Biostructural chemistry of magnesium ion: characterization of the weak binding sites on tRNAPhe(yeast). Implications for conformational change and activity

Biochemistry ◽  
1990 ◽  
Vol 29 (25) ◽  
pp. 6025-6032 ◽  
Author(s):  
Susan S. Reid ◽  
James A. Cowan
Keyword(s):  
Conformational Change ◽  
Binding Sites ◽  
Magnesium Ion ◽  
Weak Binding

Isolation And Characterization Of The Vitamin K Dependent Domain Of Human Prothrombin

1981 ◽  
Author(s):  
C Dodé ◽  
A Thiesce ◽  
D Labie ◽  
J Elion
Keyword(s):  
Conformational Change ◽  
Vitamin K ◽  
Disulfide Bond ◽  
Binding Sites ◽  
Terminal Portion ◽  
Isolation And Characterization ◽  
Cooperative Process ◽  
Dependent Domain ◽  
Stimulation Of

Chymotryptic cleavage of human prothrombin (hPt) produces two fragments of 68000 and 5000 MW respectively. These two species were separated by Ba citrate adsorption and Sephadex G100 chromatography. The 68000 MW species corresponds to hPt (des 1-44) and has lost all the vitamin K dependent properties of hPt : adsorption on Ba citrate, Ca+2 and phospholipid dependent stimulation of the activation by FXa, presence of strong Ca+2 binding sites (as shown by dialysis equilibrium) and Ca+2 induced fluorescence quenshing. The 5000 MW species corresponds to the N-terminal portion of hPt (residues 1-41). It contains the 10 Gla residues and the 17-22 disulfide bond. This peptide is quantitatively adsorbed on Ba citrate. Activation of hPt in a mixture containing FXa, Ca+2 and phospholipid is drastically inhibited by the addition of peptide 1-41 (≃ 50 % inhibition for a 1/1 peptide/Pt ratio, ≃ 7 % for a 40/1 ratio).Ca+2 produces a quenshing of the intrinsic fluorescence of peptide 1-41 (λ emission = 355 nm). This quenshing plateausat 40 % of the initial fluorescence at 3 mM Ca+2. The plot of the fluorescence quenshing versus Ca+2 concentration however is not cooperative. Mn+2 and Mg+2 also induced fluorescence quenshing but to a lesser extent. Hence peptide 1-44 represents a functionnal domain in itself interacting with Ca+2 and phospholipid. It contains only one Trp (residue 41), showing directly the involvment of this residue in the Ca+2 induced fluorescence quenshing observed for Pt fragment (FI) and Pt. This isolated vitamin K dependent domain therefore retains many of the vitamin K dependent properties of FI or Pt, but shows differences in the Ca+2 induced conformational change in that it is no longer a cooperative process.

Characterization of a Mode of Specific Binding of Fibrin Monomer through its Amino-Terminal Domain by Macrophages and Macrophage Cell-Lines

1990 ◽  
Vol 63 (02) ◽  
pp. 193-203 ◽  
Author(s):  
John R Shainoff ◽  
Deborah J Stearns ◽  
Patricia M DiBello ◽  
Youko Hishikawa-Itoh
Keyword(s):  
Peritoneal Macrophages ◽  
Affinity Binding ◽  
Macrophage Cell ◽  
High Affinity Binding ◽  
Amino Terminal ◽  
High Affinity ◽  
Terminal Domain ◽  
Weak Binding ◽  
Amino Terminal Domain

SummaryThe studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (α-fibrin) or both fibrinopeptides A and B (αβ-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX ≃ 200–800 molecules/cell, KD ≃ 10−12 M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX ≥ 105 molecules/cell, KD ≥ 10−6 M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD ≃ 10−10 M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (β-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the α- and the β-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with elec-trophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

Characterization of Thromboxane A2/Prostaglandin H2 Receptors in Porcine Coronary Artery -The Inhibitory Effect of a Novel Dibenzoxepin Derivative, KW-3635

1992 ◽  
Vol 67 (05) ◽  
pp. 582-584 ◽  
Author(s):  
Ichiro Miki ◽  
Akio Ishii
Keyword(s):  
Coronary Artery ◽  
Binding Sites ◽  
Thromboxane A2 ◽  
Inhibitory Effect ◽  
Scatchard Analysis ◽  
Single Class ◽  
Porcine Coronary Artery ◽  
Equilibrium Binding ◽  
H2 Receptors

SummaryWe characterized the thromboxane A2/prostaglandin H2 receptors in porcine coronary artery. The binding of [3H]SQ 29,548, a thromboxane A2 antagonist, to coronary arterial membranes was saturable and displaceable. Scatchard analysis of equilibrium binding showed a single class of high affinity binding sites with a dissociation constant of 18.5 ±1.0 nM and the maximum binding of 80.7 ± 5.2 fmol/mg protein. [3H]SQ 29,548 binding was concentration-dependently inhibited by thromboxane A2 antagonists such as SQ 29,548, BM13505 and BM13177 or the thromboxane A2 agonists such as U46619 and U44069. KW-3635, a novel dibenzoxepin derivative, concentration-dependently inhibited the [3H]SQ 29,548 binding to thromboxane A2/prosta-glandin H2 receptors in coronary artery with an inhibition constant of 6.0 ± 0.69 nM (mean ± S.E.M.).

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