-induced fluorescence drop was also determined by the stopped-flow spectrofluorometry. The Ca.ATP-induced fluorescence drop manifested a biphasic time course. The first phase reflects the conformational change which was previously shown [Suzuki, H., Obara, M., Kuwayama, H., & Kanazawa, T. (1987) J. Biol. Chem. 262, 15448-15456] to occur upon formation of the calcium-enzyme-substrate complex. The amplitude of the second phase was 2.5% of the fluorescence before addition of Ca.ATP. This phase of the fluorescence drop coincided with the phosphoenzyme formation, which was determined by the continuous flow-rapid quenching method. The phosphoenzyme formed was largely sensitive to ADP. When adenosine 5'-(beta,gamma-methylenetriphosphate) (a nonhydrolyzable ATP analog incapable of phosphorylating the enzyme) was added in the presence of 5 mM CaCl2 without added MgCl2 the steady-state fluorescence decreased rapidly by 20%. However, this drop lacked the second phase. When phosphoenzyme isomerization from the ADP-sensitive form to the ADP-insensitive form was prevented by the N-ethylmaleimide treatment, the second phase of the Ca.ATP-induced fluorescence drop again coincided with the phosphoenzyme formation.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Diphosphate↗
Transbilayer redistribution of phosphatidylglycerol in small, unilamellar vesicles induced by specific divalent cations.
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Cations, Divalent↗
Modulation of membrane fusion by membrane fluidity: temperature dependence of divalent cation induced fusion of phosphatidylserine vesicles.
We have investigated the temperature dependence of the fusion of phospholipid vesicles composed of pure bovine brain phosphatidylserine (PS) induced by Ca2+ or Mg2+. Aggregation of the vesicles was monitored by 90 degrees light-scattering measurements, fusion by the terbium/dipicolinic acid assay for mixing of internal aqueous volumes, and release of vesicle contents by carboxyfluorescein fluorescence. Membrane fluidity was determined by diphenylhexatriene fluorescence polarization measurements. Small unilamellar vesicles (SUV, diameter 250 A) or large unilamellar vesicles (LUV, diameter 1000 A) were used, and the measurements were done in 0.1 M NaCl at pH 7.4. The following results were obtained: (1) At temperatures (0-5 degrees C) below the phase transition temperature (Tc) of the lipid, LUV (PS) show very little fusion in the presence of Ca2+, although vesicle aggregation is rapid and extensive. With increasing temperature, the initial rate of fusion increases dramatically. Leakage of contents at the higher temperatures remains limited initially, but subsequently complete release occurs as a result of collapse of the internal aqueous space of the fusion products. (2) SUV (PS) are still in the fluid state down to 0 degree C, due to the effect of bilayer curvature, and fuse rapidly in the entire temperature range from 0 to 35 degrees C in the presence of Ca2+. The initial rate of leakage is low relative to the rate of fusion. At higher temperatures (15 degrees C and above), subsequent collapse of the vesicles' internal space causes complete release.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Divalent cations regulate glucagon binding. Evidence for actions on receptor-Ns complexes and on receptors uncoupled from Ns.
The effects of Mg2+ or ethylenediaminetetraacetic acid (EDTA) on 125I-glucagon binding to rat liver plasma membranes have been characterized. In the absence of guanosine 5'-triphosphate (GTP), maximal binding of 125I-glucagon occurs in the absence of added Mg2+. Addition of EDTA or Mg2+ diminishes binding in a dose-dependent manner. In the presence of GTP, maximal binding occurs in the presence of 2.5 mM Mg2+ (EC50 = 0.3 mM) while EDTA or higher concentrations of Mg2+ diminish binding. Response to exogenous Mg2+ or EDTA depends on the concentration of Mg2+ in the membranes and may vary with the method used for membrane isolation. Solubilized 125I-glucagon-receptor complexes fractionate on gel filtration columns as high molecular weight, GTP-sensitive complexes in which receptors are coupled to regulatory proteins and lower molecular weight, GTP-insensitive complexes in which receptors are not coupled to other components of the adenylyl cyclase system. In the absence of GTP, 40 mM Mg2+ or 5 mM EDTA diminishes receptor affinity for hormone (from KD = 1.2 +/- 0.1 nM to KD = 2.6 +/- 0.3 nM) and the fraction of 125I-glucagon in high molecular weight receptor-Ns complexes without affecting site number (Bmax = 1.8 +/- 0.1 pmol/mg of protein). Thus, while GTP promotes disaggregation of receptor-Ns complexes, Mg2+ or EDTA diminishes the affinity with which these species bind hormone. In the presence of GTP, hormone binds to lower affinity (KD = 9.0 +/- 3.0 nM), low molecular weight receptors uncoupled from Ns.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
In vitro microtubule assembly regulation by divalent cations and nucleotides.
Mg2+ and guanosine 5'-triphosphate (GTP) are usually used to assemble microtubule protein (tubulin + microtubule-associated proteins) or tubulin into microtubules in vitro. Recently, it has been shown that Mn2+ will substitute for Mg2+ in inducing pure tubulin + GTP to assemble into microtubules. We find that Mn2+ also substitute for Mg2+ in inducing two-cycle calf microtubule protein (MTP) + GTP to assemble into microtubules. Zn2+ or Co2+ induces MTP + GTP to form sheets with more than 13 protofilaments. We find that Co2+ also substitutes for Zn2+ in inducing tubulin to form 200-nm tubules similar to those reported with Zn2+. To learn whether metal-induced assembly is due to direct binding and/or a metal-GTP complex, metal-induced assembly of MTP and tubulin was studied in the presence of CrIIIGTP or taxol, two probes which promote assembly in the absence of added GTP. With CrGTP, Mg2+ and ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) were required for optimal assembly into microtubules, and Mn2+ could substitute for Mg2+. MTP incubated with Zn2+ and CrGTP assembled into sheets, shorter than but similar to those induced by Zn2+ + GTP. Mg2+-induced microtubules and Zn2+-induced sheets contained 0.45 mol of [8-3H]GTP/mol of tubulin if assembled from [8-3H]GTP and a mixture of 0.25 mol of [8-3H]CrGTP, 0.1 mol of [8-3H]GDP, and 0.05 mol of [8-3H]CrGDP/mol of tubulin if assembled from [8-3H]CrGTP. Zn2+ induced taxol-treated MTP to form sheets. Sheets were also induced from tubulin + Zn2+ and either CrGTP or taxol. These studies suggest that the Zn2+-induced structures are not due to a Zn-GTP complex and that Mg2+ does not promote assembly only through a Mg-GTP complex.
Animals↗
Binding of divalent cations of dipalmitoylphosphatidylcholine bilayers and its effect on bilayer interaction.
We have confirmed that CaCl2 swells the multilayer lattice formed by dipalmitolyphosphatidylcholine (DPPC) in an aqueous solution. Specifically, at room temperature 1 mM CaCl2 causes these lipid bilayers to increase their separation, dw, from 19 A in pure water to greater than 90 A. CaCl2 concentrations greater than 4 mM cause less swelling. We have measured the net repulsive force between the bilayers in 30 mM CaCl2 at T = 25 degrees C (below the acyl chain freezing temperature). For interbilayer separations between 30 and 90 A, the dominant repulsion between bilayers is probably electrostatic; Ca2+ binds to DPPc lecithin bilayers, imparting a charge to them. The addition of NaCl to CaCl2 solutions decreases this repulsion. For dw less than 20 A, the bilayer repulsion appears to be dominated by the "hydration forces" observed previously between both neutral and charged phospholipids. From the electrostatic repulsive force, we estimate the extent of Ca2+ binding to the bilayer surface. The desorption and bound Ca2+, apparent when bilayers are pushed together, is more rapid than one would expect if an association constant governed Ca2+ binding. The association affinity does not appear to be a fixed quantity but rather a sensitive function of ionic strength and bilayer separation.
Calcium↗