scholarly journals Calcium binding to human platelet integrin GPIIb/IIIa and to its constituent glycoproteins. Effects of lipids and temperature

1991 ◽  
Vol 276 (1) ◽  
pp. 35-40 ◽  
Author(s):  
G A Rivas ◽  
J González-Rodríguez
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Calcium Binding ◽  
Equilibrium Dialysis ◽  
Degree Of Saturation ◽  
Membrane Glycoproteins ◽  
Platelet Surface ◽  
Alpha Chain ◽  
High Affinity ◽  
Adhesive Proteins

Platelet plasma membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa) form a Ca(2+)-dependent heterodimer. GPIIb/IIIa, which serves as the receptor for fibrinogen and other adhesive proteins at the surface of activated platelets. Using equilibrium dialysis measurements, it was established that both GPIIb and GPIIIa in solution have low-affinity Ca(2-)-binding sites (Kd0.2-0.3 mM), five in GPIIb and two in GPIIIa, and it was confirmed that only the alpha-chain of GPIIb (GPIIb alpha) binds Ca2+. Furthermore, Ca2+ binding was found with two CNBr fragments of GPIIb, GPIIb alpha-(1-285) and GPIIb alpha-(314-489), which carry three out of the four putative Ca(2+)-binding sites. GPIIb/IIIa in solution has a single high-affinity Ca(2+)-binding site (Kd1 80 +/- 30 nM at 21 degrees C), whose degree of saturation regulates the state of association of GPIIb and GPIIIa in the GPIIb/IIIa heterodimer at room temperature, and 3-4 medium-affinity Ca(2+)-binding sites (Kd2 40 +/- 15 microM at 21 degrees C). When GPIIb/IIIa was incorporated into liposomes, Kd1 decreased by an order of magnitude (9 +/- 3 nM at 21 degrees C) and reached the dissociation constant estimated for the high-affinity Ca(2+)-binding sites at the platelet surface [Brass & Shattil (1982) J. Biol. Chem. 257, 1400-1405], whereas Kd2 remained unchanged. The high-affinity Ca(2+)-binding site of GPIIb/IIIa in solution at 4 degrees C has almost the same affinity (Kd1 65 +/- 20 nM) as at 21 degrees C; however, at 37 degrees C, either its affinity decreases enough so as to become experimentally indistinguishable from the medium-affinity Ca(2+)-binding sites determined at this temperature (number of binding sites 3.9 +/- 1.2 mol of Ca2+/mol of GP, Kd 25 +/- 11 microM), or vanishes altogether. Studies on Ca(2+)-dependent dissociation of GPIIIb/IIIa at 37 degrees C in solution seem to support the former interpretation. Further work will be necessary to decide whether the dissociation of GPIIb/IIIa in the platelet membrane at 37 degrees C is regulated by the degree of saturation of the high-affinity Ca(2+)-binding site, as occurs in solution. It is suggested that the high-affinity Ca(2+)-binding site could be related to the putative GPIIIa-binding region in GPIIb (residues 558-747 of the alpha chain).

scholarly journals Binding of Calcium to Fibrinogen: Some Related Properties

1977 ◽  
Author(s):  
G. Marguerie
Keyword(s):  
Binding Sites ◽  
Calcium Binding ◽  
Specific Binding ◽  
Equilibrium Dialysis ◽  
Structural Features ◽  
Salt Bridges ◽  
High Affinity ◽  
Direct Titration ◽  
Specific Binding Sites ◽  
Calcium Binding Sites

The calcium binding properties of bovin fibrinogen have been studied using equilibrium dialysis method. At pH 7.5 fibrinogen has 3 specific calcium binding sites of high affinity and several non specific binding sites of low affinity. Direct titration of the calcium induced proton release indicates that the binding center is a chelate. Thermal an acid denaturation is found to be markedly influenced by the presence of Ca++, suggesting that structural features are related to the binding. However the circular dichroism spectra show that no generalized conformational change is induced when Ca++ is bound to the protein.The plasminic digestion of fibrinogen is also found to be specificaly influenced by Ca++. The velocity of the initial cleavages is slightly reduced in the presence of calcium. It is therefore suggested that the C-terminal part of the Aα chain is involved in the binding.Considering the dimeric structure of the fibrinogen molecule, the presence of only 3 calcium binding sites of high affinity suggests the existence of “salt bridges” between the constitutive polypeptide chains.

scholarly journals Characterization of Ca2+ interactions with matrix metallopeptidase-12: implications for matrix metallopeptidase regulation

2006 ◽  
Vol 398 (3) ◽  
pp. 393-398 ◽  
Author(s):  
Thomas Gossas ◽  
U. Helena Danielson
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Calcium Binding ◽  
Three Dimensional ◽  
Zinc Ion ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
High Affinity Binding Site ◽  
Matrix Metallopeptidase

Matrix metallopeptidase-12 (MMP-12) binds three calcium ions and a zinc ion, in addition to the catalytic zinc ion. These ions are thought to have a structural role, stabilizing the active conformation of the enzyme. To characterize the importance of Ca2+ binding for MMP-12 activity and the properties of the different Ca2+ sites, the activity as a function of [Ca2+] and the effect of pH was investigated. The enzymatic activity was directly correlated to calcium binding and a Langmuir isotherm for three binding sites described the activity as a function of [Ca2+]. The affinities for two of the binding sites were quantified at several pH values. At pH 7.5, the KD was 0.1 mM for the high-affinity binding site, 5 mM for the intermediate-affinity binding site and >100 mM for the low-affinity binding site. For all three sites, the affinity for calcium decreased with reduced pH, in accordance with the loss of interactions upon protonation of the calcium-co-ordinating aspartate and glutamate carboxylates at acidic pH. The pKa values of the calcium binding sites with the highest and intermediate affinities were determined to be 4.3 and 6.5 respectively. Optimal pH for catalysis was above 7.5. The low-, intermediate- and high-affinity binding sites were assigned on the basis of analysis of three-dimensional-structures of MMP-12. The strong correlation between MMP-12 activity and calcium binding for the physiologically relevant [Ca2+] and pH ranges studied suggest that Ca2+ may be involved in controlling the activity of MMP-12.

BINDING OF CALCIUM TO HUMAN AND BOVINE FACTOR X

1987 ◽  
Author(s):  
D M Monroe ◽  
D W Deerfield ◽  
D L Olson ◽  
T N Stewart ◽  
H R Roberts ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Calcium Binding ◽  
Human Factor ◽  
Equilibrium Dialysis ◽  
Equilibrium Constants ◽  
Scatchard Analysis ◽  
Factor X ◽  
Amino Terminal ◽  
Bovine Factor

Human and bovine factor X contain 11 and 12 glutamyl residues respectively within the first forty amino terminal residues that are posttranslationally modified to y-carboxyglutamyl (Gla) residues. Calcium binding to these Gla residues and at other sites is critical for activity in factor X. We have measured calcium binding to human factor X by equilibrium dialysis for the first time. We have also re-examined calcium binding to bovine factor X in order to compare the two species. Factor X (10 μM) was incubated with 45Ca in 20 mM Tris (pH 7.5), 100 mM NaCl in a half cell separated by a 12-14000 molecular weight fast-equilib-rium disk membrane at 25°C for 24 hours. Four aliquots (100 μL each) were removed from each side of the cell and counted. Data were analyzed with a variety of models that allow for more than one class of binding site and for cooperativity among binding sites. Calcium binding to bovine factor X was best simulated by a model that assumes 1 very tight site, 3 cooperative tight sites, and 18 equivalent, non-interacting sites. Based on data from des(Gla)factor X, the first site is probably a high affinity non-Gla binding site. Our results differ from two previously published reports that indicated either 1 tight and 39 loose noncooperative sites (R.H. Yue & M.M. Gertler (1978) Thrombos. Haemostas. (Stuttg.) 40, 350) or 20 calcium binding sites with the first 4 being cooperative (M.J. Lindhout & H.C. Hemker (1978) Biochimica Biophysica Acta 533, 318). Our data on human factor X fit the same model as used for bovine factor X; however, coop-erativity is less in the 3 cooperative sites. Shown below are the first six thermodynamic equilibrium constants derived from a Scatchard analysis of binding data (values are M−1).Both proteins demonstrate the same total number of binding sites and essentially the same value for the first, tight binding site. Bovine factor X exhibits cooperativity, whereas human factor X has reduced cooperativity.

The determination of calcium-binding sites of human erythrocyte membranes

1984 ◽  
Vol 62 (6) ◽  
pp. 398-408 ◽  
Author(s):  
R. Blaine Moore ◽  
E. E. Dryden ◽  
D. I. C. Kells ◽  
J. F. Manery
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Calcium Binding ◽  
Plasma Membranes ◽  
Equilibrium Dialysis ◽  
Association Constants ◽  
Erythrocyte Membranes ◽  
Least Squares Regression ◽  
Calcium Binding Site ◽  
Calcium Binding Sites

Calcium binding to leaky erythrocyte plasma membranes was measured by three different procedures: Millipore filtration, equilibrium dialysis, and partition centrifugation. The curve derived from the binding equation, which best fit the means of the raw data, was used to estimate the association constants and capacities of the binding sites. A computer program (Gaushaus) which uses a nonlinear, least-squares regression protocol was also used to confirm these estimates. On the basis of these analyses we propose the presence of three classes of calcium-binding sites with the following apparent association constants and capacities: site 1, Ka = 3 × 104 M−1 and n = 30 nmol/mg protein; site 2, Ka = 3 × 103 M−1 and n = 200 nmol/mg protein; site 3, Ka = ~102 M−1 and n = ~200 nmol/mg protein. Calcium binding to erythrocyte membranes sealed in a high-salt solution showed the presence of site 3, but not site 2. The influence of phospholipids on the binding of calcium was evaluated by pretreating ghosts with phospholipase C (Clostridium welchii, EC 3.1.4.3). Treatment with this enzyme removed 80% of the total membrane phosphorus, predominantly from sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine. By the method of partition centrifugation two classes of binding sites were identified by computer analysis. Their association constants and capacities are, respectively, 1.1 × 105 M−1 and 20 nmol/mg protein for site 1 and 4.4 × 103 M−1 and 200 nmol/mg protein for site 2. We speculate that calcium-binding site 1 is composed of acidic phospholipids, calcium-binding site 2 is composed of spectrin and actin, and calcium-binding site 3 is composed of sialic acid.

Calcium-binding sites of the thrombin-thrombomodulin-protein C complex: Possible implications for the effect of platelet factor 4 on the activation of vitamin K-dependent coagulation factors

2007 ◽  
Vol 97 (06) ◽  
pp. 899-906 ◽  
Author(s):  
Likui Yang ◽  
Alireza Rezaie
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Calcium Binding ◽  
Protein C ◽  
Catalytic Domain ◽  
Coagulation Factors ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
High Affinity ◽  
Gla Domain

SummaryThe Ca2+-dependence of protein C activation by thrombin in complex with thrombomodulin (TM) containing chondroitin sulfate (CS) exhibits saturation at ~0.5–1 mM Ca2+, but withTM lacking CS, it has a distinct optimum at ~0.1 mM Ca2+. Since the substrate protein C has multiple Ca2+-binding sites, and the cofactor TM also interacts with Ca2+, the basis for differences in Ca2+ effect on protein C activation by thrombin in complex with TM containing or lacking CS is not known. In this study, by using full-length and Gla-domainless mutants of protein C whose activation by thrombin is independent of either Ca2+ or both Ca2+ and TM, we demonstrate that i) the Ca2+ occupancy of a high-affinity binding site in TM is essential for the high-affinity interaction of the cofactor with thrombin, ii) the Ca2+ occupancy of a binding site (KD ~50 μM) in the catalytic domain of protein C is required for the substrate recognition by the thrombin-TM complex, however, at this concentration of Ca2+ the Gla domain of protein C is not folded properly and thus interacts with exosite-2 of thrombin in complex with TM that lacks CS but not withTM that contains CS, and finally iii) platelet factor 4 can nonspecifically interact with the Gla domain of protein C and other coagulation factors to influence their activation only at subphysiological concentrations of Ca2+.

scholarly journals Mutation of the high affinity calcium binding sites in cardiac troponin C.

1992 ◽  
Vol 267 (2) ◽  
pp. 825-831 ◽  
Author(s):  
J C Negele ◽  
D G Dotson ◽  
W Liu ◽  
H L Sweeney ◽  
J A Putkey
Keyword(s):  
Binding Sites ◽  
Calcium Binding ◽  
Cardiac Troponin ◽  
Troponin C ◽  
High Affinity ◽  
Cardiac Troponin C ◽  
Calcium Binding Sites

Rat GTP cyclohydrolase I is a homodecameric protein complex containing high-affinity calcium-binding sites 1 1Edited by W. Baumeister

1998 ◽  
Vol 279 (1) ◽  
pp. 189-199 ◽  
Author(s):  
Michel O Steinmetz ◽  
Christoph Plüss ◽  
Urs Christen ◽  
Bettina Wolpensinger ◽  
Ariel Lustig ◽  
...  
Keyword(s):  
Protein Complex ◽  
Binding Sites ◽  
Calcium Binding ◽  
Gtp Cyclohydrolase I ◽  
Gtp Cyclohydrolase ◽  
High Affinity ◽  
Calcium Binding Sites

Generation of an agonistic binding site for blockers of the M3 muscarinic acetylcholine receptor

2008 ◽  
Vol 412 (1) ◽  
pp. 103-112 ◽  
Author(s):  
Doreen Thor ◽  
Angela Schulz ◽  
Thomas Hermsdorf ◽  
Torsten Schöneberg
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
G Protein ◽  
Binding Sites ◽  
Expression System ◽  
Muscarinic Acetylcholine Receptor ◽  
Intracellular Loop ◽  
Inverse Agonists ◽  
Yeast Expression System ◽  
High Affinity

GPCRs (G-protein-coupled receptors) exist in a spontaneous equilibrium between active and inactive conformations that are stabilized by agonists and inverse agonists respectively. Because ligand binding of agonists and inverse agonists often occurs in a competitive manner, one can assume an overlap between both binding sites. Only a few studies report mutations in GPCRs that convert receptor blockers into agonists by unknown mechanisms. Taking advantage of a genetically modified yeast strain, we screened libraries of mutant M3Rs {M3 mAChRs [muscarinic ACh (acetylcholine) receptors)]} and identified 13 mutants which could be activated by atropine (EC50 0.3–10 μM), an inverse agonist on wild-type M3R. Many of the mutations sensitizing M3R to atropine activation were located at the junction of intracellular loop 3 and helix 6, a region known to be involved in G-protein coupling. In addition to atropine, the pharmacological switch was found for other M3R blockers such as scopolamine, pirenzepine and oxybutynine. However, atropine functions as an agonist on the mutant M3R only when expressed in yeast, but not in mammalian COS-7 cells, although high-affinity ligand binding was comparable in both expression systems. Interestingly, we found that atropine still blocks carbachol-induced activation of the M3R mutants in the yeast expression system by binding at the high-affinity-binding site (Ki ∼10 nM). Our results indicate that blocker-to-agonist converting mutations enable atropine to function as both agonist and antagonist by interaction with two functionally distinct binding sites.

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