Iron protein reticulocytes

The dwell time of a transferrin molecule with the reticulocyte may be only a minute or two (52, 80) or possibly as long as 10 min (86), after which the protein is released for another cycle of iron transport. The affinity of reticulocyte receptors for apotransferrin appears less than for iron-loaded molecules (80). This property may facilitate the release of the protein from the receptor after it has donated its iron and ensures that the apoprotein will not impede the delivery of iron to reticulocytes by competing with iron-bearing molecules for available receptors on the cell surface. 

Schade reviewed (114) the earlier studies on the role of serum transferrin in iron transport. Various early investigators had observed that the blood serum transferrin rapidly bound iron administered either through the gastrointestinal tract or by intravenous injection. There was a rapid turnover of iron in the blood serum and the degree of saturation of the transferrin was related to the amount of iron administered. In no instances, however, was the blood serum transferrin ever saturated with iron. Jandl et al. (71) have shown that both ovotransferrin and serum transferrin can transport plasma iron into red cells and that the transport is dependent on the concentration of transferrin. Iron taken up by the blood cells could not be eluted by subsequent incubation with iron-free transferrin solutions. More recently Morgan and Laurel (99) reported that iron uptake in reticulocytes is independent of the transferrin concentration. The iron complex of serum transferrin has a higher affinity for immature red cells than does the iron-free protein (72). Both bind specifically to immature red cells and the attachment permits the cells to remove the iron. Once the iron is removed, however, the iron-free transferrin can be replaced by an iron-transferrin complex. 

Jandl, J. H., J. K. Inman, R. L. Simmons, and D. W. Allen Transfer of iron from serum-iron-binding protein to human reticulocytes. J. Clin. Invest. 38, 161 (1959). 

A low molecular weight protein, different from metallothionine which reversibly binds iron with high affinity has been isolated from rabbit reticulocyte cytosol (54, 55, 56). Although very little is yet known about its physiological properties, the molecular weight is around 6000, and iron appears to be reversibly bound under physiological conditions. This protein may be able to mobilize iron from the plasma membrane and donate it for heme and ferritin biosynthesis (56), but no definitive physiological role for siderochelin has been established. 

The parenchymatous liver cells (hepatocytes) hold a key position in the overall metabolism of iron (57, 58, 59, 60), and since functionally intact liver mitochondria can be conveniently prepared at high yield, these mitochondria have been most extensively studied so far. The iron transporting system discussed above for liver mitochondria is present also in mitochondria from other tissues and animal species (Table III). Quantitatively, erythroid cells of the bone marrow play the most important role in the overall metabolism of iron (61), and it was therefore not unexpected to find that the energized uptake of iron by isolated reticulocyte mitochondria exceeds that of mitochondria isolated from, for example, liver, kidney, and heart (Table IV). Thus, a relationship appears to exist between the rate and extent of heme protein turnover in mitochondria isolated from different tissues and their energized iron accumulation (30). Thus, it is evident that cellular differentiation is expressed at the mitochondrial level by modulation of the activity of essential functions related to iron transport and heme biosynthesis. 

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... 

The uptake of iron from transferrin by reticulocytes is a time-, temperature-, and energy-dependent process in which integrity of both protein and cells is required (64, 65). Synthetic iron chelates, once thought to be effective iron donors (66), appear to depend on membrane-bound transferrin as an intermediate agent cells depleted of the protein by preincubation and washing no longer accept iron from such complexes (67). When such cells are reincubated with transferrin, their capacity to accept iron initially bound to synthetic chelators is largely restored. 

Other mechanisms are also involved in the reticulocyte-mediated release of iron from transferrin, and its subsequent incorporation into heme. Intact oxidation-reduction pathways are essential and seem to operate at a step following the attack on the anion (90). Heme exerts an inhibitory influence on iron transfer from protein to cell, seemingly by interfering at an early stage in the release of the metal from transferrin (89). Non-transferrin carriers for iron have also been identified in the reticulocyte and may participate in its incorporation into heme (81). 

The iron inserted into apo-heme is acquired from the transferrin circulating in the bloodstream. Transferrin is taken up by cells according to their need for iron. This transport is mediated by a membrane-boimd protein of the plasma membrane called the transferrin receptor. The erythropoietic cell contains a large number of transferrin receptors in its plasma membrane. Stem cells contain very few transferrin receptors because of their lack of hemoglobin synthesis. The normoblast contains 0.3 to 0.8 million transferrin receptors, whereas the more mature reticulocyte contains 0.1 million receptors. After release of iron, transferrin returns to the bloodstream. Most of the transferrin contains iron derived from recently catabolized red blood cells, as shown in Figure 10.33. About 99% of the transferrin contains iron acquired from macrophages, where old red blood cells are dismantled only 1% is derived from recently absorbed dietary iron. 

Under normal conditions, almost all of the iron bound to transferrin is rapidly taken up by the marrow (35, 36, 37). The observed half-life of Fe(III)-transferrin in vivo was reported to be 1.3 hours, whereas the half-life for the protein moiety was much slower, 7 days (38). Any er) throid stimulation leads to a faster turnover of iron in vivo, and ery-throid suppression has the opposite eflFect (38). Only the reticulocytes are capable of utilizing the Fe(III) bound to transferrin although both the reticulocytes and the mature red cell can take up free Fe(III). Katz and Jandl (38) concluded that the transferrin directs the entry of iron into those cells which are still actively making hemoglobin and prevents any accumulation of iron by the mature cell. 

Chloramphenicol can cause two forms of bone marrow depression. One is serious and irreversible, and can result in fatal aplastic anaemia, whereas the other is probably unrelated, milder and reversible, and appears to occur at chloramphenicol serum levels of 25 micrograms/mL or more. This occurs because chloramphenicol can inhibit protein synthesis, the first sign of which is a fall in the reticulocyte count, which reflects inadequate red cell maturation. This response to chloramphenicol has been seen in animals healthy individuals, a series of patients with liver disease," and in anaemic patients being treated with iron dextran or vitamin Bj2. 

An interesting lipoxygenase has been characterized in reticulocytes from rabbits [202j. The molecular weight (78000), isoelectric point (5.5) and sugar content (5%), as well as the association of two iron atoms per enzyme molecule were reported. This lipoxygenase was found to inactivate respiratory proteins of the developing reticulocyte. 

A unique lipoxygenase was isolated and purified from rabbit reticulocytes [212], The purified enzyme with a molecular weight of 78000 contains two moles of iron per enzyme molecule. The enzyme is identical with the previously known protein factors which inhibit the respiratory chain. The purified lipoxygenase causes a loss of acid-labile sulfur from mitochondrial electron transfer particles and acts preferably on the mitochondrial membrane. The enzyme is considered to work in the lysis of mitochondria during the maturation of erythrocytes. The major product from arachidonic acid is 15S-hydroperoxy-5,8,l 1,13-eicosatetraenoic acid, although the 12S-hydroperoxy derivative is produced as a minor product [213]. 

While the reticulocyte synthesizes equal amounts of both a and /J chains, the production of both of these proteins comes to a halt if the cells are incubated in vitro in a medium without iron (for heme synthesis) or without heme itself (Waxman and Rabinovitz, 1965 for further references see Rabinovitz et al., 1969). The decrease in globin synthesis during heme deficiency is associated with a disaggregation of polysomes to 80 S monomers, which is rapidly reversed on readdition of heme. This suggests that the supply of heme controls the rate at which ribosomes can attach to mRNA and initiate protein synthesis and has no effect, for example, on the stability of mRNA. Reticulocyte lysates also require heme to synthesize globin for longer than 5-10 minutes, and lack of heme leads to polysome disaggregation. 

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