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

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.

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

Biochemical Journal ◽

10.1042/bj2450103 ◽

1987 ◽

Vol 245 (1) ◽

pp. 103-109 ◽

Cited By ~ 13

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.

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Binding of various ovotransferrin fragments to chick-embryo red cells

Biochemical Journal ◽

10.1042/bj2570301 ◽

1989 ◽

Vol 257 (1) ◽

pp. 301-304 ◽

Cited By ~ 6

Author(s):

A Oratore ◽

G D'Andrea ◽

K Moreton ◽

J Williams

Keyword(s):

Amino Acid ◽

Chick Embryo ◽

Red Blood Cells ◽

Receptor Binding ◽

Blood Cells ◽

Tight Binding ◽

Red Cells ◽

Transferrin Receptors ◽

Amino Acid Residues

1. The ability of N- and C-terminal half-molecule fragments of hen ovotransferrin to interact with chick red blood cells (CERBC) has been studied under conditions that allow binding of the transferrin to transferrin receptors to take place, but not the delivery of iron to the cell. Two kinds of half-molecule fragments were used: (a) those which can associate with one another to give a dimer resembling native transferrin and (b) those which cannot associate in this way because they lack a few amino acid residues from their C-terminal ends. 2. Neither N nor C half-molecules alone can bind to the CERBC, but, when both are present, tight binding occurs. 3. Whether or not the half-molecules can associate with one another makes little difference to receptor binding. 4. Given that one of the half-molecules is iron-saturated, the presence or absence of iron in the contralateral half-molecule again makes little difference to receptor binding.

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Influence of Heat Stress on Development of Chick Embryo (in ovo)

Journal of Biotechnology Research Center ◽

10.24126/jobrc.2012.6.1.191 ◽

2012 ◽

Vol 6 (1) ◽

pp. 10-18

Author(s):

Ahlam Al-Kharusi ◽

Sumaya Al-Mahrouqi ◽

Esmail K. Shubber

Keyword(s):

Heat Stress ◽

Chick Embryo ◽

Red Blood Cells ◽

Blood Cells ◽

Genome Stability ◽

Control Group ◽

Poultry Production ◽

In Ovo ◽

Group 2 ◽

Group 1

The present study was conducted to determine the adverse effects of high incubation temperature on growth, development and genome stability of broiler chick embryo in ovo). One hundred twenty broiler eggs from Cobb Company, USA were weighted and divided into two groups. The first group was incubated at 37oC ± 0.5oC, and the second group was incubated at 41oC ± 0.5oC from 0 to 18th day. Starting on day 4th and every other day; three eggs from each group were examined following performed measurements as weight of eggs post incubation, embryo, yolk, and egg shell for measuring growth index. Blood smear was also prepared for counting heterophiles, and lymphocytes to determine H/L ratio. Micronucleus formation and presence of binucleated red blood cells were investigated as genome stability parameters, in 2000 cells. Significant reduction (P<0.01) in growth indices was observed in embryos grown at 41oC compared to those grown at 37oC ± 0.5oC. Reduction in H/L ratio was statistically significant (p≤0.01) in embryos of 2nd group comparing to 1st group embryos. Blood of embryo from heat stress group group (2) showed Red blood cells with micronuclei and binucleated cells while no such phenomenon could be seen in embryos from control group group (1). These results suggested that heat stress is influencing cell division at telophase and induces chromosomal damage. 88% of chicks from group (1) were hatched on day 21st; only 18% of chicks from group (2) were hatched lately on day 23rd, while the others were found dead. These results indicate that heat stress not only adversely affects growth and development of embryo stem cells but also induces genome instability which intern resulted in poultry production losses.

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