Ferric-transferrin

Transferrin model compounds and 44 related iron(III) crystal structures were used to modify the AMBER force field for subsequent studies of ferric transferrin. Eneigy minimization was conducted both in vacuo and, more interestingly, with the generalized Bom/surface area (GB/SA) continuum treatment described in Chapter 2, Section 2.712201. 

At least three types of proton channel systems are recognized in animal cells. These include the Na+/H+ exchanger, the H+-ATPase, and the HCOj/Cl- exchanger. It is clear that a major part of proton release by some cells in response to transplasma membrane electron transport is by activation of the Na+/H+ exchanger. This is clear from the characteristics of the proton movement elicited and the magnitude of H+ release in relation to electron flow when electron transport is activated. Activation of electron transport can be elicited by addition of di-ferric transferrin to activate the transmembrane NADH oxidase activity or by electron flow to external ferricyanide from internal NADH. Addition of di-ferric transferrin to certain cells, especially pineal cells, elicits a remarkable proton release and internal alkaliniza-tion. The stoichiometry of H+ release to iron reduced is more than 100 to 1 (Sun et... 

Figure 6. Scheme to represent known aspects of the plasma membrane NADH oxidase and its association with proton release. The oxidase is activated when hormones or ferric transferrin bind receptors. Oxidase may activate tyrosine kinase which can activate MAP kinases to result in phosphorylation of the exchanger leading to Na+/H+ exchange. Oxidation of quinol in the membrane can also release protons to the outside equal to the number of electrons transferred. External ferricyanide can activate electron flow by accepting electrons at the quinone. G proteins (GTP binding proteins) such as ras-activate electron transport and proton release in some way and may be a link to kinase activation (McCormick, 1993). Semiquinone formation in the membrane could lead to superoxide and peroxide formation by one electron reduction of oxygen. 

Note Reduction and proton release measured under the same conditions in 100 mM NaCI, 83.5 mM sucrose, 1.5 mM Tris HCI at starting pH 7.4 at 22 °C, 0.1 mM ferricyanide or 17 pM di-ferric transferrin added to start the reactions. 

Note A problem in the interpretation of the amiloride inhibition of proton release is the amiloride inhibition of di-ferric transferrin and ferricyanide reduction (Sun et al., 1987). The question arises does inhibition of the exchanger inhibit electron transport or does inhibition of electron transport inhibit a H release not dependent on the exchanger Exchanger inhibition is most likely because of the Na+ dependence for part of the H release (Sun et al., 1988). As an alternative, proton transport may be necessary for electron transport (Stahl and Anst, 1993). 

In HeLa cells, the di-ferric transferrin-stimulated proton release is accompanied by an increase in the pH inside the cell as measured by change in fluorescence of the internal indicator BCECF incorporated into the cells (Figure 7). This alkalinization is consistent with exchanger activation to export protons. Most of the internal pH increase is inhibited by amiloride or dimethylamiloride (Mrkic et al., 1992 Yun et al., 1993). The internal change in pH is prevented if Cs+ is substituted for Na+ or Li+ in the external media (Toole-Simms, 1988). Ferricyanide-stimulated proton release by HeLa cells is not accompanied by an internal pH increase. The basis for this difference from di-ferric transferrin has not been investigated. It is possible that the rapid electron transport in response to ferricyanide leads to oxidation of sufficient NADH, which produces protons inside the cell, to counter... 

Table 5. Effects of Insulin on Cell Response to Ferricyanide and Bombesin on Response to Di-ferric Transferrin...

Note HeLa cells grown in aMEM with 10% FCS. Assay in absence of serum. Swiss 3T3 cells also assayed in absence of serum. Bombesin effects are discussed by Rozengurt (1986). Fe(CN>6 is potassium ferricyanide and Fe2Tf is di-ferric transferrin. Fe2Tf and Fe(CN>6 reduction rate is in nmole min"1 gww of cells-1. Cell growth is measured after 48 hr as cells x 10s/25 cm2 flask. For proton release, cells are equilibrated first with insulin or bombesin to set a baseline, then oxidant is added. Insulin alone can activate the exchanger (Ives and Rector, 1984). 

Table 6. Effect of Retinoic Acid on Proton Release from Pineal Cells in Absence and Presence of Di-ferric Transferrin...

The dramatic role of the anion can perhaps best be appreciated from simple quantitative considerations. In the absence of a suitable anion, specific binding of iron to transferrin does not occur at all the effective binding constant is zero. At physiologic pH and bicarbonate concentrations, however, the effective binding constant is about 5 X 1023 M"1 24, 50). This means that in 1 L of blood plasma, in which the transferrin is only about 30% saturated with iron, there will be less than one free ferric ion or that a molecule of the ferric—transferrin complex will spontaneously dissociate only about once in 10,000 years. Since iron is readily removed from the transferrin molecule during its interaction with the reticulocyte without disrupting protein structure 51, 52), a... 

Studies of ferric transferrins with a wide range of spectroscopic methods have been reported but here we focus on their optical and EPR properties. Ferric transferrins are a salmon pink color, resulting from a weak Fe(lll)-phenolate charge transfer absorption at 470 nm (for lactoferrin = 2, 070Tmol cm per Fe), which has been used to... 

A prolate shape also appears to explain better the hydrodynamic properties of iron-free (142) and of iron-saturated (143) transferrin. Ferric transferrin (a/b = 3) would, however, be more elongated than the iron-free form (a/b =2) while the effective hydrodynamic volume (Ve) would be higher for the iron complex than for the apoprotein. These results not only differ from those given in Table 3 for conalbumin but are also in partial disagreement with dielectric dispersion and viscosity measurements (144) which have indicated that human transferrin assumes a more spherical shape with iron-saturation, the axial ratio decreasing from 2.5 (apo) to 2.0 (ferric). This latter investigation also indicates a slight expansion (15.4 16.9) of the hydrated volume... 

Similarly, the lower accessibility of tyrosyl residues to base titration in ferric lactoferrin compared to ferric transferrin has been suggested to be linked to their relative stabilities as metallocomplexes 67, 163). Ferric conalbumin also appears to be more stable than ferric transferrin 157). These stability differences may be of biological significance given that a role of the serum protein is to distribute iron in the body rather than to sequester the metal. 

Figure 15 Transferrin-mediated iron uptake by bacteria. One lobe of ferric transferrin binds to the OM transmembrane receptor protein TbpA TbpB facilitates bmdmg.The unchelated ferric ion passes through the membrane and is then bound by the ferric-binding protein (FbpA), located in the periplasm. A TonB-ExbBD-like protein system provides the energy required for this process. FbpA shuttles the iron to the CM transmembrane protein FbpB. The iron moves into the cytoplasm following hydrolysis of ATP by FbpC.

Alcantara O, Obeid L, Hannun Y, Ponka P, Boldt DH. 1994. Regulation of protein kinase C (PKC) expression by iron Effect of different iron compounds on PKC-beta and PKC-alpha gene expression and role of the 5 -flanking region of the PKC-beta gene in the response to ferric transferrin. Blood 84 3510-3517. 

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