Monoclonal antibodies to either domain of ovotransferrin block binding to transferrin receptors on chick reticulocytes

Monoclonal antibodies produced to both chicken ovotransferrin and to the isolated N- and C-terminal half-molecule domains of ovotransferrin have been used to probe the interaction of ovotransferrin with its specific receptor on chick embryo red blood cells. Two antibodies to epitopes on the N-terminal domain and one antibody to an epitope on the C-terminal domain were able to block the binding of 125I-labeled diferric ovotransferrin to the receptor. When the cellular surface receptors were first saturated with ovotransferrin at 0 degrees C, none of these antibodies bound to the cell-associated ovotransferrin. This suggests that the antibodies are to epitopes which lie very near to, or in the regions of, the two domains which interact with receptor. The same three antibodies also blocked the binding to the receptor of ovotransferrin associated in situ from the isolated N- and C-terminal half-molecule domains. A fourth antibody did not block binding to receptor of 125I-labeled diferric ovotransferrin or the associated domains; furthermore, it was able to bind to ovotransferrin bound to the cell surface at 0 degrees C. This antibody thus appears to recognize an epitope remote from the receptor binding region of ovotransferrin. Additional evidence for the requirement of the presence of both domains of ovotransferrin to effect binding to the transferrin receptor on chick reticulocytes was obtained with a fifth antibody which recognized only the N-terminal half-molecule domain but not holo-ovotransferrin. Although this antibody had no effect on the binding of 125I-labeled ovotransferrin to cells, it blocked binding to receptor of the associated domains of ovotransferrin, presumably by inhibiting the association of the two domains.     

Physiological levels of binding and iron donation by complementary half-molecules of ovotransferrin to transferrin receptors of chick reticulocytes

Two fragments, each corresponding to approximately half of the ovotransferrin (OTf) molecule and containing an iron-binding site were produced by digestion with affinity bound trypsin and were purified by isoelectric focusing and gel filtration chromatography. The immunologically distinct "half-molecules" individually have little ability to bind to transferrin receptors on chick embryo red blood cells or to donate iron to them. Combining them, however, leads to both binding and iron donation approaching that found for holo-OTf. Furthermore, similar amounts of radiolabeled iron can be extracted into the putative heme fraction from Fe2OTf and from the various combined half-molecules. These findings conflict with those reported by Keung and Azari ( (1982) J. Biol. Chem. 257, 1184-1188) for subtilisin-derived half-molecules of OTf examined in a similar system. They found that each half-molecule appeared to bind at a level of approximately one-third that of Fe2OTf and that the half-molecules competed with each other for binding sites. In contrast, our equilibrium binding studies, in the presence of 2,4-dinitrophenol to prevent iron removal, led to the determination of 4.79 X 10(4) binding sites/cell for Fe2OTf, 4.44 X 10(4) for the NH2-terminal half-molecules in the presence of excess COOH-terminal half-molecules and 4.17 X 10(4) for COOH-terminal half-molecules in the presence of NH2-terminal half-molecules; apparent binding constants were estimated to be 3.29 X 10(6), 1.19 X 10(6), and 0.67 X 10(6) M-1 for these same samples. Problems associated with equilibrium binding studies in which a narrow range of concentrations of ligand is used and/or iron is being removed are discussed. Labeled combined half-molecules were half as effective as labeled Fe2OTf in competition with unlabeled Fe2OTf. These findings are consistent with the lower apparent binding constant found in the equilibrium binding studies. Equimolar apo-OTf had no effect on binding of either Fe2OTf or the combined half-molecules. It seems apparent from our studies that the NH2- and COOH-terminal half-molecules each contain a recognition region both of which are necessary for binding to the transferrin receptor and iron donation to the chick embryo red blood cell.     

Nerve growth factor receptors. Characterization of two distinct classes of binding sites on chick embryo sensory ganglia cells

Steady state and kinetic studies on the binding of 125I-beta nerve growth factor (NGF) to single cells from sensory ganglia of 8-day-old chick embryos show two distinct, saturable binding sites with dissociation constants of Kd(I) = 2.3 X 10(-11) M and Kd(II) = 1.7 X 10(-9) M. The difference in the affinities is due to different rate constants of dissociation (k-1(I) = 10(-3) s-1, k-1(II) = 2 X 10(-1 s-1). The association to both sites is apparently diffusion controlled (k+1(I) = 4.8 X 10(7) M-1s-1, k+2(II) = 10(7) to 10(8) M-1s-1). The binding of betaNGF to both sites is specific, since none of a number of hormones or proteins tested compete for the binding of 125I-betaNGF to either of those two sites. The heterogeneity of the binding of 125I-betaNGF is not due to heterogeneity of the 125I-betaNGF preparation nor to a negatively cooperative binding. In experiments where the dissociation of 125I-betaNGF is induced by the addition of saturating amounts of unlabeled betaNGF, the ratio of the 125I-betaNGF released with either of the two dissociation rate constants is solely dependent on the occupancy of the two sites before dissociation is started and is independent of the total occupancy of the sites during dissociation. The rate of dissociation of 125I-betaNGF from the higher affinity binding site I is accelerated by unlabeled betaNGF under conditions where the occupancy is both increased and decreased. Although the dissociation characteristics of 125I-beta NGF change with increasing times of exposure of the cells to the ligand, and 125I-beta NGF is degraded after it binds to the cells, these secondary processes do not interfere with the analysis of the binding data. At the lowest concentration of 125I-beta NGF used for the analysis less than 10% of the 125I-beta NGF is degraded. Both kinetic and steady state binding data reveal the two NGF binding sites at 2 degrees C as well as at 37 degrees C.     

Effects of anion binding on the conformations of the two domains of ovotransferrin

A previous paper (Harris (1985) Biochemistry 24, 7412-7418) reported the occurrence of two classes of anion binding sites in transferrin. To evaluate the locations of the two anion binding sites in relation to the two major domains of transferrin we determined the binding constants of whole ovotransferrin and its two half-molecules by means of the difference UV spectroscopic technique. Anions induced strong negative absorbance at 245 nm in the order: citrate greater than phosphate greater than bicarbonate for whole ovotransferrin and the N-terminal half-molecule; and: phosphate greater than citrate greater than bicarbonate for the C-terminal half-molecule. The anion dissociation constants of the N-terminal half-molecule were consistent with lower dissociation constants, and those of the C-terminal half-molecule, with higher dissociation constants of whole ovotransferrin, indicating that the two classes of anion binding sites correspond to the binding sites in individual structural domains. Anion binding markedly protected the N-terminal half-molecule, but not the C-terminal half-molecule from digestion with trypsin and disulfide reduction with dithiothreitol. As to the far and near ultraviolet CD spectra data, however, there was no significant difference between in the presence and absence of an anion. Therefore, the binding of an anion would induce some conformational changes which were not reflected by the CD spectrum.     

Differential effect of hormones on macromolecular synthesis and mitosis in chick embryo cells

This study describes the selection and preliminary characterization of mammalian cells resistant to 100 mug Tevenel/ml. Tevenel, the sulfamoyl analog of chloramphenicol, is a specific inhibitor of mitochondrial protein synthesis. After growth in suspension culture for 5 days in 100 mug Tevenel/ml and subsequent plating in 100 mug Tevenel/ml, LMTK- cells yielded resistant clones. As a control, L cells treated identically yielded no clones. Three resistant clones were chosen for study. Each resistant cell line had an identical growth rate in the presence and absence of 100 mug Tevenel/ml. By plating efficiency analysis, the resistant cells were found to be cross-resistant to D-chloramphenicol. The change responsible for resistance was found to be stable for at least 100 generations in the absence of the drug. Protein synthesis by isolated mitochondria of resistant cells was found to be less inhibited by concentrations of both Tevenel and D-chloramphenicol up to 200 mug/ml than the protein synthesis by LMTK- mitochondria. This resistance in vitro was not changed by incubation of the mitochondria in 0.01% Triton X-100.     

Insulin binding to cells of several tissues of the early chick embryo.

The binding of I¹²⁵-labeled insulin to isolated cells from several tissues of the 3- and 4-day chick embryo was determined over a concentration range of insulin from 2 × 10^?11 to 2 × 10^?7M. The cells were obtained from limb bud and nonlimb bud tissues of the 4-day chick, from the headless 3-day chick embryo, and from cartilage of the 12-day embryo. The amount of bound insulin was found to be similar for the cells from the different embryonic tissues. Some implications of these findings for the interpretation of the nature of the binding sites and the teratogenic effect of insulin are discussed.     

Effect of pH and iron content of transferrin on its binding to reticulocyte receptors

The effect of pH on the binding of apotransferrin and diferric transferrin to reticulocyte membrane receptors was investigated using rabbit transferrin and rabbit reticulocyte ghosts, intact cells and a detergent-solubilized extract of reticulocyte membranes. The studies were performed within the pH range 4.5–8.0. The binding of apotransferrin to ghosts and membrane extracts and its uptake by intact reticulocytes was high at pH levels below 6.5 but decreased to very low values as the pH was raised above 6.5. By contrast, diferric transferrin showed a high level of binding and uptake between pH 7.0 and 8.0 in addition to binding only slightly less than did apotransferrin at pH values below 6.5. It is proposed that the high affinity of apotransferrin for its receptor at lower pH values and low affinity at pH 7.0 or above allow transferrin to remain bound to the receptor when it is within acidic intracellular vesicles, even after loss of its iron, but also allow ready release from the cell membrane when it is exteriorized by exocytosis after iron uptake. The binding of transferrin to the receptor throughout the endocytosis-exocytosis cycle may protect it from proteolytic breakdown and aid in its recycling to the outer cell membrane     

Sensitivity of cultured chick embryo heart cells to acetylcholine. Evidence for nicotinic and muscarinic receptors

Sensitivity of cultured chick embryo heart cells to acetylcholine changes with time in culture. In 24 h cultures, about 25% of the cells exhibit a positive chronotropic response to acetylcholine. This effect is no longer observed after 48 h in culture. Positive and negative chronotropic effects of acetylcholine can be related to the presence of nicotinic and muscarinic receptors evidenced by autoradiography. Some data suggest a possible relationship between the type of sensitivity to acetylcholine and the changes in cell membrane properties occurring in culture.     

Differential binding of human interferon-alpha subtypes to receptors on lymphoblastoid cells

Highly purified human interferon-alpha subtype A (HuIFN alpha A) was iodinated for use in direct ligand binding studies on human lymphoblastoid (Daudi) cells. Unlabelled preparations of HuIFN alpha subtypes A, C, D, and hybrid molecules AD (Bgl II), AD (Pvu II), and DA (Bgl II) showed different responses in competition experiments with labelled alpha A probe. Specifically, IFNs alpha D and alpha DA were unable to displace the probe, whereas IFNS alpha A, alpha C, and the hybrid alpha ADs showed similar competition curves. These results support a two-idiotope model of IFN recognition by its receptor. IFN effects on [3H]-thymidine incorporation and cell growth (long term effects) did not reflect the apparent affinities of HuIFN alpha subtypes for cell surface receptor.     

Differential effect of hypertonic initiation block on the synthesis of collagen chains by cultured chick embryo cells

The major type of collagen synthesized by fibroblasts or bone cells, type I collagen, consists of two chains normally found in a 2:1 ratio designated alpha 1(I)2 alpha 2(I) or more simply alpha 1(I)2 alpha 2. I have analyzed the relative synthesis of type I chains in these cells under conditions which reduce the initiation of protein synthesis. It was found that in bone cells, which make a large amount of collagen, the alpha 1(I):alpha 2 ratio is unaltered whereas in fibroblasts, which make smaller amounts of collagen, the alpha 2 chain is particularly sensitive to these same conditions. Examination of the collagen secreted into the medium, under these same conditions, also revealed an altered chain ratio from cells making low amounts of collagen.     

Antioxidant effect of red wine polyphenols on red blood cells

The protective effect of red wine polyphenols against hydrogen peroxide (H(2)O(2))-induced oxidation was investigated in normal human erythrocytes (RBCs). RBCs, preincubated with micromolar amounts of wine extract and challenged with H(2)O(2), were analyzed for reactive oxygen species (ROS), hemolysis, methemoglobin production, and lipid peroxidation. All these oxidative modifications were prevented by incubating the RBCs with oak barrel aged red wine extract (SD95) containing 3.5 mM gallic acid equivalent (GAE) of phenolic compounds. The protective effect was less apparent when RBCs were incubated with wines containing lower levels of polyphenols. Furthermore, resveratrol and quercetin, well known red wine antioxidants, showed lower antioxidant properties compared with SD95, indicating that interaction between constituents may bring about effects that are not necessarily properties of the singular components. Our findings demonstrate that the nonalcoholic components of red wine, mainly polyphenols, have potent antioxidant properties, supporting the hypothesis of a beneficial effect of red wine in oxidative stress in human system.     

In vitro triiodothyronine binding to cytoplasmic proteins from human red blood cells.

In vitro incubations of cytosol proteins from human red blood cells with [¹²⁵I] labelled L-3,5,3′ triiodothyronine demonstrated the existence of high affinity and limited capacity binding sites for T₃. At 4°C, the rate constant of association was 3 × 10⁷ M^?1h^?1, and the rate constant of dissociation was 9.10^?3h^?1. The dissociation constant K_d was calculated from these data or measured by Scatchard analysis and found to be between 3 and 7.10^?10M. The maximum binding capacity was 1.4 f moles of L-3,5,3′ triiodothyronine per mg cytosol proteins. A close parallel between the biological pontency of the analogs of L-T₃ was observed.     

Efficient production and isolation of recombinant amino-terminal half-molecule of human serum transferrin from baby hamster kidney cells.

Expression of the amino-terminal lobe of human serum transferrin secreted into the culture medium by transformed baby hamster kidney (BHK) cells has been increased from the levels reported originally of 10-15 micrograms/ml to 55-120 micrograms/ml. Use of the serum substitute, Ultraser G, has facilitated isolation of the recombinant protein, resulting in approximately 80% recovery of expressed hTF/2N from the culture medium. In the three experiments described, 300-750 mg of recombinant protein was collected over a period of 25 days from five expanded surface roller bottles each containing 200 ml of medium (seven to nine collections). The use of alginate beads to encapsulate the transformed BHK cells provided no advantage over normal culturing over 25 days. A lag in production resulting in 30% less recombinant protein over this time period was observed. The production and isolation procedures described are easily handled by one person. The system is amenable to incorporation of isotopically substituted amino acids useful in NMR studies.     

The effect of anions on Mg-ATPase in red blood cells.

Anions exert an influence on the passive permeability of Na+ and K+ in erythrocytes. THE EFFECT ON Mg-ATPase activity has been studied in human erythrocytes. 40 mM bicarbonate increased the activity as compared to the effect of 40 mM chloride; 20 mM sulphate inhibited it. Salicylate acted first as an activator then as an inhibitor of Mg-ATPase; maximum activity was reached at 60 mM CONCENTRATION. Thiocyanate inhibited saponin-stimulated Mg-ATPase, Ki = 1.85 X 10(-2)M. The probable mechanisms of action of the above anions on Mg-ATPase and possible relation to passive permeability of Na+ and K+ ions are discussed.     

Disparity between expression of transferrin receptor ligand binding and non-ligand binding domains on human lymphocytes

We compared transferrin receptor (TfR) expression on human peripheral blood lymphocytes (PBL) activated by phorbol myristate acetate (PMA) or L-phytohemagglutinin (LPHA) using two techniques: (1) 125I-iron-saturated transferrin (FeTf) binding, (2) reactivity with monoclonal anti-TfR antibodies--OKT9 and B3/25. These monoclonal antibodies do not block FeTf binding, and therefore bind to TfR domains separate from the ligand binding site. Unstimulated PBL bound fewer than 1,000 molecules of 125I-FeTf per cell, and less than 5% of cells expressed TfR antigens detected by OKT9 or B3/25. 125I-FeTf binding and antibody binding increased in parallel on LPHA-activated PBL. After exposure to LPHA for 72 hr, 125I-FeTf binding increased 100-fold to 10(5) molecules per cell and greater than 50% of cells expressed TfR antigens. By contrast, PMA activation of PBL markedly increased binding of OKT9 and B3/25 but not the binding of 125I-FeTf. Cell surface expression of TfR antigens seen by OKT9 and B3/25 did not differ between LPHA- and PMA-activated PBL. However, after 72 hr with PMA, 125I-FeTf binding increased only 6-fold and consistently remained at less than 10(4) molecules per cell. Therefore, PMA induced a disparity between expression of TfR ligand binding domains and immunological domains at the cell surface. Cell proliferation assessed by fluorescent DNA analysis was similar in cultures stimulated by LPHA or PMA. These data indicate that lymphoid cells may possess a mechanism for modulating TfR expression in which down-regulation of FeTf binding occurs without receptor internalization. Alternatively, it is possible that this observation may reflect a membrane perturbation effect of PMA.     

Tryptose phosphate broth confers to chick embryo cells resistance to the inhibitory effect of chloramphenicol on growth

Tryptose phosphate broth (10% V/V) prevents the inhibitory effect of chloramphenicol (80 ug/ml) on the growth of chick embryo fibroblasts. Cellular cytochrome $\text{c}$ oxidase activity is rapidly lost indicating that chloramphenicol inhibits the mitochondrial protein-synthesizing system in the presence of the broth. To compensate for the apparent decreased mitochondrial ATP production, pyruvate kinase and lactate dehydrogenase activities, 2-deoxyglucose uptake and lactic acid produced in treated cells are greater than those in the control cells during the first few generations. These fermentation values tend to revert to normal during later cell generations.     

Differential function of the two nucleotide binding domains on cystic fibrosis transmembrane conductance regulator

The genetic disease cystic fibrosis is caused by defects in the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). CFTR belongs to the family of ABC transporters. In contrast to most other members of this family which transport substrates actively across a membrane, the main function of CFTR is to regulate passive flux of substrates across the plasma membrane. Chloride channel activity of CFTR is dependent on protein phosphorylation and presence of nucleoside triphosphates. From electrophysiological studies of CFTR detailed models of its regulation by phosphorylation and nucleotide interaction have evolved. These investigations provide ample evidence that ATP hydrolysis is crucial for CFTR gating. It becomes apparent that the two nucleotide binding domains on CFTR not only diverge strongly in sequence, but also in function. Based on previous models and taking into account new data from pre-steady-state experiments, a refined model for the action of nucleotides at two nucleotide binding domains was recently proposed.