Redox properties of human transferrin bound to its receptor

Virtually all organisms require iron, and iron-dependent cells of vertebrates (and some more ancient species) depend on the Fe(3+)-binding protein of the circulation, transferrin, to meet their needs. In its iron-donating cycle, transferrin is first captured by the transferrin receptor on the cell membrane, and then internalized to a proton-pumping endosome where iron is released. Iron exits the endosome to enter the cytoplasm via the ferrous iron transporter DMT1, a molecule that accepts only Fe(2+), but the reduction potential of ferric iron in free transferrin at endosomal pH (approximately 5.6) is below -500 mV, too low for reduction by physiological agents such as the reduced pyridine nucleotides with reduction potentials of -284 mV. We now show that in its complex with the transferrin receptor, which persists throughout the transferrin-to-cell cycle of iron uptake, the potential is raised by more than 200 mV. Reductive release of iron from transferrin, which binds Fe(2+) very weakly, is therefore physiologically feasible, a further indication that the transferrin receptor is more than a passive conveyor of transferrin and its iron.     

Rapid endocytosis of the transferrin receptor in the absence of bound transferrin

The rate of endocytosis of transferrin receptors, occupied or unoccupied with transferrin, was measured on the cell line K562. At 37 degrees C, receptors, radioiodinated on the cell surface at 4 degrees C, were internalized equally rapidly in the presence or absence of transferrin. In both cases, 50% of the labeled receptors became resistant to externally added trypsin in 5 min. An antitransferrin antibody was used to show directly that the receptors had entered the cells without bound transferrin. The distribution of the receptors on the cell surface was revealed by antibody and protein A-gold staining after prolonged incubation in the presence or absence of transferrin. The receptors were concentrated in coated pits under both conditions. The data suggest that endocytosis of transferrin receptors is not "triggered" by ligand binding and raise the possibility that ligand-induced down-regulation of surface receptors may not occur by this mechanism. Instead receptors may be recognized as being ligand-occupied, not at the cell surface, but at some other site in the recycling pathway such as the endosome.     

Comaprative binding study of aluminum and chromium to human transferrin

The characteristics of aluminum and chromium binding to apotransferrin (apo-tf) have been investigated and compared. Both metal ions were taken up by human transferrin forming complexes with the maximum absorbances at 405 nm for chromium-transferrin (cr-tf) and 240 nm for aluminum-transferrin (Al-tf). In the presence of citric acid, chromium binding to transferrin is five times more than aluminum. The binding of aluminum or chromium to apo-transferrin was reduced by 18 and 22% in the presence of 200 ng/mL of iron. The binding of both metals to apo-tf appears to be pH dependent. In acidic pHs, less chromium and more aluminum binding occurred.     

Direct spectrophotometric determination of the two site binding of aluminum to transferrin

Difference UV spectrophotometry is used to determine the conditional binding constants for aluminum to human transferrin in the presence of HCO3- of initial concentration 18 mM according to a two site model. The values obtained at 37 degrees C and pH 7.4 are: K1 = 3.0 (+/- 0.8) X 10(15) M-2, K2 = 2.9 (+/- 0.7) X 10(15) M-2 for the reactions: Tr + Al3+ + HCO3- = Tr-Al-HCO3 and Tr-Al-HCO3 + Al3+ + HCO3- = Tr-Al2-(HCO3)2 respectively. Possible consequences arising for the transport of aluminum in human serum are discussed.     

Functional equivalence of iron bound to human transferrin at low pH or high pH

Human transferrin was labeled with ⁵⁹Fe at one of its two metal-binding sites (designated A) at pH 6.0. ⁵⁵Fe was then added to site B at pH 7.5. Both isotopes of iron were taken up in equal proportions by human reticulocytes. These experiments do not support the hypothesis that each binding site of transferrin has a different physiologic function.     

Preferential utilization in vitro of iron bound to diferric transferrin by rabbit reticulocytes.

59Fe uptake by rabbit reticulocytes from human transferrin-bound iron was studied by using transferrin solutions (35, 50, 65, 80 and 100% saturated with iron) whose only common characteristic was their content of diferric transferrin. During the early incubation period, 59Fe uptake from each preparation by reticulocytes was identical despite wide variations in amounts of total transferrin, total iron, monoferric transferrin and apotransferrin in solution. During the later phase of incubation, rate of uptake declined and was proportional to each solution's monoferric transferrin content. Uptake was also studied in a comparative experiment which used two identical, partially saturated transferrin preparations, one uniformly 59Fe-labelled and the other tracer-labelled with [59Fe]diferric transferrin. In both experiments, iron uptake by reticulocytes corresponded to utilization of a ferric ion from diferric transferrin before utilization of iron from monoferric transferrin.     

S-adenosylhomocysteine is bound to pineal hydroxyindole o-methyltransferase

A substance with maximal absorbance at 260 nm was co-chromatographed with hydroxyindole O-methyltransferase (EC 2.1.1.4) by immunoaffinity chromatography. The co-chromatographed substance was separated from the transmethylase by stepwise elution and was identified as S-adenosylhomocysteine by spectrophotometrical analysis, and by thin-layer chromatography. Identity of S-adenosylhomocysteine was confirmed by determination of demethylated product by using a mixture of [carboxyl-¹⁴C]S-adenosylmethionine and [methyl-³H]S-adenosylmethionine as a substrate. The immunoaffinity chromatography provides direct evidence for a presence of the enzyme-product complex $\text{in vivo}$ and $\text{in vitro}$. At low concentration of S-adenosylmethionine enzymatic activity was inhibited by the co-purified S-adenosylhomocysteine. The endogenous S-adenosylhomocysteine bound to the enzyme probably regulates the melatonin biosynthesis $\text{in vivo}$.     

Kinetics of the release of aluminum from human serum dialuminum transferrin to citrate

The kinetics of release of Al3+ from human serum dialuminum transferrin (Al2Tf) to citrate were investigated at 37 degrees C, pH 7.4, mu = 0.7 M, by difference UV spectrophotometry. The two metal-binding sites are not identical but behave in a kinetically similar manner to give apparent second-order rate constants of 0.60 and 0.38 M-1 s-1, respectively, for release of the first Al3+ from Al2Tf. The rate constants for release of the second metal ion from the monoaluminum transferrins are 0.27 and 0.12 M-1 s-1. The kinetic scheme for release of A13+ from Al2Tf is therefore similar to that for release of Fe3+ from Fe2Tf, but the rate of constants for metal ion release are between two and four orders of magnitude larger.     

Functional equivalence of iron bound to human transferrin at low pH or high pH.

Human transferrin was labeled with 59Fe at one of its two metal-binding sites (designated A) at pH 6.0. 55Fe was then added to site B at pH 7.5. Both isotopes of iron were taken up in equal proportions by human reticulocytes. These experiments do not support the hypothesis that each binding site of transferrin has a different physiologic function.     

A protease is bound to rat liver nucleosomes

Nucleosome prepared from pure rat liver nuclei contain protease activity. No protease is associated with nucleosome core particle and the protease-containing nucleosome has considerably higher sedimentation coefficient than the bulk nucleosomes. Only H1 histone is susceptible to the nucleosome-bound protease.     

The region of human transferrin involved in binding to bacterial transferrin receptors is iocalized in the C-lobe

Iron-saturated human transferrin was digested with either chymotrypsin or trypsin to produce C-lobe and N-lobe protein fragments. Individual protein fragments were purified by a combination of gel filtration and Concanavalin A affinity chromatographic procedures. The C-lobe and N-lobe fragments of human transferrin were then used in binding assays to assess their ability in binding to the bacterial transferrin receptors. Competitive binding assays demonstrated that the C-lobe fragment of human transferrin binds as well as intact human transferrin to bacterial transterrin receptors from Neisseria meningitidis, Neisseria gonorrhoeae and Haemophlius influenzae. Using isogenic mutants of N. meningitidis deficient in either of the transferrin-binding proteins (Tbps), we demonstrated that both transferrin-binding proteins were able to bind to the C-lobe fragment of human transferrin.     

Arc repressor is tetrameric when bound to operator DNA

The Arc repressor of bacteriophage P22 is a member of a family of DNA-binding proteins that use N-terminal residues in a beta-sheet conformation for operator recognition. Here, Arc is shown to bind to its operator site as a tetramer. When mixtures of Arc (53 residues) and an active variant of Arc (78 residues) are used in gel retardation experiments, five discrete protein-DNA complexes are observed. This result is as expected for operators bearing heterotetramers containing 4:0, 3:1, 2:2, 1:3, and 0:4 ratios of the two proteins. Direct measurements of binding stoichiometry support the conclusion that Arc binds to a single 21-base-pair operator site as a tetramer. The Arc-operator binding reaction is highly cooperative (Hill constant = 3.5) and involves at least two coupled equilibria. In the first reaction, two unfolded monomers interact to form a folded dimer (Bowie & Sauer, 1989a). Rapid dilution experiments indicate that the Arc dimer is the kinetically significant DNA-binding species and allow an estimate of the equilibrium dissociation constant for dimerization [K1 = 5 (+/- 3) x 10(-9) M]. The rate of association of Arc-operator complexes shows the expected second-order dependence on the concentration of free Arc dimers, with k2 = 2.8 (+/- 0.7) x 10(18) M-2 s-1. The dissociation of Arc-operator complexes is a first-order process with k-2 = 1.6 (+/- 0.6) x 10(-4) s-1. The ratio of these kinetic constants [K2 = 5.7 (+/- 2.3) x 10(-23) M2] provides an estimate for the equilibrium constant for dissociation of the DNA-bound tetramer to two free Arc dimers and the operator. An independent determination of this complex equilibrium constant [K2 = 7.8 (+/- 4.8) x 10(-23) M2] was obtained from equilibrium binding experiments.     

The erythroblast antigen: a membrane protein bound to the erythroid form of the transferrin receptor?]

An interspecific marker of mammalian erythroid cells, which was called the erythroblast antigen, was identified in 1974, using polyclonal monospecific antibodies. Further studies have demonstrated the expression of this antigen in a variety of nonhemopoietic organs and tissues, which have the following common feature: they have a barrier location; that is, they are located at the boundary. It has been proposed that the erythroblast antigen participates directly or indirectly in the transport of various substances and specifically transport of iron. The present review deals with this topic.     

Comparison of the kinetics of cycling of the transferrin receptor in the presence or absence of bound diferric transferrin.

The kinetics of cycling of the transferrin receptor in A431 human epidermoid-carcinoma cells was examined in the presence or absence of bound diferric transferrin. In order to investigate the properties of the receptor in the absence of transferrin, the cells were maintained in defined medium without transferrin. It was demonstrated that Fab fragments of a monoclonal anti-(transferrin receptor) antibody (OKT9) did not alter the binding of diferric 125I-transferrin to the receptor or change the accumulation of [59Fe]diferric transferrin by cells. OKT9 125I-Fab fragments were prepared and used as a probe for the function of the receptor. The first-order rate constants for endocytosis (0.16 +/- 0.02 min-1) and exocytosis (0.056 +/- 0.003 min-1) were found to be significantly lower for control cells than the corresponding rate constants for endocytosis (0.22 +/- 0.02 min-1) and exocytosis (0.065 +/- 0.004 min-1) measured for cells incubated with 1 microM-diferric transferrin (mean +/- S.D., n = 3). The cycling of the transferrin receptor is therefore regulated by diferric transferrin via an increase in both the rate of endocytosis and exocytosis. Examination of the accumulation of OKT9 125I-Fab fragments indicated that diferric transferrin caused a marked decrease in the amount of internalized 125I-Fab fragments associated with the cells after 60 min of incubation at 37 degrees C. Diferric transferrin therefore increases the efficiency of the release of internalized 125I-Fab fragments compared with cells incubated without diferric transferrin. These data indicate that transferrin regulates the sorting of the transferrin receptor at the cell surface and within endosomal membrane compartments.