Anion binding by human lactoferrin: results from crystallographic and physicochemical studies.

The anion-binding properties of lactoferrin (Lf), with Fe3+ or Cu2+ as the associated metal ion, have been investigated by physicochemical and crystallographic techniques. These highlight differences between the two sites and in the anion-binding behavior when different metals are bound. Carbonate, oxalate, and hybrid carbonate-oxalate complexes have been prepared and their characteristic electronic and EPR spectra recorded. Oxalate can displace carbonate from either one or both anion sites of Cu2(CO3)2Lf, depending on the oxalate concentration, but no such displacement occurs for Fe2(CO3)2Lf. Addition of oxalate and the appropriate metal ion to apoLf under carbonate-free conditions gives dioxalate complexes with both Fe3+ and Cu2+, except when traces of EDTA remain associated with the protein, when hybrid complexes M2(CO3)(C2O4)Lf can result. The anion sites in the crystal structures of Fe2(CO3)2Lf, Cu2-(CO3)2Lf, and Cu2(CO3)(C2O4)Lf, refined at 2.2, 2.1, and 2.2 A, respectively, have been compared. In every case, the anion is hydrogen bonded to the N-terminus of helix 5, an associated arginine side chain, and a nearby threonine side chain. The carbonate ion binds in bidentate fashion to the metal, except in the N-lobe site of dicupric lactoferrin, where it is monodentate; the difference arises from slight movement of the metal ion. The hybrid complex shows that the oxalate ion binds preferentially in the C-lobe site, in 1,2-bidentate mode, but with the displacement of several nearby side chains. These observations lead to a generalized model for synergistic anion binding by transferrins.     

Effect of Lactoferrin on the Ferroxidase Activity of Ceruloplasmin

The effects of various forms of lactoferrin (Lf) interacting with ceruloplasmin (Cp, ferro-O2-oxidoreductase, EC 1.16.3.1) on oxidase activity of the latter were studied. Comparing the incorporation of Fe3+ oxidized by Cp into Lf and serum transferrin (Tf) showed that at pH 5.5 apo-Lf binds the oxidized iron seven times and at pH 7.4 four times faster than apo-Tf under the same conditions. Apo-Lf increased the oxidation rate of Fe2+ by Cp 1.25 times when Cp/Lf ratio was 1 : 1. Lf saturated with Fe3+ or Cu2+ increased the oxidation rate of iron 1.6 and 2 times when Cp to holo-Lf ratios were 1 : 1 and 1 : 2, respectively. Upon adding to Cp the excess amounts of apo-Lf (Cp/apo-Lf < 1 : 1) or of holo-Lf (Cp/holo-Lf < 1 : 2) the oxidation rate of iron no longer changed. Complex Cp-Lf demonstrating ferroxidase activity was discovered in breast milk.     

Metal substitution in transferrins: the crystal structure of human copper-lactoferrin at 2.1-A resolution.

The structural consequences of binding a metal other than iron to a transferrin have been examined by crystallographic analysis of human copper-lactoferrin, Cu2Lf. X-ray diffraction data were collected from crystals of Cu2Lf, using a diffractometer, to 2.6-A resolution, and oscillation photography on a synchrotron source, to 2.1-A resolution. The structure was refined crystallographically, by restrained least-squares methods, starting with a model based on the isomorphous diferric structure from which the ligands, metal ions, anions, and solvent molecules had been deleted. The final model, comprising 5321 protein atoms (691 residues), 2 Cu2+ ions, 2 (bi)carbonate ions, and 308 solvent molecules has good stereochemistry (rms deviation of bond lengths from standard values of 0.018 A) and gives a crystallographic R value of 0.196 for 43,525 reflections in the range 7.5-2.1-A resolution. The copper coordination is different in the two binding sites. In the N-terminal site, the geometry is square pyramidal, with equatorial bonds to Asp 60, Tyr 192, His 253, and a monodentate anion and a longer apical bond to Tyr 92. In the C-terminal site, the geometry is distorted octahedral, with bonds to Asp 395, Tyr 435, Tyr 528, and His 597 and an asymmetrically bidentate anion. The protein structure is the same as for the diferric protein, Fe2Lf, demonstrating that the closure of the protein domains over the metal is the same in each case irrespective of whether Fe3+ or Cu2+ is bound and that copper could be transported and delivered to cells equally well as iron. The differences in metal coordination are achieved by small movements of the metal ion and anion within each binding site, which do not affect the protein structure.     

Metal substitution in transferrins: specific binding of cerium(IV) revealed by the crystal structure of cerium-substituted human lactoferrin

Proteins of the transferrin family play a key role in iron homeostasis through their extremely strong binding of iron, as Fe3+. They are nevertheless able to bind a surprisingly wide variety of other metal ions. To investigate how metal ions of different size, charge and coordination characteristics are accommodated, we have determined the crystal structure of human lactoferrin (Lf) complexed with Ce4+. The structure, refined at 2.2 A resolution (R=20.2%, Rfree=25.7%) shows that the two Ce4+ ions occupy essentially the same positions as do Fe3+, and that the overall protein structure is unchanged; the same closed structure is formed for Ce2Lf as for Fe2Lf. The larger metal ion is accommodated by small shifts in the protein ligands, made possible by the presence of water molecules adjacent to each binding site. The two Ce4+ sites are equally occupied, indicating that the known difference in the pH-dependent release of Ce4+ arises from a specific protonation event, possibly of the His ligand in one of the binding sites. Comparing the effects of binding Ce4+ with those for the binding of other metal ions, we conclude that the ability of transferrins to accommodate metal ions other than Fe3+ depends on an interplay of charge, size, coordination and geometrical preferences of the bound metal ion. However, it is the ability to accept the six-coordinate, approximately octahedral, site provided by the protein that is of greatest importance.     

Synergistic anion-directed coordination of ferric and cupric ions to bovine serum transferrin--an inorganic perspective

A series of new iron(III) and copper(II) complexes of bovine serum transferrin (BTf), with carbonate and/or oxalate as the synergistic anion, are presented. The complexes [Fe(2)(CO(3))(2)BTf], [Fe(2)(C(2)O(4))(2)BTf], [Cu(2)(CO(3))(2)BTf] and [Cu(C(2)O(4))BTf] were prepared by standard titrimetric techniques. The oxalate derivatives were also obtained from the corresponding carbonate complexes by anion-displacement. The site-preference of the transition metal-oxalate synergism has facilitated the preparation and isolation of the mononuclear complex [Cu(C(2)O(4))BTf], the mixed-anion complexes [Cu(2)(CO(3))(C(2)O(4))BTf] and [Fe(2)(CO(3))(C(2)O(4))BTf] and the mixed-metal complex [FeCu(C(2)O(4))(2)BTf]. The sensitivity of electron paramagnetic resonance (EPR) spectroscopy to the nature of the synergistic anions at the specific-binding sites of the transferrins has made this physical technique particularly indispensable to this study. None of the other members of the transferrin family of proteins has ever been demonstrated to bind the ferric and cupric ions one after the other, each occupying a separate specific-binding site of the same transferrin molecule, as a response to the coordination restrictions imposed by the oxalate ion. The bathochromic shift of the visible p(pi)-d(pi*) CT band for iron(III)-BTf and the hypsochromic shift of the p(pi)-d(sigma*) CT band for copper(II)-BTf, on replacing carbonate by oxalate as the associated anion, are consistent with the relative positions of these anionic ligands in the spectrochemical series and the nature of the d-type acceptor orbitals involved in the CT transitions. The binding and spectroscopic properties of bovine serum transferrin--a serum transferrin--very nearly mirror those of human serum transferrin, but differ significantly from those of human lactoferrin.     

Three-dimensional structure of a new form of mare lactoferrin in 70% PEG 400 at 3.8 A resolution.

Three-dimensional (3D) structure of a new form of diferric mare lactoferrin has been determined at 3.8 A resolution. The protein was crystallized in a space group P2(1)2(1)2(1) with a = 80.1 A, b = 103.7 A, c = 112.2 A with a solvent content of 57%. The structure was solved by molecular replacement method using the model of native mare lactoferrin. The structure has been refined using X-PLOR to a final R-factor of 22.6% for all the data in 15.0-3.8 A resolution range. The final refined model comprises 5281 protein atoms, 2Fe3+ and 2CO3(2-) ions. The protein folds into two globular N- and C-lobes. The two lobes are further divided into two domains N1, N2 in the N-lobe and C1, C2 in the C-lobe. The overall folding of the protein is similar to that observed for the native protein. The superposition of Calpha traces of native mare lactoferrin and the present structure gives an r.m.s shift of 0.7 A. There is a slight variation in the orientation of two lobes but the domain orientations in the present structure are identical to those observed in the native mare lactoferrin.     

Crystallization and structure determination of goat lactoferrin at 4.0 A resolution: a new form of packing in lactoferrins with a high solvent content in crystals

Lactoferrin was purified from fresh samples of goat colostrums, saturated with Fe3+ and CO3(2-) ions and crystallized by microdialysis method. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with a=104.6 A, b=153.8 A, c=155.1 A and Z=4. The quality of crystals was poor, thus the intensity data were restricted to 4.0 A resolution only. The structure was determined by molecular replacement method using diferric buffalo lactoferrin as a model. The solution clearly indicated the presence of one molecule in the asymmetric unit, which corresponds to a Vm value of 7.1 A3/Da. The structure was refined with stringent constraints to an R-factor of 0.246 using all the reflections 15,870 to 4.0 A resolution. The overall structure of goat lactoferrin is essentially similar to those of buffalo and bovine lactoferrins. However, the iron-binding environment in goat lactoferrin is somewhat different, in which 2 CO3(2-). ions have low occupancies. The solvent content of approximately 84% was very high in the present case which explains the fragility of the crystals of goat lactoferrin. In a way, it is very surprising that the crystals grow at all, although crystals with solvent as high as 89% have been reported.     

The identification of 14 alpha,17 beta-dihydroxyandrost-4-en-3-one monohydrate and 14 alpha,17 beta-dihydroxyandrosta-1,4-dien-3-one monohydrate, metabolites of androstenedione in Mucor piriformis.

The microorganism Mucor piriformis transforms androst-4-ene-3,17-dione into a major and several minor metabolites. X-ray crystallographic analysis of two of these metabolites was undertaken to determine unambiguously their composition and chirality. Crystals belong to the orthorhombic space-group P2(1)2(1)2(1), with a = 7.199(4) A and a = 6.023(3) A, b = 11.719(3) A and b = 13.455(4) A, c = 20.409(3) A and c = 20.702(4) A for the two title compounds, respectively. The structures have been refined to final R values of 0.060 and 0.040, respectively.     

Crystal structure of human seminal diferric lactoferrin at 3.4 Angstrom resolution

Lactoferrin was purified from human seminal fluid obtained from the semen bank. The purified samples were saturated with Fe3+ and crystallized by microdialysis method. The crystals belong to orthorhombic space group P21212, with a = 55.9 Angstrom. b = 97.2 Angstrom, c = 156.1 Angstrom and Z = 4. The structure was determined with molecular replacement method and refined to an R factor of 18.7% for all the data to 3.4 Angstrom resolution. The overall structure of seminal lactoferrin is similar to human colostrum lactoferrin. The amino acid sequence of seminal lactoferrin shows that it has one amino acid less than human colostrum lactoferrin and the structure of its N-terminal region is far more ordered than other lactoferrins. The structure of the iron-binding site and its immediate surroundings indicate well defined features.     

The effect of zinc on [59Fe]lactoferrin binding to blood neutrophilic leucocytes and colostral cells

Zn2+ inhibits the binding of [59Fe]lactoferrin to neutrophilic leucocytes. The inhibiting effect is proportional to zinc concentration in the range 10-330 mumol/l. Zn2+ inhibits the [59Fe]lactoferrin binding to the colostral cells in the same degree as PMN. The inhibiting effect of Zn2+ on [59Fe]lactoferrin binding to neutrophilic leucocytes is equal to those of non-labelled lactoferrin and transferrin. Fe2+ and Cu2+ does not have such effect on binding of [59Fe]lactoferrin to the PMN leucocytes.     

Comparison of the electron spin echo envelope modulation (ESEEM) for human lactoferrin and transferrin complexes of copper(II) and vanadyl ion

Copper(II) and vanadyl ions were bound to human milk lactoferrin or serum transferrin with carbonate or oxalate as the synergistic anion. Electron spin echo envelope modulation (ESEEM) due to nitrogen of a coordinated histidine imidazole was observed for both the copper and vanadyl complexes. For both metals, the modulation frequencies in the Fourier transforms of the data were similar for the two proteins and were weakly dependent on anion. When data in D2O/glycerol-d3 were compared with data in H2O/glycerol, the deep deuterium modulation indicated multiple exchangeable protons in the vicinity of the metals with at most one proton within about 2.9 A of the metal. The distribution of exchangeable protons around the metals as probed by ESEEM was the same, within experimental uncertainty, for the copper or vanadyl complexes with either carbonate or oxalate as the anion. When 13C-labeled oxalate was used as the synergistic anion, 13C-ESEEM was observed for both the copper and vanadyl complexes of lactoferrin and transferrin. The deeper 13C modulation for copper and vanadyl transferrin [13C]oxalate than for vanadyl transferrin [13C]carbonate suggests that both ends of the oxalate are bound to the metal in the transferrin and lactoferrin complexes.     

X-ray structure and the reaction mechanism of bovine heart cytochrome c oxidase

X-ray structure of bovine heart cytochrome c oxidase in the fully oxidized state shows a peroxide bridging between Fe2+ and Cu2+ in the O2 reduction site. The bond distances for Fe-O and Cu-O are 2.52 and 2.16 A, respectively. The structure is consistent with antiferromagnetic coupling between the two metals, which has long been known and to recent redox titration results [J. Biol. Chem. 274 (1999) 33403]. The trigonal planer coordination of Cu1+ in the O2 reduction site is consistent with the very weak interaction between Cu1+ and O2 bound at Fe2+ revealed by time-resolved resonance Raman investigations. One of the three histidine imidazoles coordinated to the Cu ion in the O2 reduction site fixes a tyrosine phenol group near the O2 reduction site with the direct covalent link between the two groups. The structure suggests that the phenol group is the site for donating protons to the bound O2. Redox-coupled conformational change in an extramembrane loop indicates that an aspartate (Asp51) in the loop apart from the O2 reduction site is the site for proton pumping.     

EPR investigation of Cu2+-substituted photosynthetic bacterial reaction centers: evidence for histidine ligation at the surface metal site

The coordination environments of two distinct metal sites on the bacterial photosynthetic reaction center (RC) protein were probed with pulsed electron paramagnetic resonance (EPR) spectroscopy. For these studies, Cu2+ was bound specifically to a surface site on native Fe2+-containing RCs from Rhodobacter sphaeroides R-26 and to the native non-heme Fe site in biochemically Fe-removed RCs. The cw and pulsed EPR results clearly indicate two spectroscopically different Cu2+ environments. In the dark, the RCs with Cu2+ bound to the surface site exhibit an axially symmetric EPR spectrum with g(parallel) = 2.24, A(parallel) = 160 G, g(perpendicular) = 2.06, whereas the values g(parallel) = 2.31, A(parallel) = 143 G, and g(perpendicular) = 2.07 were observed when Cu(2+) was substituted in the Fe site. Examination of the light-induced spectral changes indicate that the surface Cu2+ is at least 23 A removed from the primary donor (P+) and reduced quinone acceptor (QA-). Electron spin-echo envelope modulation (ESEEM) spectra of these Cu-RC proteins have been obtained and provide the first direct solution structural information about the ligands in the surface metal site. From these pulsed EPR experiments, modulations were observed that are consistent with multiple weakly hyperfine coupled 14N nuclei in close proximity to Cu2+, indicating that two or more histidines ligate the Cu2+ at the surface site. Thus, metal and EPR analyses confirm that we have developed reliable methods for stoichiometrically and specifically binding Cu2+ to a surface site that is distinct from the well characterized Fe site and support the view that Cu2+ is bound at or near the Zn site that modulates electron transfer between the quinones QA and QB (QA-QB --> QAQB-) (Utschig, L. M., Ohigashi, Y., Thurnauer, M. C., and Tiede, D. M (1998) Biochemistry 37, 8278-8281) and proton uptake by QB- (Paddock, M. L., Graige, M. S., Feher, G., and Okamura, M. Y. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 6183-6188). Detailed EPR spectroscopic characterization of these Cu2+-RCs will provide a means to investigate the role of local protein environments in modulating electron and proton transfer.     

Spectroscopic studies on copper (II) complexes of human lactoferrin

The interaction of Cu(II) with human lactoferrin has been studied as a function of pH, using electronic and electron spin resonance spectroscopy. Specific Cu(II) binding, with bicarbonate as the co-anion, occurs over the pH range 6 to 9. In the presence of a fiftyfold molar excess of oxalate, a monocopper(II) lactoferrin oxalate complex forms when the Cu(II) to protein is 1:1. If this ratio is increased to 2:1, a hybrid complex forms, in which the second copper utilizes bicarbonate as the co-anion, thus demonstrating, as for serum transferrin, a difference in the anion binding sites. The quenching of the intrinsic fluorescence of apolactoferrin is significantly less in the presence of oxalate than bicarbonate. The interaction of Cu(II) with apolactoferrin in the presence of the malonate, glycolate, thioglycolate, glycinate, and ethylenediaminetetraacetate ions has been examined.     

Infrared evidence of cyanide binding to iron and copper sites in bovine heart cytochrome c oxidase. Implications regarding oxygen reduction

Cyanide binding to bovine heart cytochrome c oxidase at five redox levels has been investigated by use of infrared and visible-Soret spectra. A C-N stretch band permits identification of the metal ion to which the CN- is bound and the oxidation state of the metal. Non-intrinsic Cu, if present, is detected as a cyanide complex. Bands can be assigned to Cu+CN at 2093 cm-1, Cu2+CN at 2151 or 2165 cm-1, Fe3+CN at 2131 cm-1, and Fe2+CN at 2058 cm-1. Fe2+CN is found only when the enzyme is fully reduced whereas the reduced Cu+CN occurs in 2-, 3-, and 4-electron reduced species. A band for Fe3+CN is not found for the complex of fully oxidized enzyme but is for all partially reduced species. Cu2+CN occurs in both fully oxidized and 1-electron-reduced oxidase. CO displaces the CN- at Fe2+ to give a C-O band at 1963.5 cm-1 but does not displace the CN- at Cu+. Another metal site, noted by a band at 2042 cm-1, is accessible only in fully reduced enzyme and may represent Zn2+ or another Cu+. Binding of either CN- or CO may induce electron redistribution among metal centers. The extraordinary narrowness of ligand infrared bands indicates very little mobility of the components that line the O2 reduction site, a property of potential advantage for enzyme catalysis. The infrared evidence that CN- can bind to both Fe and Cu supports the possibility of an O2 reduction mechanism in which an intermediate with a mu-peroxo bridge between Fe and Cu is formed. On the other hand, the apparent independence of Fe and Cu ligand-binding sites makes a heme hydroperoxide (Fe-O-O-H) intermediate an attractive alternative to the formation an Fe-O-O-Cu linkage.     

Heparin-binding properties of lactoferrin and lysozyme.

1. Binding of biotin-heparin to immobilized lactoferrin and lysozyme was optimum at pH 6.0, 100 mM NaCl. Complex interactions between NaCl and CaCl2 concentrations were observed for heparin binding to both proteins. 2. The metal ions Cu2+, Zn2+, Fe2+ and Fe3+ inhibited heparin binding, with half-maximal inhibition of binding to lactoferrin occurring between 600 microM and 1 mM and for lysozyme between 500 and 800 microM. 3. Binding of biotin-heparin to both proteins was inhibited to varying degrees by heparin, heparan sulfate, chondroitin sulfate A, dextran sulfate and DNA.     

Lactoferrin-protector against oxidative stress and regulator of glycolysis in human erythrocytes

Binding of lactoferrin (Lf) to its membrane receptors requires an electron for the reduction of Fe(3+)LF to Fe(2+)LF. It is possible that glyceraldehyde -3-phosphate dehydrogenase, a glycolytic enzyme part of the erythrocyte membrane, delivers that electron. Then Lf, obtaining an electron from the coenzyme NADH, might stimulate glycolysis, which requires the oxidised state of the coenzyme NAD+. Such possibility is supported by the finding that another extracellular e- acceptor--potassium ferricyanide activates glycolysis by the similar mechanism. Present results show that ferricyanide inhibited the specific 59Fe-lactoferrin binding to its erythrocyte membrane receptors. It may be assumed that ferricyanide competes with lactoferrin for an electron which leads to decrease of the binding of 59Fe-lactoferrin to its receptors. Lactoferrin (50 and 100 nM), similar to ferricyanide, increased the accumulation of lactate (respectively by 25% and 30%). These results support the assumption that ferricyanide and lactoferrin are final acceptors of a common electron transport chain connected with the regulation of glycolysis. We established an antioxidative effect of lactoferrin on erythrocytes, which was expressed as: a) an influence on content and on activity of intracellular antioxidants--namely an enhancement of the content of reduced glutathione; b) a decreased content both of products of lipid peroxidation (thiobarbituric acid reactive substances) and hemolysis under normal conditions and oxidative stress. Lactoferrin is capable to bind metal ions and thus to block their catalytic participation in the oxidative disturbances of the membrane. In most of our experiments there were no metal ions in the incubation mixtures (except those stimulating oxidative stress). Our results showed that Lf limited both the generation of thiobarbituric acid reactive substances and hemolysis in the absence of metal ions in the media, as well as in their presence. These facts suggest that probably the antioxidative property of lactoferrin is glycolysis stimulation, leading to increased formation of ATP, which is necessary to maintain the ion gradient, membrane potential and morphology of the erythrocyte.     

Certain metal ions are inhibitors of cytochrome b6f complex 'Rieske' iron-sulfur protein domain movements

Many current models of the Q cycle for the cytochrome (cyt) b6f and the cyt bc1 complexes incorporate 'Rieske' iron-sulfur protein (ISP) domain movements to gate electron transfer and to ensure high yields of proton shuttling. It was previously proposed that copper ions, which bind at a site distant from the quinol oxidase (Q(o)) site, inhibit plastoquinol (PQH2) binding by restraining the hydrophilic head domain of the ISP [Rao B. K., S., Tyryshkin, A. M., Roberts, A. G., Bowman, M. K., and Kramer, D. M. (1999) Biochemistry 38, 3285-3296]. The present work presents evidence that this is indeed the case for both copper ions and Zn2+, which appear to inhibit by similar mechanisms. Electron paramagnetic resonance (EPR) spectra show that Cu2+ and Zn2+ binding to the cyt b6f complex displaces the Q(o) site inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB). At high concentrations, both DBMIB and Cu2+ or Zn2+ can bind simultaneously, altering the Rieske 2Fe2S cluster and Cu2+ EPR spectra, suggesting perturbations in their respective binding sites. Both Zn2+ and Cu1+ altered the orientations of the Rieske 2Fe2S cluster with respect to the membrane plane, but had no effect on that of the cyt b6 hemes. Cu2+ was found to change the orientation of the cyt f heme plane, consistent with binding on the cyt f protein. Within conservative constraints, the data suggest that the ISP is shifted into a position intermediate between the ISP(C) position, when the Q(o) site is unoccupied, and the ISP(B) position, when the Q(o) site is occupied by inhibitors such as DBMIB or stigmatellin. These results support the role of ISP domain movements in Q(o) site catalysis.     

Binding and endocytosis of apo- and holo-lactoferrin by isolated rat hepatocytes.

We characterized binding and endocytosis of 125I-bovine lactoferrin by isolated rat hepatocytes. Iron-depleted (apo-Lf), approximately 30% saturated (Lf), and iron-saturated (holo-Lf) lactoferrin were used. At 4 degrees C, cells bound 125I-apo-Lf and 125I-holo-Lf with nearly identical apparent first order kinetics (t1/2 = approximately 42 min). Holo-Lf and apo-Lf competed with each other for binding. Hepatocytes bound lactoferrin optimally at pH greater than or equal to 7 but poorly at pH less than or equal to 6. Ca2+ (greater than or equal to 100 microM) enhanced Lf binding to cells, and holo-Lf remained monomeric with Ca2+ present as determined by gel filtration chromatography. With Ca2+, cells exhibited approximately 10(6) high affinity sites (Kd approximately 20 nM) and approximately 10(7) low affinity sites (Kd approximately 700 nM) for both apo- and holo-Lf. Without Ca2+, cells bound 125I-holo-Lf by the low affinity component only. EGTA and dextran sulfate together released greater than or equal to 90% 125I-Lf prebound at 4 degrees C, but individually removed separate populations of surface-bound 125I-Lf. Cells bound 125I-Lf in a Ca(2+)-dependent manner with dextran sulfate present. We conclude that the high affinity but not the low affinity sites require Ca2+; only the low affinity sites are dextran sulfate-sensitive. Neither transferrin nor asialo-orosomucoid blocked lactoferrin binding to hepatocytes. Some cationic proteins but not others inhibited lactoferrin binding. At 37 degrees C, hepatocytes endocytosed 125I-apo-Lf and 125I-holo-Lf similarly, and hyperosmolality (greater than 500 mmol/kg) blocked uptake by approximately 90%. These data support the proposal that hepatocytes regulate blood lactoferrin concentration by receptor-mediated endocytosis.     

In vitro interaction between ceruloplasmin and human serum transferrin

The thermodynamics of the interactions of serum apotransferrin (T) and holotransferrin (TFe(2)) with ceruloplasmin (Cp), as well as those of human lactoferrin (Lf), were assessed by fluorescence emission spectroscopy. Cp interacts with two Lf molecules. The first interaction depends on pH and μ, whereas the second does not. Dissociation constants were as follows: K(11Lf) = 1.5 ± 0.2 μM, and K(12Lf) = 11 ± 2 μM. Two slightly different interactions of T or TFe(2) with Cp are detected for the first time. They are both independent of pH and μ and occur with 1:1 stoichiometry: K(1T) = 19 ± 7 μM, and K(1TFe2) = 12 ± 4 μM. These results can improve our understanding of the probable process of the transfer of iron from Cp to T in iron and copper transport and homeostasis.