scholarly journals Reversible association of half-molecules of ovotransferrin in solution. Basis of co-operative binding to reticulocytes

1987 ◽  
Vol 245 (1) ◽  
pp. 103-109 ◽  
Author(s):  
A Brown-Mason ◽  
S A Brown ◽  
N D Butcher ◽  
R C Woodworth
Keyword(s):  
Chick Embryo ◽  
Red Blood Cells ◽  
Dissociation Constant ◽  
Blood Cells ◽  
Gel Filtration ◽  
Free Ligand ◽  
Least Squares Regression ◽  
Binding Studies ◽  
Apparent Dissociation Constant

In the present paper, gel-filtration studies of diferric-ovotransferrin (Fe2OTf), the individual half-molecules of ovotransferrin (OTf) and equimolar mixtures of half-molecules have been interpreted according to the Gilbert theory as developed by Ackers & Thompson [(1965) Proc. Natl. Acad. Sci. U.S.A. 53, 342-349]. The data indicate that the half-molecules associate reversibly in solution and allow determination of a dissociation constant, Kd' = 8.0 (+/- 2.7) microM. Equilibrium binding studies have been performed using NH4Cl to block removal of iron from equimolar differentially iodine-labelled half-molecules (125I and 131I), in order to evaluate the binding of each to chick-embryo red blood cells under identical conditions. The amount of associated half-molecules over a range of concentrations has been calculated using the constant derived from the gel-filtration experiments described above. A computerized non-linear least-squares regression analysis of the data leads to determination of Kd* (the apparent dissociation constant for the interaction between OTf or half-molecules and the transferrin (Tf) receptors of chick-embryo red blood cells) and Bmax (binding at infinite free-ligand concentration) for the half-molecules similar to those found for Fe2OTf. Recent reports confirm that the two iron-binding domains of both OTf and human lactotransferrin associate non-covalently in solution. Our work shows that the isolated half-molecules of OTf are able to reassociate in solution and that this reassociation has functional significance by allowing the complex to be recognized by the Tf receptor.

scholarly journals Differential effect of iodination of ovotransferrin and its two half-molecule domains on binding to transferrin receptors on chick embryo red blood cells

1987 ◽  
Vol 247 (2) ◽  
pp. 417-425 ◽  
Author(s):  
A B Mason ◽  
S A Brown
Keyword(s):  
Chick Embryo ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Solid Phase ◽  
Free Ligand ◽  
Transferrin Receptors ◽  
Apparent Dissociation Constant ◽  
Receptor Recognition ◽  
Chloramine T ◽  
Peptide Maps

Iodination of the C-terminal half-molecule domain of ovotransferrin (OTF) causes a significant reduction in binding to transferrin receptors on chick reticulocytes when compared to the binding observed with holo-OTF or the N-terminal half-molecule domain. (In such studies binding of iodinated half-molecule is measured in the presence of equimolar unlabelled complementary half-molecule). In particular iodination of the C-terminal half-molecule domain by the solid-phase reagent Iodogen resulted in half the binding found when ICl was used. The iodinated N-terminal half-molecule domain labelled by either Iodogen or ICl showed consistently higher binding than was observed with the C-terminal half-molecule or Fe2OTF. Although the molecular basis for the reduced binding of these proteins relative to the N-terminal half-molecule has not been definitively established, the implication is that there is a Tyr in the C-terminal domain which is involved in receptor recognition and binding. Addition of one or more bulky iodine atoms to the Tyr interferes with the interaction. Tryptic peptide maps of unlabelled holo-OTF and half-molecule domains and of the half-molecule domains labelled by both ICl and Iodogen are presented. The maps indicate limited access of the tyrosine residues to iodination especially in the C-terminal half-molecule domain. Equilibrium binding experiments have been carried out to compare the Kd (the apparent dissociation constant for the interaction between OTF and the transferrin receptors on chick-embryo red blood cells) with the Bmax, (binding at infinite free-ligand concentration) for Fe2OTF labelled using ICl, Iodogen, Enzymobeads and Chloramine-T. The effect of labelling Fe2OTF by Bolton-Hunter reagent has also been assessed. These studies show that ICl appears to be the reagent of choice for labelling Fe2OTF and its half-molecule domains.

Chloride (or bicarbonate)-dependent copper uptake through the anion exchanger in human red blood cells

1990 ◽  
Vol 259 (4) ◽  
pp. C570-C576 ◽  
Author(s):  
J. O. Alda ◽  
R. Garay
Keyword(s):  
Red Blood Cells ◽  
Dissociation Constant ◽  
Anion Exchanger ◽  
Blood Cells ◽  
Initial Rate ◽  
Disulfonic Acid ◽  
Copper Uptake ◽  
Selective Electrode ◽  
Apparent Dissociation Constant ◽  
Human Red Blood Cells

The initial rate of Cu2+ uptake in human red blood cells was measured by atomic absorption. About 80% of Cu2+ uptake was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) concentrations greater than 5-10 microM. DIDS-sensitive Cu2+ uptake required the presence of external HCO3- or external Cl-. Cl- strongly stimulated Cu2+ uptake following a Michaelis-like function, with apparent dissociation constant (KCl) of 72 +/- 9.4 (SD) mM (n = 6 experiments). HCO3- stimulated DIDS-sensitive Cu2+ uptake following a Michaelis-like function, with apparent dissociation constant (Kbic) of 10 +/- 1.9 (SD) mM (n = 4 experiments). Maximal rates (of Cl(-)- or HCO3(-)-stimulated Cu2+ uptake) were nonadditive. DIDS-sensitive Cu2+ uptake was not modified by physiological concentrations of phosphate or sulfate. Conversely, it was strongly inhibited by physiological concentrations of L-histidine and cysteine (at a Cu2+ concentration of 100 microM, these physiological ligands exhibited KHis and KCys of 50 and 80 microM, respectively). By using a copper-selective electrode, we found that at pH 7-7.4 copper is associated with OH-, particularly in the form of Cu(OH)2 complexes. In conclusion, the anion exchanger is the major transport mechanism for red blood cell Cu2+ uptake. The translocating species can be the monovalent anion complexes of copper with OH-, Cl-, and/or HCO3-.

Measurement of hemoglobin oxygen saturation in capillaries

1987 ◽  
Vol 252 (5) ◽  
pp. H1031-H1040 ◽  
Author(s):  
M. L. Ellsworth ◽  
R. N. Pittman ◽  
C. G. Ellis
Keyword(s):  
Light Intensity ◽  
Oxygen Saturation ◽  
Red Blood Cells ◽  
Optical Density ◽  
Blood Cells ◽  
Striated Muscle ◽  
Cheek Pouch ◽  
Retractor Muscle ◽  
Hamster Cheek Pouch

We present a computer-aided videodensitometric method for the determination of oxygen saturation in red blood cells flowing through capillaries of the hamster cheek pouch retractor muscle. The optical density (OD) of red blood cells is determined at two wavelengths. At the first, 431 nm, there is a maximum difference between absorption by oxygen deoxyhemoglobin. At the second, 420 nm, absorption is equal for the two absorbing species (isosbestic wavelength). In capillaries of the retractor muscle a relationship between oxygen saturation (S) and the following OD ratio was obtained as S = -1.71 (OD431/OD420) + 2.20. The error (95% confidence interval) in oxygen saturation associated with a determination of the OD ratio is estimated to be +/- 4.8%. The computerization of the method employs a frame-by-frame analysis of the light intensity over a selected capillary segment. The light intensity waveform along the segment is digitized and the minimum (I) and maximum (I0) light intensities are used to compute an optical density (OD = log10 [I0/I]). These minimum and maximum intensities correspond to the presence and absence of a red blood cell, respectively. The method permits the off-line analysis of videotaped scenes and provides a means of assessing the extent of temporal and spatial heterogeneity of oxygen saturation in selected capillary networks. The method has been developed for use in capillaries in transilluminated striated muscle but should be generally applicable to the measurement of capillary oxygen saturation in other tissues.

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