Manganese, calcium, and saccharide binding to concanavalin A, as studied by ultrafiltration

The binding of the ligands Mn2+, Ca2+, and methyl alpha-D-glucopyranoside to concanavalin A, purified as described (A.J. Sophianopoulos and J.A. Sophianopoulos (1981) Prep. Biochem. 11, 413-435), was studied by ultrafiltration in 0.2 M NaCl, pH 5.2 and pH 6.5 to 7, and at 23 to 25 degrees C. The association constant (Ka) of methyl alpha-D-glucopyranoside to concanavalin A was (2 +/- 0.2) X 10(3) M-1, both at pH 5.2 and 7. At pH 5.2 and in the absence of Ca2+, the Ka of Mn2+ to concanavalin A was (5 +/- 1) X 10(3) M-1, and in the presence of 1 mM Ca2+, the Ka was (9.1 +/- 2.1) X 10(5) M-1. At pH 6.5 Mn2+ bound to concanavalin A with a Ka of (7.3 +/- 1.8) X 10(5) M-1, and the binding affinity was virtually independent of the presence of Ca2+. Experiments of binding of 4-methylumbelliferyl alpha-D-mannopyranoside to concanavalin A indicated that at pH 5.2, binding of a single Mn2+ per concanavalin A monomer was sufficient to induce a fully active saccharide binding site. Ca2+ is not necessary for such activation, but rather it increases the affinity of concanavalin A for binding Mn2+.     

Stoichiometry of manganese and calcium ion binding to concanavalin A

Using measurements of solvent nuclear (proton) magnetic relaxation dispersion (NMRD), we have previously shown that concanavalin A (Con A) can exist in two conformational forms and that, in the absence of Ca2+, Mn2+ can bind to both the S1 and S2 sites of each monomer of Con A of at least one conformer [Brown, R.D., III, Brewer, C.F., & Koenig, S.H. (1977) Biochemistry 16, 3883-3896]. Recently other investigators have claimed that the stoichiometry of Mn2+ binding to Con A is only 1:1 for this conformational state, both in the absence and presence of saccharide; the same was claimed for Ca2+ under similar conditions. We now present titration and equilibrium dialysis experiments, both in the absence and presence of saccharide, using NMRD and atomic absorption spectroscopy, to investigate the stoichiometry of Mn2+ and Ca2+ binding to Con A. We have extended the NMRD method to include the determination of the total concentration of Mn2+ in samples of Con A. This, coupled with our previous use of NMRD to measure the concentration of free Mn2+ in protein solutions as well as the distribution of bound Mn2+ among different sites, allows us to measure the stoichiometry of binding with precision. We reconfirm that, at equilibrium in the presence of excess Mn2+, the binding stoichiometry of Mn2+ to Con A is 2:1, both in the absence and presence of saccharide. Addition of Ca2+ to a solution of Mn2+-Con A results in stoichiometric displacement of Mn2+ from the S2 site under the conditions investigated. Under nonequilibrium conditions, Mn2+ forms a metastable binary complex with the protein that persists for days at 5 degrees C. We also report, for the first time, values for all of the dissociation constants of binary and ternary complexes of Mn2+ with both conformations of Con A in solution. Atomic absorption measurements also indicate that Ca2+, in the absence of Mn2+, binds to both S1 and S2 sites in the absence and presence of saccharides.     

Calorimetric experiments of Mn2+ -binding to alpha-lactalbumin

We measured by batch microcalorimetry the standard enthalpy change delta H degrees of the binding of Mn2+ to apo-bovine alpha-lactalbumin; delta H degrees = -90 +/- k J.mol-1. The binding constants, KMn2+, calculated from the calorimetric and circular dichroism titration curves, are (4.6 +/- 1).10(5) M-1 and (2.1 +/- 0.4).10(5) M-1, respectively. Batch calorimetry confirms the competitive binding Ca2+, Mn2+ and Na+ to the same site. The relatively small enthalpy change for Mn2+ binding compared to Ca2+ binding favours a model of a rigid and almost ideal Ca2+-complexating site, different from the well-known EF-hand structures. Cation binding to the high-affinity site most probably triggers the movement of an alpha-helix which is directly connected to the complexating loop.     

Remarkable affinity and selectivity for Cs+ and uranyl (UO22+) binding to the manganese site of the apo-water oxidation complex of photosystem II.

The size and charge density requirements for metal ion binding to the high-affinity Mn2+ site of the apo-water oxidizing complex (WOC) of spinach photosystem II (PSII) were studied by comparing the relative binding affinities of alkali metal cations, divalent metals (Mg2+, Ca2+, Mn2+, Sr2+), and the oxo-cation UO22+. Cation binding to the apo-WOC-PSII protein was measured by: (1) inhibition of the rate and yield of photoactivation, the light-induced recovery of O2 evolution by assembly of the functional Mn4Ca1Clx, core from its constituent inorganic cofactors (Mn2+, Ca2+, and Cl-); and by (2) inhibition of the PSII-mediated light-induced electron transfer from Mn2+ to an electron acceptor (DCIP). Together, these methods enable discrimination between inhibition at the high- and low-affinity Mn2+ sites and the Ca2+ site of the apo-WOC-PSII. Unexpectedly strong binding of large alkali cations (Cs+ > Rb+ > K+ > Na+ > Li+) was found to smoothly correlate with decreasing cation charge density, exhibiting one of the largest Cs+/Li+ selectivities (>/=5000) for any known chelator. Both photoactivation and electron-transfer measurements at selected Mn2+ and Ca2+ concentrations reveal that Cs+ binds to the high-affinity Mn2+ site with a slightly greater affinity (2-3-fold at pH 6.0) than Mn2+, while binding about 10(4)-fold more weakly to the Ca2+-specific site required for reassembly of functional O2 evolving centers. In contrast to Cs+, divalent cations larger than Mn2+ bind considerably more weakly to the high-affinity Mn2+ site (Mn2+ > Ca2+ > Sr2+). Their affinities correlate with the hydrolysis constant for formation of the metal hydroxide by hydrolysis of water: Me2+aq --> [MeOH]+aq + H+aq. Along with the strong stimulation of the rate of photoactivation by alkaline pH, these metal cation trends support the interpretation that [MnOH]+ is the active species that forms upon binding of Mn2+aq to apo-WOC. Further support for this interpretation is found by the unusually strong inhibition of Mn2+ photooxidation by the linear uranyl cation (UO22+). The intrinsic binding constant for [MnOH]+ to apo-WOC was determined using a thermodynamic cycle to be K = 4.0 x 10(15) M-1 (at pH 6.0), consistent with a high-affinity, preorganized, multidentate coordination site. We propose that the selectivity for binding [MnOH]+, a linear low charge-density monocation, vs symmetrical Me2+ dications is functionally important for assembly of the WOC by enabling: (1) discrimination against higher charge density alkaline earth cations (Mg2+ and Ca2+) and smaller alkali metal cations (Na+ and K+) that are present in considerably greater abundance in vivo, and thus would suppress photoactivation; and (2) higher affinity binding of the one Ca2+ ion or the remaining three Mn2+ ions via coordination to form mu-hydroxo-bridged intermediates, apo-WOC-[Mn(mu-OH)2Mn]3+ or apo-WOC-[Mn(mu-OH)Ca]3+, during subsequent assembly steps of the native Mn4Ca1Clx core. In contrast to more acidic Me2+ divalent ion inhibitors of the high-affinity Mn2+ site, like Ca2+ and Sr2+, Cs+ does not accelerate the decay of the first light-induced intermediate, IM1, formed during photoactivation (attributed to apo-WOC-[Mn(OH)2]+). The inability of Cs+ to promote decay of IM1, despite having comparable affinity as Mn2+, is consistent with its considerably weaker Lewis acidity, resulting in the reprotonation of IM1 by water becoming the rate-limiting step for decay prior to displacement of Mn2+. All four different lines of evidence provide a self-consistent picture indicating that the initial step in assembly of the WOC involves high-affinity binding of [MnOH]+.     

Binding of lanthanum and gadolinium ions to concanavalin A studied calorimetrically at 25 degrees C

The interaction between Concanavalin A (ConA) and the lanthanide ions La3+ and Gd3+ has been studied calorimetrically at 25 degrees C. The measurements were carried out at a pH of 4.5, where the protein exists prevailingly as a dimer. Calorimetry allows the direct determination of the binding enthalpy and the evaluation of both the apparent association constant, and the apparent free energy and entropy. Three groups of data were collected. The first concerns the interaction of the 'native' protein, i.e., fully metallized with Mn2+ and Ca2+, with the lanthanides. The second concerns the interaction of the completely demetallized protein with La3+ and Gd3+. Finally, the affinity of each complex was tested for the specific sugar alpha-methylmannopyranoside. The analysis of the thermodynamic parameters obtained, led to the following conclusions: 1) a specific site, named S3, exists on the protein for the lanthanides, distinct from the S1 site of the transition metal and from the S2 site, specific for calcium. There is only one S3 site per protomer when the protein has Mn2+ in S1 and Ca2+ in S2. Moreover, there is no appreciable competition for S1 and S2 from the lanthanides. The 'native' protein, metallized with La3+ or Gd3+, is a fully functional protein. 2) The demetallized protein (ApoCon A) has at least two sites per protomer for the lanthanides. The hypothesis is that, besides the S3 site, the lanthanides, in the absence of Mn2+, can also occupy the S1, but not the S2, site. The protein metallized only with gadolinium ion is completely inactive toward the interaction with the mannoside. The same happens when, along with gadolinium, only calcium or manganese is present. Hence, in the absence of the transition metal in S1 or of calcium in S2, the protein is not in the conformation suitable to interact with its specific substrate.     

Interaction of monolayers of concanavalin A with mono- and polysaccharides. A study by means of infrared-attenuated total reflectance spectroscopy

The effects of pH, Mn2+ and Ca2+ and urea denaturation on the interaction of monolayers of concanavalin A on saline with the polysaccharide dextran B-1355 and the monosaccharides methyl alpha-D-mannopyranoside and D-galactose have been investigated. Infrared absorption spectra of compressed monolayers of the protein and the protein-dextran complex coated on a germanium plate have been obtained by means of attenuated total reflectance spectroscopy. Except in one case of denaturation, the amide I absorption of concanavalin A peaked around 1631 cm-1, indicating a predominance of the beta-pleated sheet conformation, in agreement with its secondary structure in the solution and crystalline phases. The contribution to the absorbance of the concanavalin A-dextran films at 3300 cm-1 due to absorption by the O-H stretching modes of the polysaccharide is a measure of its binding. Increasing the pH from 6.1 to 7.5 appreciably reduced the dextran binding, at pH 9.3 the binding was zero. Adding 1 mM Mn2+ and Ca2+ to the subphase at pH 7.5 restored both the dextran binding and the affinity of concanavalin A for methyl alpha-D-mannopyranoside to that of the native protein at pH 6.1. At this latter pH, the weak binding of dextran to monolayers of demetallized concanavalin A (apo-concanavalin A) was also restored to that for the native molecule by the addition of these divalents. This indicates the requirement of concanavalin A for these ions to maintain the integrity of the saccharide-binding site. The loss of dextran binding with urea denaturation was also observed. These results parallel those for solutions of the protein, indicating the validity of the monolayer system for the study of these interactions.     

Rapid induction of acetylcholine receptor aggregates by a neural factor and extracellular Ca2+

A soluble fetal brain extract (EBX) induces acetylcholine receptor (AChR) aggregation in cultured rat myotubes within 4 hr at 36 degrees C in a defined medium containing 1.8 mM (normal) extracellular Ca2+ (Olek et al., 1983). The activity of EBX was Ca2+ dependent; reducing extracellular Ca2+ significantly inhibited EBX-induced AChR aggregation and a 15-50% increase in extracellular Ca2+ synergistically enhanced the activity of EBX. Synergism was specific for Ca2+ as increases in other divalent cations (Ba2+, Co2+, Mg2+, Mn2+, Sr2+) had no effect. A large increase (300-500%) in extracellular Ca2+ alone also induced AChR aggregation within 4 hr at 36 degrees C. An equivalent increase in other cations (Ba2+, Co2+, Mg2+, Mn2+, Sr2+) did not promote AChR aggregation. An initial 15-min pulse of increased extracellular Ca2+ alone or with EBX was adequate to induce AChR aggregation. Aggregates induced by EBX, Ca2+ alone, or EBX/Ca2+ were found predominantly on the top surface of the myotube. These treatments did not detectably alter preexisting aggregates present at substrate contact sites on the bottom surface of myotubes. AChR aggregation induced by any treatment was not inhibited by cycloheximide, Ca2+ channel blockers, or protease inhibitors but was blocked by Co2+ and sodium azide.     

Interaction of calcium with bovine plasma protein C

The binding of 45Ca2+ to bovine plasma protein C (PC) and to activated bovine plasma protein C (APC) has been examined by equilibrium ultrafiltration at pH 7.4 and 25 degrees C. Under these conditions, PC possesses 16.0 plus or minus 2.0 equivalent Ca2+ binding sites, of average KD (8.7 plus or minus 1.5) x 10(-4) M, and APC contains 9.0 plus or minus 1.0 equivalent Ca2+ binding sites, with an average KD of (4.3 plus or minus 1.1) x 10(-4) M. Both Mn2+ and Sr2+ were capable of ready displacement of Ca2+ from a Ca2+-PC complex, while Mg2+ was less effective in this regard. The alpha-thrombin-catalyzed activation of PC was inhibited by the presence of Ca2+. A kinetic analysis of this effect demonstrated that it was, in large part, due to an increase in the Km of the reaction. Addition of other divalent cations, e.g. Mn2+, Sr2+, and Mg2+, in place of Ca2+ also resulted in inhibition of the alpha-thrombin-catalyzed activation of PC in a manner which paralleled their ability to displace Ca2+ from a Ca2+-PC complex. On the other hand, the activation of PC by the coagulant protein from Russell's Viper venom was augmented by the presence of Ca2+. Other divalent metal ions, such as Sr2+ and Mn2+, in the absence of Ca2+, also weakly stimulated this reaction. Mg2+ was without notable effect.     

Characterization of calcium binding to brush-border membranes from rat duodenum.

The Ca2+-binding properties of isolated brush-border membranes at physiological ionic strength and pH were examined by rapid Millipore filtration. A comprehensive analysis of the binding data suggested the presence of two types of Ca2+-binding sites. The high-affinity sites, Ka = (6.3 +/- 3.3) X 10(5) M-1 (mean +/- S.E.M.), bound 0.8 +/- 0.1 nmol of Ca2+/mg of protein and the low-affinity sites, Ka = (2.8 +/- 0.3) X 10(2) M-1, bound 33 +/- 3.5 nmol of Ca2+/mg of protein. The high-affinity site exhibited a selectivity for Ca2+, since high concentrations of competing bivalent cations were required to inhibit Ca2+ binding. The relative effectiveness of the competing cations (1 and 10 mM) for the high-affinity site was Mn2+ approximately equal to Sr2+ greater than Ba2+ greater than Mg2+. Data from the pH studies, treatment of the membranes with carbodi-imide and extraction of phospholipids with aqueous acetone and NH3 provided evidence that the low-affinity sites were primarily phospholipids and the high-affinity sites were either phosphoprotein or protein with associated phospholipid. Two possible roles for the high-affinity binding sites are suggested. Either high-affinity Ca2+ binding is involved with specific enzyme activities or Ca2+ transport across the luminal membrane occurs via a Ca2+ channel which contains a high-affinity Ca2+-specific binding site that may regulate the intracellular Ca2+ concentration and gating of the channel.     

The effect of divalent metal cations on the inhibition of human coagulation factor Xa by plasma proteinase inhibitors

The inactivation of human coagulation factor Xa by the plasma proteinase inhibitors alpha 1-antitrypsin, antithrombin III and alpha 2-macroglobulin in purified systems was found to be accelerated by the divalent cations Ca2+, Mn2+ and Mg2+. The rate constant for the inhibition of factor Xa by antithrombin III rose from 2.62 X 10(4) M-1 X min-1 in the absence of divalent cations to a maximum of 6.40 X 10(4) M-1 X min-1 at 5 mM Ca2+, 8.10 X 10(4) M-1 X min-1 at 5 mM Mn2+, with a slight decrease in rate at higher cation concentrations. Mg2+ caused a gradual rise in rate constant to 5.65 X 10(4) M-1 X min-1 at 20 mM. The rate constant for the inhibition of factor Xa by alpha 1-antitrypsin in the absence of divalent cations was 5.80 X 10(3) M-1 X min-1. Ca2+ increased the rate to 1.50 X 10(4) M-1 X min-1 at 5 mM and Mn2+ to 2.40 X 10(4) M-1 X min-1 at 6 mM. The rate constant for these cations again decreased at higher concentrations. Mg2+ caused a gradual rise in rate constant to 1.08 X 10(4) M-1 X min-1 at 10 mM. The rate constant for the factor Xa-alpha 2-macroglobulin reaction was raised from 6.70 X 10(3) M-1 X min-1 in the absence of divalent cations to a maximum of 4.15 X 10(4) M-1 X min-1 at 4 mM Ca2+, with a decrease to 3.05 X 10(4) M-1 at 10 mM. These increases in reaction rate were correlated to the binding of divalent cations to factor Xa by studying changes in the intrinsic fluorescence and dimerization of factor Xa. The changes in fluorescence suggested a conformational change in factor Xa which may be responsible for the increased rate of reaction, whilst the decrease in rate constant at higher concentrations of Ca2+ and Mn2+ may be due to factor Xa dimerization.     

Microcalorimetric investigation of the interaction of calmodulin with seminalplasmin and myosin light chain kinase

Flow microcalorimetric titrations of calmodulin with seminalplasmin at 25 degrees C revealed that the high affinity one-to-one complex in the presence of Ca2+ (Comte, M., Malnoe, A., and Cox, J. A. (1986) Biochem. J. 240, 567-573) is entirely enthalpy-driven (delta H0 = -50 kJ.mol-1; delta S0 = O J.K-1.mol-1; delta Cp0 = O J.K-1.mol-1) and is not influenced by the proton or Mg2+ concentration. The Sr2+- and Cd2+-promoted high affinity complexes are also exothermic for -49 and -45 kJ.mol-1, respectively. The observed low affinity interaction in the absence of divalent ions displays no enthalpy change. No enthalpy changes are observed when calmodulin and seminalplasmin are mixed in the presence of millimolar concentrations of Mg2+, Zn2+, or Mn2+. Enthalpy titrations of the 1:1 calmodulin-seminalplasmin complex with Ca2+ and of partly Ca2+-saturated calmodulin with seminalplasmin revealed that only the species calmodulin.Can greater than or equal to 2 is fully competent for high affinity interaction with seminalplasmin. Binding of the second Ca2+ is strongly enhanced (K2 greater than or equal to 5 X 10(7) M-1) as compared to that in free calmodulin (K2 = 2.6 X 10(5) M-1). This is essentially due to the concomitant strongly exothermic step of isomerization of the calmodulin-seminalplasmin complex from its low to its high affinity form. Binding of the remaining two Ca2+ to the high affinity seminalplasmin-calmodulin complex displays the same affinity constants and endothermic enthalpy change as in free calmodulin. A microcalorimetric study on the complex formation between Ca2+-saturated calmodulin and turkey gizzard myosin light chain kinase revealed that the interaction is strongly exothermic with an important overall gain of order (delta H0 = -85 kJ.mol-1; delta S0 = -122 J.K-1.mol-1) and occurs with significant proton uptake (0.44 H+ per mol at pH 7.5). The observed low affinity interaction (K = 2.2 X 10(5) M-1) in the absence of Ca2+ (Mamar-Bachi, A., and Cox, J. A. (1987) Cell Calcium 8, 473-482) displays neither a change in enthalpy nor in protonation.     

Characteristic changes of the S2/S1 difference FTIR spectrum induced by Ca2+ depletion and metal cation substitution in the photosynthetic oxygen-evolving complex

Effects of Ca2+ depletion and substitution with other metal cations on the structure of the protein matrices of the oxygen-evolving complex (OEC) and their corresponding changes upon the S1 to S2 transition were examined using Fourier transform infrared (FTIR) spectroscopy. Ca2+ depletion and further supplementation with Li+, Na+, Mg2+, Ca2+, or Sr2+ did not significantly affect the typical vibrational features in the double difference S2/S1 spectrum, including the symmetric [1365(+)/1404(-) cm(-1)] and the asymmetric [1587(+)/1566(-) cm(-1)] stretching modes of the carboxylate ligand and the amide I and II modes of the backbone polypeptides. On the other hand, supplementation with K+, Rb+, Cs+, or Ba2+ significantly modified the S2/S1 spectrum, in which the carboxylate modes disappeared and the amide I and II modes were modified. Results indicate that the binding of metal cations that have ionic radii larger than that of Ca2+ to the Ca2+ site induces perturbations in the protein matrices in the vicinity of the Mn cluster to interrupt the characteristic structural and/or conformational changes upon the oxidation of the Mn cluster accompanied with the S1 to S2 transition. The spectrum was also altered by the supplementation of Cd2+, which has an ionic radius comparable to that of Ca2+. A single-pulse-induced S2/S1 difference spectrum revealed that bands that have been assigned to the vibrational modes for the Y(Z) tyrosine and the histidine ligand for the Mn cluster were not induced in the K+-supplemented membranes, although the histidine band is likely to be preserved in the Ca2+-depleted membranes. The Y(Z) band was considerably small in the double difference S2/S1 spectrum in the Ca2+-depleted and the cation-substituted membranes but distinctively present in the Sr2+- or Ca2+-replenished membranes. Furthermore, cation supplementation induced several new bands that disappeared following the Ca2+ replenishment. These results suggest that the proper organization of the hydrogen bond network within OEC for the water oxidation chemistry requires the Ca2+ ion and indicate that the role of Ca2+ is not purely structurally defined by the physical properties of the ion, such as valence and ionic radius. On the basis of these and other findings, we propose that Ca2+ is necessary for the formation of the hydrogen bond network that is involved in the reaction step of water oxidation.     

Protective effect of divalent cations in the plasmin degradation of fibrinogen

Calcium limits the plasmic proteolysis of fibrinogen fragment D by binding to a specific site on the carboxy-terminal segment of the D gamma chain. Employing sodium dodecyl sulfate-polyacrylamide gel electrophoresis to visualize plasmic fragments, Sr2+, Ba2+, and Mn2+ were found to have an equivalent capacity to limit the degradation of fibrinogen fragment D (Mr 94,000). Mg2+, Fe2+, Co2+, and Zn2+ did not comparably limit the digestion of fragment D. Equilibrium dialysis demonstrated that Ba2+ competitively inhibited Ca2+ binding to fibrinogen, suggesting that the ions occupied the Ca2+ binding site of fibrinogen and thereby limited the plasmic digestion of fragment D. The results suggest that Ca2+, Sr2+, Ba2+, and Mn2+ limit plasmin digestion of fragment D by interacting with a Ca2+ binding site in the D domain of the fibrinogen molecule.     

Reaction mechanism of calcium-ATPase of sarcoplasmic reticulum. Substrates for phosphorylation reaction and back reaction, and further resolution of phosphorylated intermediates

Reaction of the purified Ca2+-ATPase of sarcoplasmic reticulum at 0 degrees C at low [gamma-32P]ATP (0.1 to 0.67 microM) and enzyme (0.025 to 0.24 microM) concentration in the presence of 0.11 to 30 mM Ca2+ without added Mg2+ has resulted in the formation of phosphorylated intermediate (EP:maximal level of EP = 0.45 mol/mol of enzyme) at a very slow rate. Under these conditions, the reaction steps in which EP decomposition takes place are completely prevented. This has permitted us to study the EP formation reaction and its reversal specifically, with a considerably improved time resolution. An apparent rate constant of EP formation (Vf) increases in parallel with the concentration of Ca . ATP, but not with those of Mg . ATP, or of protonated or fully ionized free ATP. This suggests that Ca . ATP is the substrate under these conditions. If Co2+ or Mn2+ are in excess over the other ions during the reaction, Vf varies in parallel with [Co . ATP] or [Mn . ATP]. Thus, it appears that either Ca2+, Co2+, or Mn2+ can be complexed with ATP to form the effective substrate. An apparent rate constant of the back reaction of EP initiated by addition of ADP to EP (Vr) increases in proportion to [ADP] or [H . ADP], but is inhibited by increasing concentrations of the ADP complex with Ca2+ or Mg2+, indicating that free ADP or protonated ADP, or both, are actual substrates for the back reaction of EP. These results suggest a new type of site to which the metal moiety of metal . ATP complex remains bound after the release of ADP from the enzyme. An acid-stable phosphorylated intermediate (EP) produced in the presence of high Ca2+ concentrations (e.g. 0.11 mM) without added Mg2+ does not decompose spontaneously, and the major portion (approximately 90%) of this EP (EPD+) reacts with ADP to form ATP (ADP-sensitive). Upon chelating Ca2+ with ethylene glycol bis(beta-amino-ethyl ether)N,N,N',N'-tetraacetic acid (EGTA), EPD+ is converted to another form of EP (EPD-), which is unreactive with ADP (or ADP-insensitive). Addition of Mg2+, after initiation of the reaction leading to EPD- by EGTA, results in rapid production of Pi from a portion of EPD- with KMg approximately equal to 3.3 x 10(3) M-1. The fraction of EPD- that is Mg2+-sensitive (EPD-,M+) increases with reaction time at a much slower rate than the Mg2+-insensitive portion of EPD- (EPD-,M-). These results suggest that the enzyme reaction involves the sequential formation of at least three forms of acid-stable EP, viz. in the order of formation, EPD+, EPD-,M-, and EPD-,M+. The equilibrium between EPD+ and EPD-,M- is shifted by higher [K+] and [Ca2+] towards EPD+.     

Binding strength of calcium ion to bovine alpha-lactalbumin

A Ca2+-sensitive electrode was used for determination of the binding strength of Ca2+ to bovine alpha-lactalbumin in 60 mM Tris buffer (pH 7.8-8.5) in the presence of various concentrations of NaCl. The dependence of the apparent binding constant on the concentration of NaCl was consistent with competitive binding of Ca2+ and Na+, and the binding constants of Ca2+ and Na+ were found to be 2.2 (+/- 0.5) X 10(7) M-1 and 99 (+/- 33) M-1, respectively, at 37 degrees C and pH 8.0. The temperature dependence of the binding constant of Ca2+ was examined between 30 and 45 degrees C; extrapolation of the dependence led to a binding constant of approximately 1 X 10(8) M-1 at pH 8.4 and 25 degrees C. The electrostatic contribution and conformational effect of the protein were also taken into consideration, and the intrinsic binding constant of Ca2+ to native alpha-lactalbumin was calculated to be (1.2-1.5) X 10(10) M-1 at 37 degrees C and pH 8.0.     

An equilibrium study of metal ion binding to human plasma coagulation factor XIII.

1. The binding of Ca2+ to plasma coagulation Factor XIII from man and from cow caused a small decrease in the intrinsic fluorescence of the protein with a dissociation constant of 0.1 mM. A similar decrease was observed with the thrombin-activated Factors (Factors XIIa). The decrease in protein fluorescence was also caused by both Ni2+ and Mn2+ but not by Mg2+. 2. 45Ca2+ binding was directly demonstrated by equilibrium dialysis. Ca2+ at 0.2 mM bound to Factor XIII (a2b2) and Factor XIIIa (a'2b2) but not to isolated b2-protein. A tight-binding site for Ca2+ is associated with the a-subunits. 3. The Ca2+ essential for the enzyme activity of Factor XIII from man, pig and cow can be replaced by Ni2+, Cu2+, La3+, Mn2+, Fe3+, Y3+, Co2+, Sr2+ or Tb3+, but not by Mg2+.     

Effects of bivalent cations on prostaglandin biosynthesis and phospholipase A2 activation in rabbit kidney medulla slices.

The bivalent cations Ca2+, Mg2+, Co2+, Mn2+, Sr2+ and Ba2+ were compared for their stimulatory or inhibitory effect on prostaglandin formation in rabbit kidney medulla slices. Ca2+, Mn2+ and Sr2+ ions stimulated prostaglandin generation up to 3--5-fold in a time- and dose-dependent manner (Ca2+ greater than Mn2+ congruent to Sr2+). The stimulation by Mn2+ (but not by Sr2+) was also observed in incubations of medulla slices in the presence of Ca2+. Mg2+ and Co2+ ions were without significant effects on either basal or Ca2+-stimulated prostaglandin synthesis. The stimulatory effects of Ca2+, Mn2+ and Sr2+ on medullary generation of prostaglandin E2 were found to correlate with their stimulatory effects on the release of arachidonic acid and linoleic acid from tissue lipids. The release of other fatty acids was unaffected, except for a small increase in oleic acid release. As both arachidonic acid and linoleic acid are predominantly found in the 2-position of the glycerol moiety of phospholipids, the stimulation by these cations of prostaglandin E2 formation appears to be mediated via stimulation of phospholipase A2 activity.     

Changes in affinity for calcium ions with the formation of two kinds of phosphoenzyme in the Ca2+,Mg2+-dependent ATPase of sarcoplasmic reticulum

The amount of Ca2+ bound to the Ca2+,Mg2+-dependent ATPase of deoxycholic acid-treated sarcoplasmic reticulum was measured during ATP hydrolysis by the double-membrane filtration method [Yamaguchi, M. & Tonomura, Y. (1979), J. Biochem. 86, 509--523]. The maximal amount of phosphorylated intermediate (EP) was adopted as the amount of active site of the ATPase. In the absence of ATP, 2 mol of Ca2+ bound cooperatively to 1 mol of active site with high affinity and were removed rapidly by addition of EGTA. AMPPNP did not affect the Ca2+ binding to the ATPase in the presence of MgCl2. Under the conditions where most EP and ADP sensitive at steady state (58 microM Ca2+, 50 microM EGTA, and 20 mM MgCl2 at pH 7.0 and 0 degrees C), bound Ca2+ increased by 0.6--0.7 mol per mol active site upon addition of ATP. The time course of decrease in the amount of bound 45Ca2+ on addition of unlabeled Ca2+ + EGTA was biphasic, and 70% of bound 45Ca2+ was slowly displaced with a rate constant similar to that of EP decomposition. Similar results were obtained for the enzyme treated with N-ethylmaleimide, which inhibits the step of conversion of ADP-sensitive EP to the ADP-insensitive one. Under the conditions where most EP was ADP insensitive at steady state (58 microM Ca2+, 30 microM EGTA, and 20 mM MgCl2 at pH 8.8 and 0 degrees C), the amount of bound Ca2+ increased slightly, then decreased slowly by 1 mol per mol of EP formed after addition of ATP. Under the conditions where about a half of EP was ADP sensitive (58 microM Ca2+, 25 microM EGTA, and 1 mM MgCl2 at pH 8.8 and 0 degrees C), the amount of bound Ca2+ did not change upon addition of ATP. These findings suggest that the Ca2+ bound to the enzyme becomes unremovable by EGTA upon formation of ADP-sensitive EP and is released upon its conversion to ADP-insensitive EP.     

Evidence that the ATP binding site of sarcoplasmic reticulum CaATPase has a Mg(2+) ion binding sub-site

The CaATPase of skeletal muscle sarcoplasmic reticulum was specifically labeled in the ATP binding site with fluorescein isothiocyanate under gentle conditions (pH 7 X 5). Fluorescence energy transfer from the attached fluorescein to Nd3+ indicated that a cation binding site was about 1 X 0 nm away from the fluorescein. Thus it appears that the ATP site includes a cation binding site. At 25 degrees C in 0 X 5 M KCl, the association constants for Nd3+, Ca2+ and Mg2+ were 3 X 3 X 10(5) M-1, 84 M-1 and 35 M-1, respectively, making it possible that, in vivo, the site binds Mg2+.     

Ca2+,Mg2+-ATPase of microsomal membranes from bovine aortic smooth muscle: effects of Sr2+ and Cd2+ on Ca2+ uptake and formation of the phosphorylated intermediate of the Ca2+,Mg2+-ATPase

The effects of various divalent cations on the Ca2+ uptake by microsomes from bovine aortic smooth muscle were studied. High concentrations (1 mM) of Co2+, Zn2+, Mn2+, Fe2+, and Ni2+ inhibited neither the Ca2+ uptake by the microsomes nor the formation of the phosphorylated intermediate (E approximately P) of the Ca2+,Mg2+-ATPase of the microsomes. The cadmium ion, however, inhibited both the Ca2+ uptake and the E approximately P formation by the microsomes. Dixon plot analysis indicated Cd2+ inhibited (Ki = 135 microM) the Ca2+ dependent E approximately P formation in a non-competitive manner. The inhibitory effect of Cd2+ was lessened by cysteine or dithiothreitol. The strontium ion inhibited the Ca2+ uptake competitively, while the E approximately P formation increased on the addition of Sr2+ at low Ca2+ concentrations. At a low Ca2+ concentration (1 microM), Sr2+ was taken up by the aortic microsomes in the presence of 1 mM ATP. It is thus suggested that Sr2+ replaces Ca2+ at the Ca2+ binding site on the ATPase.