Transferrins substitution

Smith, C.A., Anderson, B.F., Baker, H.M., and Baker, E.N. 1992. Metal substitution in transferrins the crystal structure of human copper-lactoferrin at 2.1-A resolution. Biochemistry 31 4527 -533. 

The Ru(IV)/Ru(III) redox potential is 0.78 V, so that Ru(III) or even Ru(II) species may be present in vivo. Indeed, the related Ru(III) complex 32 is also active (171), and the pendant arms in these octahedral polyaminocarboxylate complexes increase the rate of substitution reactions. Complex 32 binds rapidly to the blood proteins albumin and transferrin (172), and the ruthenium ion appears to remain in the... 

An order of effectiveness has been established and a mechanism proposed for the removal of iron from ferritin by several 3-hydroxy-4-pyridinone chelators. The removal of iron from ferritin is, as one would expect, considerably slower than from calcein or doxorubicin (cf. above) or from transferrin. Rate constants are between 1.5 x 10 s and 7.5 x 10 s for removal of iron from ferritin by a series of hexadentate ligands each consisting of three substituted A-hydroxypyrimidinone or A-hydroxypyrazinone units, the rate decreasing with increasing substituent bulk. The slowest rate approximates to that for removal of iron from ferritin by desferrioxamine. The influence of chirality on the kinetic barrier provides insight into the detailed mechanism of removal in these systems.Slow removal of iron from ferritin by chelators should be contrasted with rapid reductive removal. ... 

Fibrillin, calcium binding, 46 473, 474, 477 Fibulin-I, calcium binding, 46 473 Field desorption mass spectroscopy, 28 6, 21 Field effects, of astatophenols, 31 66 Fine structure, 13 193-204 Fingerprinting of polymetalates, 19 246-248 Finite perturbation theory, 22 211, 212 First transition series, substitution, transferrins, 41 423 26... 

Ser Se isotopic substitution, 38 105-107 Serum albumin, 46 470 Serum transferrins, 41 390 biological role, 41 391-392 half-molecules, 41 396 recombinant, 41 453 structure, 41 397... 

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... 

In this chapter, we provide protocols to determine the ability of a peptide to mediate DNA internalization in cultured human tumor cells. Fluorescence-assisted cell sorting (FACS) analysis is used to obtain quantitative data on the time and temperature dependence of macromolecular delivery. Confocal microscopy is used to study the subcellular localization in both fixed and live cells. Fluorescently labeled transferrin and dextran are used to label the clathrin-dependent (15) and the non-clathrin, non-caveolar (16) endocytic compartments, respectively. Expression of a caveolin-l-YFP fusion protein is used to label cell surface caveolae and intracellular caveosomes (17). Finally a protocol, for the overexpression of dominant-negative dynamin [GTPase deficient dynamin-2 containing the amino acid substitution K44A (18)] is provided to evaluate the dynamin dependence of the uptake mechanism. 

Some tentative conclusions about the nature of these conformational differences may be drawn from the crystallographic studies of Cu2+ and oxalate-substituted lactoferrins (26,192,193). Anions which gave class A spectra with V02+-substituted transferrins are those that can... 

Fig. 28. The pH dependence of iron release from human serum transferrin (Tf), human lactoferrin (Lf), and the recombinant N-terminal half-molecule of human lactoferrin (Lfm). Also shown is a plot (dashed line) for the release of cerium from Ce4+-substituted lactoferrin, demonstrating the increased difference between the two sites for metal ions other than Fe3+.

Distinct differences are also seen when anions other than C032 are used. The crystal structure of oxalate-substituted diferric lactoferrin shows differences in the anion binding in the two sites in the C-site the oxalate is symmetric bidentate, whereas in the N-site it is asymmetric (193). When Cu2+ is the metal ion the oxalate binding differences become even more pronounced. Copper-transferrin binds oxalate only in its N-terminal site (91). Copper-lactoferrin and copper-ovotransfer-rin each bind two oxalate ions but binding occurs preferentially in the C-lobe (157,192). These different affinities mean that hybrid complexes can be prepared with oxalate in one site and carbonate in the other (92, 157, 192). The use of oxalate as synergistic anion gives rise to spectroscopically distinct sites for other metal ions also (171). 

ESI mass spectrometry was performed on approximately 32 pmol of peptide mixture in decylglucopyranoside buffer without success (data not shown). PVDF-bound transferrin was digested in lower concentrations of detergent, and substitution of ammonium bicarbonate for Tris, although these conditions are optimum for ESI mass spectrometric analysis the digestion was not successful as determined by HPLC analysis (7). 

Differences between the two sites become more pronounced for metal ions other than Fe " and anions other than COa . The differences are most pronounced for larger metal ions such as lanthanides. For transferrin some of the larger lanthanides appear to bind in only one of the two sites (Section IV.B.3), and for lactoferrin, although binding occurs in both sites, the second metal ion binds much more weakly, as shown by the curvature of the UV difference titration graph (Fig. 18) the biphasic release of Ce from lactoferrin contrasts with that of Fe (Fig. 28). Even metal ions of the first transition series, of similar size to Fe "", enhance the differences between the two sites. When Cr is bound to either transferrin 134) or lactoferrin (154), different EPR signals are seen for the two sites, and one Cr " ion is much more readily displaced by Fe than the other. Likewise, the EPR spectra of VO " -substituted transferrin indicate different metal configurations in the two sites (207), as do NMR studies of Co -substituted ovotransfer-rin (139). In these cases one metal ion is also released much more readily than the other as the pH is lowered. 

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