scholarly journals The binding of calcium to a salivary phosphoprotein, protein C, and comparison with calcium binding to protein A, a related salivary phosphoprotein

1977 ◽  
Vol 163 (2) ◽  
pp. 241-245 ◽  
Author(s):  
A Bennick
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Alkaline Phosphatase ◽  
Magnetic Resonance ◽  
Conformational Changes ◽  
Calcium Binding ◽  
Protein C ◽  
Equilibrium Dialysis ◽  
Protein A ◽  
Apparent Dissociation Constant ◽  
Protein Molecules

The binding of Ca2+ to a salivary phosphoprotein, protein C, was studied by equilibrium dialysis. In 5mM-Tris/HCl buffer, pH 7.5, protein C bound 190 nmol of Ca2+/mg of protein. The apparent dissociation constant, K, was determined to be 1.9 x 10(-4)M and the binding of Ca2+ to the protein was non-co-operative. The binding of Ca2+ to protein C apparently depends on groups which ionize above pH 5.0. Ca2+ binding decreased with increased concentration of the dialysis buffer and on addition of SrCL2, MgCl2 and MnCl2 to the dialysis buffer. Digestion of protein C with trypsin or collagenase or heating of the protein to 60 degrees or 100 degrees C had little or no effect on the Ca2+ binding. Digestion of protein C with alkaline phosphatase caused a decrease in the amount of protein-bound Ca2+. This was also found for another salivary phosphoprotein, protein A. In the absence of Ca2+ the S020,w for protein C was 1.29 S and in the presence of Ca2+ it was 1.46S. Ca2+ may cause a conformational change in the protein or an aggregation of the protein molecules. No conformational changes of protein C in the presence of Ca2+ could be detected by circular dichroism or nuclear magnetic resonance.

scholarly journals The binding of calcium to a salivary phosphoprotein, protein A, common to human parotid and submandibular secretions

1976 ◽  
Vol 155 (1) ◽  
pp. 163-169 ◽  
Author(s):  
A Bennick
Keyword(s):  
Calcium Binding ◽  
Protein C ◽  
Equilibrium Dialysis ◽  
Protein A ◽  
Side Chain ◽  
Carboxyl Groups ◽  
Parotid Saliva ◽  
Submandibular Saliva ◽  
Hydrolysis Of ◽  
Human Parotid Saliva

The binding of Ca2+ to a previously described phosphoprotein from human parotid saliva, protein A [Bennick (1975) Biochem J. 145, 557-567] was studied by means of equilibrium dialysis. In 5 mM-Tris/HC1 buffer, pH7.5, protein A bound 664nmol of Ca/mg of protein. Km was determined to be 181 muM and the binding of Ca2+ to the protein was non-co-operative. The binding of Ca2+ apparently occurs to side-chain carboxyl groups in the protein, but protein phosphate is of minor if any importance in calcium binding. Hydrolysis of protein A by trypsin and collagenase or heating of the protein at 60 degrees or 100 degrees C did not affect Ca2+ binding. The Ca2+ binding decreases with increased concentration of the dialysis buffer and on the addition of SrCl2, or MgCl2 or MnCl2 to the dialysis buffer. Protein A does not aggregate in the presence of Ca2+, since the s20,w was identical when determined in the presence (1.30S) and absence (1.35S) of CaCl2. By use of a specific antiserum to protein A it was found that protein C [Bennick & Connell (1971) Biochem. J. 123, 455-464] and perhaps minor related components cross-reacted with protein A. No other salivary proteins showed immunological similarity. Proteins A and C were also present in submandibular saliva. The possible functions of protein A are discussed.

scholarly journals EF-hand motifs of α, β and γ isoforms of diacylglycerol kinase bind calcium with different affinities and conformational changes

1997 ◽  
Vol 321 (1) ◽  
pp. 59-64 ◽  
Author(s):  
Keiko YAMADA ◽  
Fumio SAKANE ◽  
Norio MATSUSHIMA ◽  
Hideo KANOH
Keyword(s):  
Conformational Changes ◽  
Calcium Binding ◽  
Equilibrium Dialysis ◽  
Diacylglycerol Kinase ◽  
Intrinsic Properties ◽  
Apparent Dissociation Constant ◽  
Ef Hand ◽  
Order Of Magnitude ◽  
Hydrophobic Amino Acids ◽  
Ef Hands

The three diacylglycerol kinase isoenzymes (DGKα, DGKβ and DGKγ) cloned so far contain in common a tandem repeat of EF-hand motifs. However, the Ca2+ dependences of the DGK activities are known to be variable between isoenzymes, and the Ca2+-binding activities of these motifs have not been tested except for those present in DGKα. We therefore attempted to define the intrinsic properties of EF-hands occurring in the DGK isoenzymes. For this purpose we bacterially expressed and purified the EF-hand motifs (termed DKE forms) of the three DGKs. Equilibrium dialysis with the purified DKE forms showed that all of the expressed proteins could bind approx. 2 mol of Ca2+ per mol. However, the apparent dissociation constant (Kd) for calcium binding to α-DKE (9.9 µM) was an order of magnitude greater than those estimated for β-DKE (0.89 µM) and γ-DKE (0.40 µM). Experiments with 2-p-toluidinylnaphthalene 6-sulphonate, a probe for hydrophobic regions of proteins, showed that the binding of Ca2+ to β-DKE resulted in the exposure of hydrophobic amino acids, whereas hydrophobic regions of α-DKE and γ-DKE were masked by the addition of Ca2+. Taken together, these results indicate that DGKα, DGKβ and DGKγ possess EF-hand structures with intrinsic properties different from each other with respect to affinities for Ca2+ and Ca2+-induced conformational changes.

Calcium ion binding to clinically relevant chemical modifications of human serum albumin

1995 ◽  
Vol 41 (11) ◽  
pp. 1654-1661 ◽  
Author(s):  
H Vorum ◽  
K Fisker ◽  
M Otagiri ◽  
A O Pedersen ◽  
U Kragh-Hansen
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Conformational Changes ◽  
Calcium Binding ◽  
Equilibrium Dialysis ◽  
Thiol Group ◽  
Protein Molecule ◽  
Penicillin G ◽  
Lysine Residues

Abstract Calcium binding to glycated, penicilloylated, acetylated, and normal defatted human serum albumin as well as to mercapt- and nonmercaptalbumin was studied by equilibrium dialysis of radioactive Ca2+. Binding was quantified by five Scatchard constants [ni = 1, (i = 1-4) and n5 = 10]. Glycation resulted in increased k1- and k2-values and unchanged k3-k5-values, whereas penicilloylation increased all five association constants. The increments were greater the more pronounced the modification, and the enhancements caused by penicilloylation were, for the same degree of modification, greater than those produced by glycation. In contrast, acetylation by acetylsalicylate did not affect calcium binding. Likewise, binding to mercapt- and nonmercaptalbumin was the same, a finding showing that the thiol group of cysteine 34 is not important for calcium binding. D-Glucose and penicillin G are known to react with lysine residues of albumin, and the enhancement of binding resulting from glycation or penicilloylation is probably brought about by unspecific electrostatic effects, possibly supplemented by conformational changes of the protein molecule. The relative importance of the three domains of human serum albumin for calcium binding is discussed.

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