Interaction of cytochrome c with reaction centers of Rhodopseudomonas sphaeroides R-26: determination of number of binding sites and dissociation constants by equilibrium dialysis

Biochemistry ◽  
1980 ◽  
Vol 19 (25) ◽  
pp. 5687-5692 ◽  
Author(s):  
D. Rosen ◽  
M. Y. Okamura ◽  
G. Feher
Keyword(s):  
Cytochrome C ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Equilibrium Dialysis ◽  
Reaction Centers ◽  
Rhodopseudomonas Sphaeroides ◽  
Number Of Binding Sites

scholarly journals Interaction of rice (Oryza sativa) lectin with N-acetylglucosaminides. Fluorescence studies

1985 ◽  
Vol 229 (3) ◽  
pp. 687-692 ◽  
Author(s):  
F Tabary ◽  
J P Frénoy
Keyword(s):  
Oryza Sativa ◽  
Binding Sites ◽  
Wheat Germ ◽  
Equilibrium Dialysis ◽  
Blue Shift ◽  
Association Constants ◽  
Fluorescence Studies ◽  
Saccharide Binding ◽  
Binding Enthalpy

The interaction of lectin isolated from rice (Oryza sativa) embryos with N-acetylglucosaminides was studied by equilibrium dialysis and fluorescence. Equilibrium dialysis with 4-methylumbelliferyl-(GlcNac)2 showed that rice lectin (Mr 38000) contains four equivalent saccharide-binding sites. Addition of the N-acetylglucosaminides GlcNac, (GlcNac)2 and (GlcNac)3 enhanced the intrinsic fluorescence of rice lectin and this was accompanied by a 10nm blue-shift of its maximum fluorescence with (GlcNac)2 and (GlcNac)3. These changes in intensity allowed determination of the association constants, which increased with the number of saccharide units: at 20 degrees C, Ka = (1.3 +/- 0.1) X 10(3), (5.1 +/- 0.4) X 10(4) and (2.6 +/- 0.1) X 10(5) M−1 for GlcNac, (GlcNac)2 and (GlcNac)3 respectively. The binding enthalpy, delta H0, for the three glucosaminides were very low and ranged from −12.1 to −20.6 kJ X mol-1. The results are compared with those obtained with wheat-germ agglutinin, another GlcNac-specific gramineaous lectin.

Adrenergic receptors on cerebral microvessels: pericyte contribution

1989 ◽  
Vol 256 (1) ◽  
pp. R224-R230 ◽  
Author(s):  
R. M. Elfont ◽  
P. R. Sundaresan ◽  
C. D. Sladek
Keyword(s):  
Endothelial Cells ◽  
Binding Sites ◽  
Adrenergic Receptors ◽  
Radioligand Binding ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Binding Studies ◽  
Cerebral Microvessels ◽  
Beta Adrenoceptor ◽  
Number Of Binding Sites

R224-R230, 1989.--[125I]iodocyanopindolol ([125I]ICYP) and [3H]rauwolscine were used to quantitate, respectively, the beta- and alpha 2-adrenergic receptors in freshly isolated bovine cerebral microvessels and in pericyte cultures derived from these microvessels. Morphological and immunocytochemical criteria distinguished the pericytes from endothelial cells. Competitive binding studies established the specificity of the radioligand binding. The maximal number of binding sites (Bmax) for [125I]ICYP in the pericytes constituted only 8% of that in the microvessels (3.5 +/- 1.3 vs. 44.4 +/- 6.6 fmol/mg protein). In contrast, the Bmax for [3H]rauwolscine in the pericytes was 50% of that in the microvessels (55.4 +/- 11.8 vs. 111.1 +/- 9.5 fmol/mg protein). The dissociation constants for both [125I]ICYP and [3H]rauwolscine were similar in the two preparations. No alpha 1-adrenergic receptors, as defined by the specific binding of [3H]prazosin, were identified either in the pericytes or microvessels. Overall, our results suggest that pericytes contribute minimally to the total beta-adrenoceptor number of cerebral microvessels, and thus the beta-adrenoceptors must be located predominantly on endothelial cells. However, the contribution of pericytes to the total alpha 2-adrenoceptor number of the microvessels may be substantial.

scholarly journals Fluorescence-polarization studies on binding of 4-methylumbelliferyl β-d-galactopyranoside to Ricinus communis (castor-bean) agglutinin

1980 ◽  
Vol 191 (2) ◽  
pp. 395-400 ◽  
Author(s):  
M I Khan ◽  
M K Mathew ◽  
P Balaram ◽  
A Surolia
Keyword(s):  
Binding Sites ◽  
Fluorescence Polarization ◽  
Castor Bean ◽  
Ricinus Communis ◽  
Equilibrium Dialysis ◽  
Scatchard Analysis ◽  
Polarization Data ◽  
Appreciable Change ◽  
Number Of Binding Sites ◽  
Polarization Studies

The binding of Ricinus communis (castor-bean) agglutinin 1 to saccharides was studied by equilibrium dialysis and fluorescence polarization by using the fluorescently labelled sugar 4-methylumbelliferyl beta-D-galactopyranoside. No appreciable change in ligand fluorescence of 4-methylumbelliferyl beta-D-galactopyranoside was considerably polarized on its binding to the lectin. The association constants obtained by Scatchard analysis of equilibrium-dialysis and fluorescence-polarization data do not differ much from each other, and at 25 degrees C, Ka = 2.4 (+/- 0.2) X 10(4)M-1. These values agree reasonably well with that reported in the literature for Ricinus agglutinin 1. The number of binding sites obtained by the different experimental procedures is 1.94 +/- 0.1 per molecule of 120 000 daltons and is equal to the reported value of 2. The consistency in the values of Ka and number of binding sites indicate the absence of additional subsites on Ricinus agglutinin 1 for its specific sugars. In addition, the excellent agreement between the binding parameters obtained by equilibrium dialysis and fluorescence polarization indicate the potential of ligand-fluorescence-polarization measurements in the investigation of lectin-sugar interactions.

Equilibrium dialysis, ultrafiltration, and ultracentrifugation compared for determining the plasma-protein-binding characteristics of valproic acid.

1985 ◽  
Vol 31 (1) ◽  
pp. 60-64 ◽  
Author(s):  
J Barré ◽  
J M Chamouard ◽  
G Houin ◽  
J P Tillement
Keyword(s):  
Valproic Acid ◽  
Human Serum Albumin ◽  
Binding Sites ◽  
Plasma Proteins ◽  
Equilibrium Dialysis ◽  
Anticonvulsant Drug ◽  
Drug Binding ◽  
Binding Characteristics ◽  
Affinity Constants ◽  
Number Of Binding Sites

Abstract Equilibrium dialysis, ultrafiltration, and ultracentrifugation were compared to determine their reliability and applicability in the study of binding of an anticonvulsant drug, valproic acid, by plasma proteins. We studied drug binding with pooled serum and with solutions of human serum albumin at physiological concentrations. We compared binding characteristics such as number of binding sites, affinity constants, and percent of binding as measured by each method in the therapeutic range for valproic acid. Results by ultracentrifugation differed from those by equilibrium dialysis and ultrafiltration, which agreed reasonably well with each other.

Binding of Ca++ to Human Prothrombin

1975 ◽  
Author(s):  
R. Benarous ◽  
J. Elion
Keyword(s):  
Binding Sites ◽  
Equilibrium Dialysis ◽  
Specific Property ◽  
Binding Properties ◽  
Binding Ability ◽  
Ph Variation ◽  
Maximum Value ◽  
Number Of Binding Sites ◽  
Scatchard Plots

The Ca++ binding properties of human prothrombin were studied by equilibrium dialysis using 45 calcium chloride at +4° C with prothrombin concentration of about 1 mg/ml equilibrated in 0.025 M Tris HCl, 0.12 M NaCl buffer pH 7.4. Scatchard plots obtained were similar to those described by Steenflo (1973) for bovine prothrombin, suggesting a positive cooperativity in the binding of Ca++ with a maximum ratio of bound Ca++/free Ca++ of 3 moles of Ca++ bound per mole of protein.The total number of binding sites was found to be at about 7, less than 10 to 12 found for bovine prothrombin. Ca++ binding was dependent on pH variation of the buffer with a maximum value for pH 8.5. Chemical modifications of carboxyl groups of prothrombin according to Hoare and Koshland (1967) abolished the Ca++ binding ability of the molecule confirming the essential role of these residues in this specific property of prothrombin.

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