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

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.

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The High Affinity Calcium-binding Sites in the Epidermal Growth Factor Module Region of Vitamin K-dependent Protein S

Journal of Biological Chemistry ◽

10.1074/jbc.272.37.23255 ◽

1997 ◽

Vol 272 (37) ◽

pp. 23255-23260 ◽

Cited By ~ 27

Author(s):

Yvonne Stenberg ◽

Sara Linse ◽

Torbjörn Drakenberg ◽

Johan Stenflo

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Binding Sites ◽

Calcium Binding ◽

Protein S ◽

High Affinity ◽

Factor Module ◽

Dependent Protein ◽

Epidermal Growth ◽

Calcium Binding Sites

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Two calcium-binding sites mediate the interconversion of liver inositol 1,4,5-trisphosphate receptors between three conformational states

Biochemical Journal ◽

10.1042/bj3010591 ◽

1994 ◽

Vol 301 (2) ◽

pp. 591-598 ◽

Cited By ~ 55

Author(s):

I C Marshall ◽

C W Taylor

Keyword(s):

Binding Sites ◽

Calcium Binding ◽

Regulatory Proteins ◽

Conformational States ◽

High Affinity ◽

Bivalent Cations ◽

Inositol 1,4,5 Trisphosphate ◽

Liver Membranes ◽

Calcium Binding Sites

Cytosolic Ca2+ biphasically regulates Ins(1,4,5)P3-stimulated Ca2+ mobilization in liver [Marshall and Taylor (1993) J. Biol. Chem. 268, 13214-13220]. We have investigated the mechanisms underlying this biphasic control of Ca2+ mobilization in permeabilized hepatocytes by comparing the effects of Sr2+, Ba2+ and Ca2+ on the liver Ins(1,4,5)P3 receptor. Both Ca2+ and Sr2+ increased the binding of [3H]Ins(1,4,5)P3 to liver membranes by converting receptors from a low-affinity (KD approximately 35 nM) to a high-affinity (KD approximately 5 nM) state. Ba2+ (< or = 20 microM) did not affect [3H]Ins(1,4,5)P3 binding. At concentrations similar to those that caused an enhancement of [3H]Ins(1,4,5)P3 binding, Sr2+ (EC50 = 570 nM) and Ca2+ (EC50 = 200 nM) increased the sensitivity of the intracellular Ca2+ stores to Ins(1,4,5)P3. Further modest elevations in [Ca2+] (EC50 = 1.5 microM) inhibited Ins(1,4,5)P3-stimulated Ca2+ mobilization, whereas Sr2+ caused inhibition only when its concentration was very substantially increased (EC50 approximately 900 microM). Sr2+ is therefore only 3-fold less potent than Ca2+ in causing sensitization of Ins(1,4,5)P3-stimulated Ca2+ release, but 600-fold less potent in causing inhibition. Ba2+ neither sensitized ([Ba2+] < or = 20 microM) nor inhibited ([Ba2+] < or = 1 mM) Ins(1,4,5)P3-stimulated Ca2+ release, and did not inhibit either the sensitization of Ca2+ release evoked by Sr2+ or the inhibition of Ca2+ release evoked by Ca2+. Our results suggest that two distinct Ca(2+)-binding sites, which differ in their selectivities for bivalent cations, mediate the interconversion of Ins(1,4,5)P3 receptors between at least three different conformational states. These two Ca(2+)-binding sites, which may reside either on the Ins(1,4,5)P3 receptor itself or on distinct regulatory proteins, can be distinguished by their different selectivities for bivalent cations.

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STRUCTURE AND FUNCTION OF VITAMIN K-DEPENDENT PROTEIN S, a cofactor to activated protein C which also interacts with the complement protein C4b-binding protein

10.1055/s-0038-1642960 ◽

1987 ◽

Author(s):

Bjorn Dahiback ◽

Ake Lundwall ◽

Andreas Hillarp ◽

Johan Malm ◽

Johan Stenflo

Keyword(s):

Binding Sites ◽

Calcium Binding ◽

Activated Protein C ◽

Protein S ◽

Central Core ◽

Complement Protein ◽

High Affinity ◽

Calcium Binding Sites ◽

Gla Domain ◽

Cofactor Activity

Protein S is a single chain (Mr 75.000) plasma protein. It is a cofactor to activated protein C (APC) in the regulation of coagulation factors Va and Villa. It has high affinity for negatively charged phospolipids and it forms a 1:1 complex with APC on phospholipid surfaces, platelets and on endothelial cells. Patients with heterozygous protein S deficiency have a high incidence of thrombosis. Protein S is cleaved by thrombin, which leads to a loss of calcium binding sites and of APC cofactor activity. Protein S has two to three high affinity (KD 20uM) calcium binding sites - unrelated to the Gla-region - that are unaffected by the thrombin cleavage. In human plasma protein S (25 mg/liter) circulates in two forms; free (approx. 40%) and in a 1:1 noncovalent complex (KD 1× 10-7M) with the complement protein C4b-binding protein (C4BP). C4BP (Mr 570.000) is composed of seven identical 70 kDa subunits that are linked by disulfide bonds. When visualized by electron microscopy, C4BP has a spiderlike structure with the single protein S binding site located close to the central core and one C4b-binding site on each of the seven tentacles. When bound to C4BP, protein S looses its APC cofactor activity, whereas the function-of C4BP is not directly affected by the protein S binding. Chymotrypsin cleaves each of the seven C4BP subunits close to the central core which results in the liberation of multiple 48 kDa “tentacte” fragments and the formation of a 160 kDa central core fragment. We have successfully isolated a 160 kDa central core fragment with essentially intact protein S binding ability.The primary structure of both bovine and human protein S has been determined and found to contain 635 and 634 amino acids, respectively, with 82 % homology to each other. Four different regions were distinguished; the N-terminal Gla-domain (position 1-45) was followed by a region which has two thrombin-sensitive bonds positioned within a disulfide loop. Position 76 to 244 was occupied by four repeats homologous to the epidermal growth factor (EGF) precursor. In the first EGF-domain a modified aspartic acid was identified at position 95, B-hydroxaspartic acid (Hya), and in corresponding positions in the three following EGF-domains (positions 136,178 and 217) we found B-hydroxyasparagine (Hyn). Hyn has not previously been identified in proteins. The C-terminal half of protein S (from position 245) shows no homology to the serine proteases but instead to human Sexual Hormon Binding Globulin (SHBG)(see separate abstract). To study the structure-function relationship we made eighteen monoclonal antibodies to human protein S. The effects of the monoclonals on the C4BP-protein S interaction and on the APC cofactor activity were analysed. Eight of the antibodies were calciumdependent, four of these were against the Gla-domain, two against the thrombin sensitive portion and two against the region bearing the high affinity calcium binding sites. Three of the monoclonals were dependent on the presence of chelating agents, EDTA or EGTA, and were probably directed against the high affinity calcium binding region. Three other monoclonals inhibited the protein S-C4BP interaction. At present, efforts are made to localize the epitopes to gain information about functionally important regions of protein S.

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Dependence on Membrane Lipids of the Effect of Vanadate on Calcium and ATP Binding to Sarcoplasmic Reticulum ATPase

Zeitschrift für Naturforschung C ◽

10.1515/znc-1984-11-1224 ◽

1984 ◽

Vol 39 (11-12) ◽

pp. 1137-1140 ◽

Cited By ~ 2

Author(s):

Pankaj Medda ◽

Wilhelm Hasselbach

Keyword(s):

Sarcoplasmic Reticulum ◽

Binding Sites ◽

Calcium Binding ◽

Membrane Lipids ◽

Nucleotide Binding ◽

Atp Binding ◽

Transport Atpase ◽

Nucleotide Binding Sites ◽

High Affinity ◽

Calcium Binding Sites

Abstract The affinity of the sarcoplasmic reticulum transport ATPase for calcium and ATP is not affected by lipid depriviation while vanadate binding is completely abolished. Lipid substitution restores vanadate binding as well as the vanadate induced disappearance of the enzyme’s high affinity calcium and nucleotide binding sites. Nucleotide binding is simultaneously restored with the displacement of vanadate from the enzyme following the occupation of its low affinity calcium binding sites.

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