scholarly journals Calcium-binding constants of trypsin and trypsinogen. A reassessment

1981 ◽  
Vol 193 (2) ◽  
pp. 655-658 ◽  
Author(s):  
S G R Cliffe ◽  
D A W Grant
Keyword(s):  
Affinity Chromatography ◽  
Calcium Binding ◽  
Cation Exchanger ◽  
Binding Constants ◽  
High Affinity ◽  
High Affinity Site ◽  
Logarithmic Unit

The Ca2+-binding constants for trypsin and trypsinogen have been reassessed by using enzyme that has been purified by affinity chromatography and measuring the distribution of 45Ca2+ between the protein and a cation exchanger. The pKCa2+ value of 4.5 for the high-affinity site on trypsin was 1 logarithmic unit greater than that previously reported.

scholarly journals The interaction of cations with lipopolysaccharide from Escherichia coli C as shown by measurement of binding constants and aggregation reactions

1989 ◽  
Vol 263 (3) ◽  
pp. 695-702 ◽  
Author(s):  
A M Field ◽  
E Rowatt ◽  
R J P Williams
Keyword(s):  
Escherichia Coli ◽  
Sodium Salt ◽  
Binding Constants ◽  
Assay Method ◽  
Aggregation State ◽  
High Affinity ◽  
A Value ◽  
High Affinity Site ◽  
Low Ionic Strength ◽  
Very High

Lipopolysaccharide from Escherichia coli C interacts with polyvalent cations at low ionic strength at more than one site. The first site has high affinity with a KD value of 10(-8) M for Ca2+ and even stronger binding for [(NH3)5CoNH2Co(NH3)5]5+ and La3+. The high-affinity site for the latter cations is beyond the sensitivity of the assay method. The second, low-affinity, site for bivalent cations has a Km of 10(-3) M, whereas, for tervalent and quinquevalent metal cations and spermine and hexacyclen (1,4,7,10,13,16-hexa-azacyclo-octadecane), this constant has a value of 10(-5) M. Binding of cations to the high-affinity site does not alter the aggregation state of the lipopolysaccharide, but combination with the low-affinity site gives particles twice the size of those of the sodium salt. Very high Ca2+ concentrations (30 mM) give particles eight times the size of those of the sodium salt.

scholarly journals Binding to antithrombin of heparin fractions with different molecular weights

1981 ◽  
Vol 193 (2) ◽  
pp. 427-433 ◽  
Author(s):  
Ȧke Danielsson ◽  
Ingemar Björk
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Binding Constants ◽  
Gel Chromatography ◽  
Molecular Weights ◽  
High Affinity ◽  
Heparin Fractions ◽  
Fluorescence Titrations ◽  
In The Beginning ◽  
Difference Absorption

The interaction between bovine antithrombin, a plasma proteinase inhibitor, and heparin species of different molecular weights was studied. A commercial heparin preparation was divided by gel chromatography into a number of fractions with average molecular weights ranging from 6000 to 34700. Each of these fractions was further fractionated by affinity chromatography on matrix-bound antithrombin. In the latter procedure, those heparin fractions that had molecular weights lower than about 14000 were separated into three peaks. The material in the first of these was not adsorbed on the column, and the other two peaks corresponded to the low-affinity and high-affinity peaks described previously. In contrast, high-molecular-weight heparin samples gave only the low-affinity and high-affinity fractions. U.v. difference absorption studies showed that the non-adsorbed heparin fraction bound to antithrombin in solution with a binding constant at physiological ionic strength only slightly lower than that of low-affinity heparin. The division between the two fractions thus is arbitrary and only dependent on the conditions selected for the affinity-chromatography experiment. Stoicheiometries and binding constants for the binding of several high-affinity heparin species to antithrombin were determined by fluorescence titrations. High-affinity heparin fractions of equal elution positions in the beginning of the peaks of the affinity chromatographies, but with different molecular weights, showed stoicheiometries that were not experimentally distinguishable from 1:1 and also had no appreciable differences in binding constants. However, the anticoagulant activities, calculated on a molar basis, of these fractions increased markedly with molecular weight, a behaviour that thus cannot be explained by differences in the binding of the fractions to antithrombin. In contrast, high-affinity samples of similar molecular weights, which were eluted at increasing ionic strengths from matrix-linked antithrombin, were found to have an increasing proportion of chains with two binding sites for antithrombin and also to have progressively higher binding constants. These binding properties at least partly explain the increasing anticoagulant activities that were observed for these fractions.

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

1982 ◽  
Vol 208 (3) ◽  
pp. 773-781 ◽  
Author(s):  
A Miller ◽  
S T Li ◽  
F Bronner
Keyword(s):  
Brush Border ◽  
Binding Sites ◽  
Calcium Binding ◽  
Specific Binding ◽  
Relative Effectiveness ◽  
Brush Border Membranes ◽  
High Affinity ◽  
High Affinity Site ◽  
Rat Duodenum ◽  
High Concentrations

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.

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
Sign in / Sign up

Export Citation Format

Share Document