Evidence for the functional equivalence of the iron-binding sites of rat transferrin

The role of the two iron-binding sites of rat transferrin in the exchange of iron with cells has been assessed using urea polyacrylamide gel electrophoresis to separate and quantitate the four possible molecular species of transferrin generated during the incubation of ¹²⁵I-labelled transferrin with rat reticulocytes and hepatocytes. Addition of diferric transferrin to reticulocytes led directly to the appearance of apotransferrin together with small and comparable amounts of the two monoferric transferrins. After 2 h 44.8% of the iron had been removed by the cells, and of the iron-depleted transferrin 71.8% was apotransferrin, the remainder being monoferric transferrin, 16.1% with N-terminal iron and 12.1% with C-terminal iron. A similar pattern emerged with hepatocytes, but the rate of iron removal was slower and the proportion of apotransferrin generated was lower. After 4 h 10.9% of the iron had been removed from the transferrin and the distribution of the iron-depleted protein was: apotransferrin 26.9% and monoferric (N-terminal) 39.2%, (C-terminal) 33.9%. The appearance of apotransferrin during each incubation and the generation of both monoferric transferrins suggest that both cell types are able to remove iron from differic transferrin in pairwise fashion and that they do not appreciably distinguish between the two iron-binding sites of the protein. Release of iron from hepatocytes to apotransferrin lead to the appearance of both monferric species and then to increasing amounts of diferric transferrin. The process of iron release did not seem to distinguish between the vacant iron-binding sites of transferrin.     

Functional heterogeneity and pH-dependent dissociation properties of human transferrin

Human diferric transferrin was partially labeled with ⁵⁹Fe at low or neutral pH (chemically labeled) and by replacement of diferric iron previously donated to rabbit reticulocytes (biologically labeled). Reticulocyte ⁵⁹ uptake experiments with chemically labeled preparations indicated that iron bound at near neutral ph was more readily incorporated by reticulocytes than iron bound at low pH. The pH-dependent iron dissociation studies of biologically labeled transferrin solutions indicated that Fe³⁺, bound at the site from which the metal was initially utilized by the cells, dissociated between pH 5.8 and 7.4. In contrast, lower pH (5.2–5.8) was required to effect dissociation of iron that had remained bound to the protein after incubation with reticulocytes. These findings suggest that each human transferrin iron-binding site has different acid-base iron-binding properties which could be related to the observed heterogenic rabbit reticulocyte iron-binding properties of human transferrin and identifies that the near neutral iron-donating site initially surrenders its iron to these cells.     

Effect of Separation Conditions on Automated Isoelectric Focusing of Carbohydrate-Deficient Transferrin and Other Human Isotransferrins Using the PhastSystem

To investigate the effect of automated isoelectric focusing conditions in the PhastSystem, e.g., the point of sample application, prerun and separation times, and minimized gels on isotransferrin band pattern, human sera were analyzed with native transferrin iron load, after iron saturation or iron depletion in vitro. Varying the focusing conditions we found (i) Point of sample application (anode, middle of the gel, cathode) strongly affected transferrin iron loss. It was greatest at the anode and least at the cathode. (ii) Without prerun, distinct transferrin iron loss also occurred. A short prerun time prevented iron loss, but increasing it did not improve transferrin iron load stability as stated by others. (iii) An inappropriately long separation time inevitably yielded iron loss. In conclusion, inappropriate isoelectric focusing conditions strongly affect iron load stability of isotransferrins (obviously via low pH within the gel), resulting in transferrin iron release and cofocusing of isotransferrins with different sialic acid or iron contents. For determination of carbohydrate-deficient transferrin, such conditions resulted in overestimation of the marker of chronic alcohol abuse. Our findings may be of guiding importance for isoelectric focusing of protein-ligand complexes. We recommend the procedure described for development of isoelectric focusing of protein-ligand complexes.     

Transferrin uptake and release by reticulocytes treated with proteolytic enzymes and neuraminidase

The mechanism of transferrin uptake by reticulocytes was investigated using rabbit transferrin labelled with ¹²⁵I and ⁵⁹Fe and rabbit reticulocytes which had been treated with trypsin, Pronase or neuraminidase. Low concentrations of the proteolytic enzymes produced a small increase in transferrin and iron uptake by the cells. However, higher concentrations or incubation of the cells with the enzymes for longer periods caused a marked fall in transferrin and iron uptake. This fall was associated with a reduction in the proportion of cellular transferrin which was bound to a cell membrane component solubilized with the non-ionic detergent, Teric 12A9. The effect of trypsin and Pronase on transferrin release from the cells was investigated in the absence and in the presence of $\text{N-}\text{ethylmaleimide}$ which inhibits the normal process of transferrin release. It was found that only a small proportion of transferrin which had been taken up by reticulocytes at 37°C but nearly all that taken up 4°C was released when the cells were subsequently incubated with trypsin plus $\text{N-}\text{ethylmaleimide}$, despite the fact that about 80% of the ⁵⁹Fe in the cells was released in both instances. Neuraminidase produced no change in transferrin and iron uptake by the cells.These experiments provide evidence that transferrin uptake by reticulocytes requires interaction with a receptor which is protein in nature and that following uptake at 37°C, most of the transferrin is located at a site unavailable to the action of proteolytic enzymes. The results support the hypothesis that transferrin enters reticulocytes by endocytosis.     

The K+-induced apparent heterogeneity of high-affinity nucleotide-binding sites in (Na+ + K+)-ATPase can only be due to the oligomeric structure of the enzyme

K⁺ induces an apparent heterogeneity among an otherwise homogeneous population of nucleotide-binding sites in (Na⁺ + K⁺)-ATPase preparations from pig kidney. With the help of ouabain we show that this heterogeneity cannot be due to a mixture of different and independent sites and conclude that each enzyme molecule must contain two nucleotide site-containing units that show interaction. Na⁺ induces an apparent heterogeneity among an otherwise homogeneous population of ouabain-binding sites. The argument is, therefore, extended to include one ouabain site on each of the structural units that bind nucleotide. All these structural units are shown to hydrolyse substrate at identical rates. Using the presently available molecular weight data, it is concluded that the enzyme is composed of two subunits each possessing one nucleotide-binding site, one ouabain-binding site, one α-peptide and the capacity for hydrolysing ATP and $\text{p-}\text{nitrophenyl}$ phosphate.     

Vanadium complexes of transferrin and ferritin in the rat

Vanadium associates with serum transferrin of rats administered vanadyl(IV) sulfate or ammonium metavanadate(V) by gastric intubation. Low molecular weight species account for only 3% of the vanadium present in plasma. The element distributes between the two major isotransferrins in proportion to their concentrations. Rat apotransferrin binds both vanadium(IV) and vanadium(V), forming 2:1 metal-protein complexes in both instances. Although the two isotransferrins apparently differ in their physiological properties, they exhibit identical vanadyl(IV) (VO2+) EPR spectra, indicating identical or very similar metal binding sites for both proteins. In contrast to other transferrins, the two sites of the rat protein are spectroscopically indistinguishable and exhibit a VO2+ EPR spectrum similar to that of the C-terminal metal binding site of human serum transferrin. VO2+ EPR signals are observed with liver, spleen, and kidney tissue samples from animals maintained on a vanadium-supplemented diet. These signals arise from a specific intracellular VO2+ complex with the iron storage protein ferritin.     

The role of calcium in transferrin and iron uptake by reticulocytes

The possible role of calcium in the uptake of transferrin and iron by rabbit reticulocytes was investigated by altering cellular calcium levels through the use of the chelating agents EDTA and $\text{ethyleneglycol-bis}\text{-(3-}\text{aminoethylether}\text{)-N,N′-}\text{tetraacetic}$ acid (EGTA) and the ionophores, A23187 and X537A. Incubation of reticuloyctes with EDTA or EGTA at 4°C had no effect on transferrin and iron uptake but incubation at 37°C resulted in an irreversible inhibition associated with decreased adsorption of transferrin to the cells and evidence of inactivation or loss of the transferrin receptors. Transferrin and iron uptake were also inhibited when the cells were incubated with A23187 or X537A. In the case of A23187 the action was primarily exerted on the temperature-sensitive stage of transferrin uptake and was associated with loss of cellular K⁺ and decrease in cell size. The effect was greater when Ca²⁺ was added to the incubation medium than its absence. X537A produced relatively greater inhibition of iron uptake than of transferrin uptake, associated with a reduction in cellular ATP concentratio. The action of X537A was unaffected by the presence of Ca²⁺ in the incubation medium.The results obtained with EDTA and EGTA indicate that cell membrane Ca²⁺ is required for the integrity or binding of transferrin receptors to the reticulocyte membrane. No evidence was obtained from the experiments with ionophores that an increase of cellular Ca²⁺ affects transferrin and iron uptake directly. The inhibition caused by A23187 was mainly due to a reduction in cell size resulting from increased membrane permeability to K⁺ and that caused by X537A appeared to result from an inhibition of energy metabolism and ATP production.     

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.     

Site of action for β-endorphin-induced changes in plasma luteinizing hormone and prolactin in the ovariectomized rat

A number of sites have been hypothesized as loci at which opioid substances act to alter the secretion of luteinizing hormone (LH) and prolactin (PRL) (1–8). The aim of the present study was to determine the site(s) at which the opioid peptide β-endorphin (β-END) acts to influence plasma LH and PRL levels in the ovariectomized (OVX) rat. β-END, administered into the third ventricle of conscious OVX rats fitted with jugular catheters, significantly decreased plasma LH in doses ? 50 ng and increased PRL levels at all doses administered (10, 50, 100 and 250 ng) in a dose dependent fashion. To identify possible central nervous system sites of action, 250 ng β-END was unilaterally infused into various brain sites. Plasma LH was significantly decreased and plasma PRL significantly increased by infusions into the ventromedial hypothalamic area, the anterior hypothalamic area, and the preoptic-septal area. There was no significant effect of β-END infusions into the lateral hypothalamic area, amygdala, midbrain central gray, or caudate nucleus. When hemipituitaries of OVX rats were incubated $\text{in}$$\text{vitro}$ with β-END (10^?7M to 10^?5M), there was no suppression of basal or LHRH-induced LH release, nor was there any alteration of basal PRL release. It is concluded that β-END acts at a medial hypothalamic and/or preoptic-septal site and not the pituitary, to alter secretion of LH and PRL.     

Ontogeny of the mammalian insulin receptor: Studies of human and rat fetal liver plasma membranes

Insulin binding to human fetal plasma liver membranes was studied in preparations segregated into three pools according to length of gestation: 15–18 weeks (Pool A), 19–25 weeks (Pool B), and 26–31 weeks (Pool C). Receptor numbers, calculated by extrapolation of Scatchard plots to the X axis, increased from 25 × 10¹⁰ sites per 100 μg protein in the youngest group (Pool A) to 46 × 10¹⁰ sites per 100 μg protein in Pool B. No further increase in receptor number was seen in Pool C. The affinity constant for insulin at tracer concentrations, $\text{K}{}_{\mathrm{<mtext>e</mtext>}}$ (“empty site”), was 1.53 × 10⁸M^?1 in Pool A and was only slightly higher than $\text{K}{}_{\mathrm{<mtext>f</mtext>}}$ (“filled site”). $\text{K}{}_{\mathrm{<mtext>e</mtext>}}$ was higher in Pool B, 1.75 × 10⁸M^?1, and in Pool C reached a value of 5.63 × 10⁸M^?1. In Pool C $\text{K}{}_{\mathrm{<mtext>f</mtext>}}$ was 2.3 × 10⁸M^?1. Insulin binding of liver plasma membranes from rat fetuses aged 14, 16, 18, and 21 (term) days and adults was also studied. Maximum binding capacity tended to increase with gestational age and was 130 × 10¹⁰ sites per 100 μg protein at term, which was in excess of that found in adult rats (89–90 × 10¹⁰). In addition, $\text{K}{}_{\mathrm{<mtext>e</mtext>}}$ increased from 0.75 × 10⁸M^?1 at 14 days to 3.02 × 10⁸M^?1 at term, a value higher than that found in pregnant and nonpregnant adults. Dissociation of insulin in the presence of high concentrations of insulin was significantly enhanced in tissues from 18-day and term fetuses and adults, but not in membranes from fetal rats aged 14 and 16 days. These data appear to indicate that site-site interactions are not present in early fetal existence. These changes in insulin binding with increased length of gestation are not ascribable to changes in relative proportions of hematopoietic and parenchymal tissue. Human fetal plasma liver membranes demonstrated elevated insulin binding with increased gestational age, but comparison of fetal and adult liver could not be done. However, newborn human infants have been shown to have a higher capacity for binding insulin to circulating monocytes than adults. Also, human fetuses apparently lack the capability to diminish monocyte receptors in the presence of hyperinsulinemia. These experiments show that an increase in insulin receptor binding capacity and affinity also occurs in the liver of the rat fetus at term as compared to the adult rat. The reasons and mechanisms underlying enhanced capacity for insulin binding by fetal and newborn members of human and rodent species are not known.     

Benzodiazepine receptor: Heterogeneity in rabbit brain

Binding characteristics of benzodiazepine receptors were studied with synaptosomal and microsomal membranes from rabbit brain $\text{in}$$\text{vitro}$ utilizing [methyl-³H]diazepam. In synaptosomal membranes, both high and low affinity binding sites were identified with the dissociation constants (K_d) of 4.92 nM and 83.8 nM, respectively. However, only the high affinity site was identified with K_d of 3.96 nM with microsomal membranes. Benzodiazepine binding sites appear to include at least two subpopulations of receptors, one with high affinity and another with low affinity binding site.     

Mössbauer studies of electrophoretically purified monoferric and diferric human transferrin

Summary Electrophoretically purified⁵⁷Fe-enriched monoferric and diferric human transferrins and selectively labeled complexes ([C-⁵⁶Fe,N-⁵⁷Fe]transferrin and [C-⁵⁷Fe,N-⁵⁶Fe]transferrin) were studied by Mössbauer spectroscopy. The data were recorded at 4.2 K over a wide range of applied magnetic fields (0.05–6 T) and were analyzed by a spin-Hamiltonian formalism. Characteristic hyperfine parameters were found and the obtained zero-field splitting parameters (D=0.25±0.05 cm^–1 andE/D = 0.30 ± 0.02) agree with previous electron paramagnetic resonance (EPR) findings. The weak-field spectra of the [N-⁵⁷Fe]transferrin are slightly broader than those of the [C-⁵⁷Fe]transferrin, indicating that the N-terminal iron site may be more heterogeneous. However, the absorption line positions and the relative intensities of the subspectra originating from the three Kramers doublets of each Fe³⁺ site are identical. Thus the electronic structures of the two iron sites can be described by the same set of spin- Hamiltonian parameters, indicating that the ligand environments for the two sites are the same, as suggested by the recent X-ray crystallographic studies. This suggestion is further supported by the observation that the strong-field spectra of the two monoferric transferrins are indistinguishable. The selectively labeled mixed-isotope transferrins exhibit spectra that are identical to those of the corresponding monoferric⁵⁷Fe-enriched transferrins, implying that the occupation of one iron site has little or no effect on the immediate envirnoment of the other site, a finding that is not surprising since the two sites are separated by approximately 4.2 nm.     

Metabolism and uptake of L-pipecolic acid by brain and heart

Metabolism and uptake of $\text{L}$-[1-¹⁴C]pipecolate was studied in the rat through tail vein injection at low (30 μg/kg) and high (30 mg/kg) doses. No radioactive compound other than $\text{L}$-pipecolate was detected in the brain or heart samples 0.5 to 60 min after injection. The contents of $\text{L}$-pipecolate in the brain dropped rapidly to reach a plateau value 2 min after injection both in the low and high dose experiments (from 0.06 to 0.05 and from 86 to 55 nmole/g brain, respectively). Similar results were observed for the heart except that the heart had $\text{L}$-pipecolate contents 2–3 fold higher than the brain at every time interval. The influx of $\text{L}$-pipecolate to the brain, as measured by the plasma/brain ratio of its contents, was 3 fold lower than the heart at each sampling point throughout the course of measurement for both dosages. The influx of $\text{L}$-pipecolate from the plasma to the heart reached an equilibrium, i.e., plasma/heart = 1, 60 min after injection for both dosages; the plasma to brain ratio was 3 at 60 min. These results indicate that there is a blood-brain transport barrier for $\text{L}$-pipecolate in the rat, which may account for the differences in neuronal effects of $\text{L}$-pipecolate when administered intracerebrally or intraperitoneally.     

Differences between human and rabbit transferrins

Rabbit reticulocyte incorporation of iron from rabbit transferrin was independent of transferrin iron saturation but uptake from human transferrin was saturation dependent. Unlike human transferrin, rabbit transferrin does not surrender its iron from any unique preferred iron-binding site and can be described as functionally homogeneic.The two proteins also differ in their acid-base iron-binding properties. One human transferrin iron binding site retains an ability to bind iron at somewhat acid pH but this property is not shared by rabbit transferrin.     

Bromoacetylcholine as an affinity label of the acetylcholine receptor from Torpedo californica

Bromoacetyl[methyl-³H]choline is a highly specific label for the reduced acetylcholine binding site on the acetylcholine receptor from $\text{Torpedo californica}$. Only one of two binding sites per receptor monomer is susceptible to labeling. The labeled site is on the α chain of the receptor.     

Ligand binding properties of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase labelled with N-cyclohexyl-N′-(4-dimethylamino-α-naphthyl)carbodiimide

The $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ of rabbit sarcoplasmic reticulum, when labelled at two Ca²⁺-protected sites with $\text{N-}\text{cyclohexyl}\text{-N′-(4-}\text{dimethylamino}\text{-α-}\text{naphthyl}\text{)}\text{carbodiimide}$ (NCD-4) retains Ca²⁺ binding capacity at the sites with $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ values of approx. 3 μM and 0.12 mM as assessed by fluorescence titration. The sites correspond to the two high-affinity Ca²⁺ binding sites present in the native ATPase. The NCD-4 labelled ATPase exhibits slow conformational changes at each site on addition of Ca²⁺. It retains the ability to form phosphoenzyme, and can most likely translocate Ca²⁺.     

Differences between human and rabbit transferrins.

Rabbit reticulocyte incorporation of iron from rabbit transferrin was independent of transferrin iron saturation but uptake from human transferrin was saturation dependent. Unlike human transferrin, rabbit transferrin does not surrender its iron from any unique preferred iron-binding site and can be described as functionally homogeneic. The two proteins also differ in their acid-base iron-binding properties. One human transferrin iron binding site retains an ability to bind iron at somewhat acid pH but this property is not shared by rabbit transferrin.     

Quantitative assessment of heterogeneous 3H-spiperone binding to rat neostriatum and frontal cortex

Anomalies of the binding of ³Hspiperone to rat cerebral membranes have been examined. By employing a very low ligand concentration ($\text{～ 25}\text{pM}{}^3\text{Hspiperone}$) we have demonstrated that even within the corpus striatum, ³Hspiperone appears to bind to multiple sites and that dopaminergic and serotonergic agents can selectively inhibit from these sites. In the corpus striatum, 75–80% of the ³Hspiperone specific binding can be inhibited with high affinity by dopaminergic drugs while some 20–30% is inhibited with high affinity by serotonergic compounds. The two ³Hspiperone sites, which we have shown to have affinities of 31 and 325 pM, may therefore represent dopaminergic and serotonergic sites. At higher concentrations of ³Hspiperone, however, the picture may be complicated by a further low affinity site. The great selectivity shown by dopaminergic agonists for the two ³Hspiperone sites explains the ‘flattened’ displacement curves reported for ³Hspiperone/agonist interactions. As dopaminergic agents show the greater affinity for the high affinity ³Hspiperone site, it is tempting to speculate that this site has the greatest association with the dopamine receptor.     

Iron release from transferrin, its C-lobe, and their complexes with transferrin receptor: presence of N-lobe accelerates release from C-lobe at endosomal pH

Human transferrin, like other members of the transferrin class of iron-binding proteins, is a bilobal structure, the product of duplication and fusion of an ancestral gene during the course of biochemical evolution. Although the two lobes exhibit 45% sequence identity and identical ligand structures of their iron-binding sites (one in each lobe), they differ in their iron-binding properties and their responsiveness to complex formation with the transferrin receptor. A variety of interlobe interactions modulating these iron-binding functions has been described. We have now studied the kinetics of iron release to pyrophosphate from the isolated recombinant C-lobe and from that lobe in the intact protein, each free and bound to receptor. The striking finding is that the rates of iron release at the pH of the endosome to which transferrin is internalized by the iron-dependent cell are similar in the free proteins but 18 times faster from full-length monoferric transferrin selectively loaded with iron in the C-lobe than from isolated C-lobe when each is complexed to the receptor. The possibility that the faster release in the receptor complex of the full-length protein at endosomal pH contributes to the evolutionary advantage of the bilobal structure is considered.