Fate of blood meal iron in mosquitoes

Iron is an essential element of living cells and organisms as a component of numerous metabolic pathways. Hemoglobin and ferric-transferrin in vertebrate host blood are the two major iron sources for female mosquitoes. We used inductively coupled plasma mass spectrometry (ICP-MS) and radioisotope labeling to quantify the fate of iron supplied from hemoglobin or as transferrin in Aedes aegypti. At the end of the first gonotrophic cycle, approximately 87% of the ingested total meal heme iron was excreted, while 7% was distributed into the eggs and 6% was stored in different tissues. In contrast, approximately 8% of the iron provided as transferrin was excreted and of that absorbed, 77% was allocated to the eggs and 15% distributed in the tissues. Further analyses indicate that of the iron supplied in a blood meal, approximately 7% appears in the eggs and of this iron 98% is from hemoglobin and 2% from ferric-transferrin. Whereas, of iron from a blood meal retained in body of the female, approximately 97% is from heme and <1% is from transferrin. Evaluation of iron-binding proteins in hemolymph and egg following intake of (59)Fe-transferrin revealed that ferritin is iron loaded in these animals, and indicate that this protein plays a critical role in meal iron transport and iron storage in eggs in A. aegypti.     

Acquisition of iron from transferrin regulates reticulocyte heme synthesis

Fe-salicylaldehyde isonicotinoylhydrazone (SIH), which can donate iron to reticulocytes without transferrin as a mediator, has been utilized to test the hypothesis that the rate of iron uptake from transferrin limits the rate of heme synthesis in erythroid cells. Reticulocytes take up 59Fe from [59Fe]SIH and incorporate it into heme to a much greater extent than from saturating concentrations of [59Fe]transferrin. Also, Fe-SIH stimulates [2-14C]glycine into heme when compared to the incorporation observed with saturating levels of Fe-transferrin. In addition, delta-aminolevulinic acid does not stimulate 59Fe incorporation into heme from either [59Fe]transferrin or [59Fe]SIH but does reverse the inhibition of 59Fe incorporation into heme caused by isoniazid, an inhibitor of delta-aminolevulinic acid synthase. Taken together, these results suggest the hypothesis that some step(s) in the pathway of iron from extracellular transferrin to intracellular protoporphyrin limits the overall rate of heme synthesis in reticulocytes.     

The competition between transferrins labeled with59Fe,65Zn, and54Mn for the binding sites on lactating mouse mammary gland cells

Using a homologous competition of⁵⁴Mn-transferrin with Mntransferrin and⁶⁵Zn-transferrin with Zn-transferrin, it was found that on the plasma membrane of lactating mouse mammary gland cells there are receptor binding Mn-transferrin and Zn-transferrin. The heterologous competition between labeled and nonlabeled Fe-transferrin, Mn-transferrin and Zn-transferrin, as well as almost equal affinity constants of cellular receptors toward the three metals by competition of Fe-transferrin suggests that one and the same receptor accepts all three metals from the transferrin molecule. The cell receptors therefore possess a polymetal binding function. A model and a mechanism for regulation of the transport metal flow toward the mammary gland cell acting like “automated switching over” are proposed.     

Effect of aluminium on iron uptake and transferrin-receptor expression by human erythroleukaemia K562 cells.

Incubation of human erythroleukaemia K562 cells with Al-transferrin inhibited iron uptake from 59Fe-transferrin by about 80%. The inhibition was greater than that produced by a similar quantity of Fe-transferrin. Preincubation of cells for 6 h with either Al-transferrin or Fe-transferrin diminished the number of surface transferrin receptors by about 40% compared with cells preincubated with apo-transferrin. Al-transferrin did not compete significantly with Fe-transferrin for transferrin receptors and, when cells were preincubated for 15 min instead of 6 h, the inhibitory effect of Al-transferrin on receptor expression was lost. Both forms of transferrin also decreased the level of transferrin receptor mRNA by about 50%, suggesting a common regulatory mechanism. Aluminium citrate had no effect on iron uptake or transferrin-receptor expression. AlCl3 also had no effect on transferrin-receptor expression, but at high concentration it caused an increase in iron uptake by an unknown, possibly non-specific, mechanism. Neither Al-transferrin nor AlCl3 caused a significant change in cell proliferation. It is proposed that aluminium, when bound to transferrin, inhibits iron uptake partly by down-regulating transferrin-receptor expression and partly by interfering with intracellular release of iron from transferrin.     

Transferrin as a donor of iron to mitochondria Effect of pyrophosphate and relationship to mitochondrial metabolism and heme synthesis

Rat liver mitochondria accumulate iron mobilized from transferrin by pyrophosphate. The uptake has a very low energy dependence, but it is highly dependent on a functioning respiratory chain. Reduction of the ferric-iron-pyrophosphate complex is not linked to any specific respiratory complex. Half of the amount of iron accumulated is passed into heme. Iron once accumulated is very little accessible to chelation by added ferric or ferrous iron chelators. Iron uptake and heme synthesis are maximal if a suitable porphyrin substrate is added simultaneously with iron. The results represent further evidence that pyrophosphate is a possible candidate for intracellular iron transport. Also, the results suggest that iron uptake is coupled to simultaneous porphyrin uptake and heme synthesis.     

Heme regulation of HeLa cell transferrin receptor number

The number of diferic transferrin receptors on HeLa cells decreases when cells are grown in iron-supplemented media. The experiments reported here suggest that heme is the iron-containing compound which serves as the signal for receptor number regulation. When HeLa cells were grown in the presence of hemin, transferrin receptor number decreased to a greater degree than when cells were grown in equivalent amounts of iron supplied as ferric ammonium citrate. Incubation of cells in conditions which increased cellular heme content resulted in a decrease in cellular transferrin receptors. Incubating cells with 5-aminolevulinic acid (thus bypassing the rate-limiting step in heme biosynthesis, 5-aminolevulinic acid synthase) led to a decrease in transferrin receptor number. Incubation of cells with an inhibitor of heme oxygenase, Sn-protoporphyrin IX, also led to a decrease in transferrin receptor number. When cellular heme content was decreased by inhibiting heme synthesis with succinylacetone (an inhibitor of 5-aminolevulinic acid dehydratase), or by depriving cells of iron with deferoxamine, an increase in HeLa cell transferrin receptor number was seen. When HeLa cells were incubated with inducers of heme oxygenase (CoCl2, SnCl2, Co-protoporphyrin IX), transferrin receptor number also increased. The effects of all compounds which alter transferrin receptor number were dependent on the concentration of the supplement, as well as the duration of the supplementation. These experiments suggest that intracellular heme content may be an important signal controlling transferrin receptor number.     

Binding of transferrin and metal ions by suspensions of reticulocyte-rich rabbit blood

Reticulocyte binding of Fe(III)_-transferrin and transferrin complexes with other metal ions have been compared by different investigators. The functional relevance of this comparison is not clear, therefore transferrin complexes with Fe(III), Cu(II), Mn(II) and Zn(II) have been studied further by DEAE-cellulose chromatography and by measurement of transferrin and metal uptakes by rabbit reticulocytes.Human Fe-transferrin behaved as a weaker anion than apotransferrin during DEAE-cellulose chromatography; since Fe-transferrin has a higher negative charge than apotransferrin and behaves a as stronger anion in electrophoretic systems, the chromatographic result was the opposite of that anticipated. The lower affinity of human Fe-transferrin for DEAE-cellulose is probably caused by a redistribution of charged groups on the surface of transferrin molecules when Fe(III) ions are bound and is therefore considered to be dependent on molecular conformation. Apotransferrin and divalent metal-transferrin complexes were found to have nearly equal affinities for DEAE-cellulose, thus the effect on surface charge of human transferrin molecules induced by binding Fe(III) appeared to be limited to that metal ion.Iron uptake by reticulocytes was associated with increased binding of transferrin to the cell surface: uptake of divalent metals occured without a concomitant increase in transferrin uptake or evidence of a specific metal-transfer process. Cu-transferrin was rapidly dissociated during incubation with cells.The effect of Fe(III)_binding on human transferrin molecules was to alter the molecular affinity for charged surfaces, namely DEAE-cellulose and reticulocyte membranes. This was less apparent with rabbit transferrin. Transferrin complexes with divalent metals behaved as apotransferrin in the process of association with reticulocytes.     

Diacytosis of human asialotransferrin type 3 in the rat liver is due to the sequential engagement of two receptors

The possible role of transferrin receptors in the diacytosis of human asialotransferrin type 3 (HAsTf-3) by the rat liver was studied in vivo. A trace dose of the ligand was allowed to compete for hepatic binding sites against diferric transferrin, the concentration of which was varied between 5 400- and 18 000-fold. Binding of HAsTf-3 was insensitive to the presence of 2Fe-transferrin in this range, and the liver bound the ligand equally efficiently, regardless of whether it was presented in the holo or apo form. In contrast, pretreating the animals with desialylated bovine submaxillary mucin (2 mg/100 g, 2 min before the dose) prevented the asialotransferrin-liver interaction. These findings indicate that endocytosis of HAsTf-3 is mediated by the Gal/GalNAc-specific lectin and not by transferrin receptors. Although 2Fe-transferrin did not affect binding, it did reduce the half-life of the ligand in the liver, thus suggesting that transferrin receptors play an important role in the exocytic leg of the diacytic cycle. Based on our present and earlier data, a model is proposed in which the engagement of lectin and transferrin receptor in the diacytic cycle is envisaged sequentially so that HAsTf-3 switches receptors at an acidified subcellular site.     

Ribonucleotide reductase inhibition by metal complexes of Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone): a combined experimental and theoretical study

Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP) is currently the most promising chemotherapeutic compound among the class of α-N-heterocyclic thiosemicarbazones. Here we report further insights into the mechanism(s) of anticancer drug activity and inhibition of mouse ribonucleotide reductase (RNR) by Triapine. In addition to the metal-free ligand, its iron(III), gallium(III), zinc(II) and copper(II) complexes were studied, aiming to correlate their cytotoxic activities with their effects on the diferric/tyrosyl radical center of the RNR enzyme in vitro. In this study we propose for the first time a potential specific binding pocket for Triapine on the surface of the mouse R2 RNR protein. In our mechanistic model, interaction with Triapine results in the labilization of the diferric center in the R2 protein. Subsequently the Triapine molecules act as iron chelators. In the absence of external reductants, and in presence of the mouse R2 RNR protein, catalytic amounts of the iron(III)–Triapine are reduced to the iron(II)–Triapine complex. In the presence of an external reductant (dithiothreitol), stoichiometric amounts of the potently reactive iron(II)–Triapine complex are formed. Formation of the iron(II)–Triapine complex, as the essential part of the reaction outcome, promotes further reactions with molecular oxygen, which give rise to reactive oxygen species (ROS) and thereby damage the RNR enzyme. Triapine affects the diferric center of the mouse R2 protein and, unlike hydroxyurea, is not a potent reductant, not likely to act directly on the tyrosyl radical.     

Effect of pH and iron content of transferrin on its binding to reticulocyte receptors

The effect of pH on the binding of apotransferrin and diferric transferrin to reticulocyte membrane receptors was investigated using rabbit transferrin and rabbit reticulocyte ghosts, intact cells and a detergent-solubilized extract of reticulocyte membranes. The studies were performed within the pH range 4.5–8.0. The binding of apotransferrin to ghosts and membrane extracts and its uptake by intact reticulocytes was high at pH levels below 6.5 but decreased to very low values as the pH was raised above 6.5. By contrast, diferric transferrin showed a high level of binding and uptake between pH 7.0 and 8.0 in addition to binding only slightly less than did apotransferrin at pH values below 6.5. It is proposed that the high affinity of apotransferrin for its receptor at lower pH values and low affinity at pH 7.0 or above allow transferrin to remain bound to the receptor when it is within acidic intracellular vesicles, even after loss of its iron, but also allow ready release from the cell membrane when it is exteriorized by exocytosis after iron uptake. The binding of transferrin to the receptor throughout the endocytosis-exocytosis cycle may protect it from proteolytic breakdown and aid in its recycling to the outer cell membrane     

Pyrophosphate as a ligand for delivery of iron to isolated rat-liver mitochondria

Rat liver mitochondria accumulate iron mobilized from transferrin by pyrophosphate. The capacity of the mitochondria to accumulate iron is higher than the capacity of pyrophosphate to mobilize iron from transferrin: with ferric-iron-pyrophosphate as iron donor, iron uptake and heme synthesis are about 10-times that at corresponding concentrations of iron-transferrin plus pyrophosphate. Uptake of iron from ferric-iron-pyrophosphate depends on a functionary respiratory chain and involves reductive cleavage of the ferric-iron-pyrophosphate complex. Apotransferrin inhibits uptake of iron from ferric-iron-pyrophosphate by competing with the mitochondria for iron. The results focus on pyrophosphate as a possible candidate for intracellular iron transport.     

Inhibition of iron uptake in HL60 cells by 2-formylpyridine monothiosemicarbazonato Cu(II).

The electron paramagnetic resonance (EPR) signal of the tyrosyl radical attributed to ribonucleoside diphosphate reductase decreases after treatment of promyelocytic leukemic HL60 cells with 2-formylpyridine thiosemicarbazonato copper(II) (CuL). According to EPR studies, CuL forms adducts with both histidine and cysteine-like Lewis bases associated with isolated membranes from HL60 cells. After the addition of CuL, the EPR signal for the cysteine-like adduct decreases substantially over a 15-min period. The reduction of signal is consistent with oxidation of thiols as shown by an analysis of sulfhydryl content. It is hypothesized that receptor-mediated transferrin internalization is inhibited by oxidation of critical thiols. Since the uptake of 59Fe-transferrin is greatly inhibited by the treatment of HL60 cells with CuL, the reduced uptake of iron by cells, in the presence of CuL, may lead to decreased iron availability for the activity of the M2 subunit of ribonucleotide reductase and a subsequent decrease in the tyrosyl radical signal of the enzyme. Moreover, the intact subunit M2 is no longer detected by EPR, even after the addition of excess iron.     

Gallium uptake by transferrin and interaction with receptor 1

The kinetics and thermodynamics of Ga(III) exchange between gallium mononitrilotriacetate and human serum transferrin as well as those of the interaction between gallium-loaded transferrin and the transferrin receptor 1 were investigated in neutral media. Gallium is exchanged between the chelate and the C-site of human serum apotransferrin in interaction with bicarbonate in about 50 s to yield an intermediate complex with an equilibrium constant K ₁ = (3.9 ± 1.2) × 10⁻², a direct second-order rate constant k ₁ = 425 ± 50 M⁻¹ s⁻¹ and a reverse second-order rate constant k ₋₁ = (1.1 ± 3) × 10⁴ M⁻¹ s⁻¹. The intermediate complex loses a single proton with proton dissociation constant K _1a = 80 ± 40 nM to yield a first kinetic product. This product then undergoes a modification in its conformation which lasts about 500 s to produce a second kinetic intermediate, which in turn undergoes a final extremely slow (several hours) modification in its conformation to yield the gallium-saturated transferrin in its final state. The mechanism of gallium uptake differs from that of iron and does not involve the same transitions in conformation reported during iron uptake. The interaction of gallium-loaded transferrin with the transferrin receptor occurs in a single very fast kinetic step with a dissociation constant K _d = 1.10 ± 0.12 μM and a second-order rate constant k _d = (1.15 ± 0.3) × 10¹⁰ M⁻¹ s⁻¹. This mechanism is different from that observed with the ferric holotransferrin and suggests that the interaction between the receptor and gallium-loaded transferrin probably takes place on the helical domain of the receptor which is specific for the C-site of transferrin and HFE. The relevance of gallium incorporation by the transferrin receptor-mediated iron-acquisition pathway is discussed.     

Regulation of intracellular iron distribution in K562 human erythroleukemia cells

Following a pulse with 59Fe-transferrin, K562 erythroleukemia cells incorporate a significant amount of 59Fe into ferritin. Conditions or manipulations which alter the supply of iron to cells result in changes in the rate of ferritin biosynthesis with consequent variations in the size of the ferritin pool. Overnight exposure to iron donors such as diferric transferrin or hemin increases the ferritin level 2-4- or 6-8-fold above that of the control, respectively. Treatment with the anti-human transferrin receptor antibody, OKT9 (which reduces the iron uptake by decreasing the number of transferrin receptors) lowers the ferritin level by approximately 70-80% with respect to the control. The fraction of total cell-associated 59Fe (given as a pulse via transferrin) that becomes ferritin bound is proportional to the actual ferritin level and is independent of the instantaneous amount of iron taken up. This has allowed us to establish a curve that correlates different levels of intracellular ferritin with corresponding percentages of incoming iron delivered to ferritin. Iron released from transferrin appears to distribute to ferritin according to a partition function; the entering load going into ferritin is set for a given ferritin level over a wide range of actual amounts of iron delivered.     

Transport of iron and transferrin synthesis by the seminiferous epithelium of the rat in vivo

The transport of radioactive iron across the seminiferous tubules was analyzed in vivo by light-microscope quantitative radioautography. At 5 min after a single intratesticular injection of 55Fe-transferrin, a strong labeling of the basal aspect of the seminiferous epithelium was observed. Between 30 min and 2 h, the labeling on the basal aspect of the seminiferous epithelium decreased. This decrease was accompanied by a substantial increase of the radioautographic reaction over the cellular elements in the adluminal compartment. These results were consistent with the demonstration of 59Fe associated with meiotic spermatocytes and differentiating spermatids isolated by velocity sedimentation from testes injected with 59Fe-transferrin. Furthermore, after a single intratesticular injection of 59Fe-labeled human transferrin, radiolabeled rat transferrin was immunoprecipitated from homogenates of isolated tubules with a specific antibody and appeared as a single radioactive band on fluorographs of urea/polyacrylamide gels. Similarly, 59Fe-labeled rat transferrin but not 125I-transferrin was immunoprecipitated from rete testis fluids of testes infused with either 59Fe- or 125I-labeled human transferrin. Finally, the synthesis of testicular transferrin in vivo was demonstrated in fluorographs of immunoprecipitated transferrin after an intratesticular injection of 35S-methionine in rats whose livers were excluded from the general circulation by ligation of both the hepatic artery and the portal vein. Thus, our results demonstrated a unidirectional system of iron transport from the basal compartment of the seminiferous epithelium to the germ cells in the adluminal compartment involving two distinct transferrins, i.e., a serum transferrin and a testicular transferrin synthesized by the seminiferous epithelium.     

Transferrin binding and iron uptake in mouse hepatocytes

Methods were developed for obtaining highly viable mouse hepatocytes in single cell suspension and for maintaining the hepatocytes in adherent static culture. The characteristics of transferrin binding and iron uptake into these hepatocytes was investigated. (1) After attachment to culture dishes for 18–24 h hepatocytes displayed an accelerating rate of iron uptake with time. Immediately after isolation mouse hepatocytes in suspension exhibited a linear iron uptake rate of 1.14·10⁵molecules/cell per min in 5 μM transferrin. Iron uptake also increased with increasing transferrin concentration both in suspension and adherent culture. Pinocytosis measured in isolated hepatocytes could account only for 10–20% of the total iron uptake. Iron uptake was completely inhibited at 4°C. (2) A transferrin binding component which saturated at 0.5 μM diferric transferrin was detected. The number of specific, saturable diferric transferrin binding sites on mouse hepatocytes was 4.4·10⁴±1.9·10⁴ for cells in suspension and 6.6·10⁴±2.3·10⁴ for adherent cultured cells. The apparent association constants were 1.23·10⁷ 1·mol^?1 and 3.4·10⁶ 1·mol^?1 for suspension and cultured cells respectively. (3) Mouse hepatocytes also displayed a large component of non-saturable transferrin binding sites. This binding increased linearly with transferrin concentration and appeared to contribute to iron uptake in mouse hepatocytes. Assuming that only saturable transferrin binding sites donate iron, the rate of iron uptake is about 2.5 molecules iron/receptor per min at 5 μM transferrin in both suspension and adherent cells and increases to 4 molecules iron/receptor per min at 10 μM transferrin in adherent cultured cells. These rates are considerably greater than the 0.5 molcules/receptor per min observed at 0.5 μM transferrin, the concentration at which the specific transferrin binding sites are fully occupied. The data suggest that either the non-saturable binding component donates some iron or that this component stimulates the saturable component to increase the rate of iron uptake. (4) During incubations at 4°C the majority of the transferrin bound to both saturable and nonsaturable binding sites lost one or more iron atoms. Incubations including 2 mM α,α′-dipyridyl (an Fe¹¹ chelator) decreased the cell associated ⁵⁹Fe at both 4 and 37°C while completely inhibiting iron uptake within 2–3 min of exposure at 37°C. These observations suggest that most if not all iron is loosened from transferrin upon interaction of transferrin with the hepatocyte membrane. There is also greater sensitivity of ⁵⁹Fe uptake compared to transferrin binding to pronase digestion, suggesting that an iron acceptor moiety on the cell surface is available to proteolysis.     

Murine cell surface transferrin receptor: Studies with an anti-receptor monoclonal antibody

A rat monoclonal antibody against the murine transferrin receptor has been identified. The receptor is a 95,000 molecular weight species that exists in the cell membrane as a disulphide-bonded dimer. Whereas 29 of 29 murine hematopoietic tumor cell lines express detectable numbers of transferrin receptors, less than 1% of adult thymocytes or spleen cells and only 5% of bone marrow cells are positive. However, fetal liver and neonatal spleen contain substantial numbers of transferrin receptor-positive cells. Induction of Friend cells in vitro with dimethyl-sulphoxide leads to an overall increase in the expression of transferrin receptors on the cell surface. The anti-transferin receptor antibody we have obtained partially blocks iron uptake from ⁵⁹Fe-transferrin by a variety of murine cell lines and inhibits the growth of a murine myeloma cell line in vitro.     

Inhibition of reticulocyte iron uptake by NH4Cl and CH3NH2

The aim of this investigation was to test the hypothesis that elevation of intracellular pH would inhibit iron uptake by reticulocytes. The experiments were performed with rabbit reticulocytes and iron bound to rabbit transferrin. Incubation of the cells with NH₄Cl, (NH₄)₂CO₃, CH₃NH₂ and (CH₃)₂NH was used in an attempt to increase intracellular pH. These substances were all found to inhibit iron uptake by reticulocytes. The mechanism of action of NH₄Cl and CH₃NH₂ was investigated in detail. Similar results were found with both reagents. They inhibited iron uptake in a concentration-dependent manner, but produced a small increase in the cellular uptake of transferrin. The onset of action was rapid and the effect was reversible. There was no decrease in the number of transferrin-binding sites per cell and their apparent affinity for transferrin increased slightly, while the efficiency of iron removal from transferrin per binding site diminished greatly. The rate of transferrin release from reticulocytes was unaffected. NH₄Cl did not affect the rate of iron release from transferrin in a cell-free system. Incubation of reticulocytes with 10 mM NH₄Cl or CH₃NH₂ was found to produce an increase in intracellular pH of 0.05—0.15 pH units. The intracellular pH determined by used of the weak acid 5,5-dimethyl-oxazolidine-2,4-dione was significantly higher than that obtained with the weak base (CH₃)₂NH. By transmission electron microscopy it was shown that reticulocytes treated with NH₄Cl or CH₃NH₂ have enlarged intracellular vesicles. The results are considered to support the hypothesis that iron release from transferrin in reticulocytes occurs as a result of protonation of the transferrin within intracellular vesicles. According to this hypothesis, weak bases such as NH₃ and CH₃NH₂ inhibit iron release by neutralizing H⁺ within the vesicles.     

Studies on the mitogenic effect of transferrin by membrane signal transduction

One of the earliest events leading to cell activation and growth is the hydrolysis of inositol phospholipids producing various membrane signals induced by an interaction between growth factors or hormones with their respective receptors on the cell membrane [1].To demonstrate the mitogenic action of transferrin,our results show that an addition of transferrin to “serum-deprived” rat hepatoma cells produced a rapid but transient rise in inositol 1,4,5-trisphosphate(IP3) level,and at the same time,an increased intracellular Ca^2 activity and a cytoplasmic alkalinization were observed.These signal transductions further lend support to the mitogenic nature of transferrin.In addition,a possible link between the receptor-mediated endocytosis of transferrin with the generation of intracellular signals is discussed herewith.     

Evidence for and consequences of chronic heme deficiency in Belgrade rat reticulocytes

The Belgrade rat has a microcytic, hypochromic anemia inherited as an autosomal recessive trait (gene symbol b). Transferrin-dependent iron uptake is defective because of a mutation in Nramp2 (now DMT1, also called DCT1), the protein responsible for endosomal iron efflux. Hence, Belgrade reticulocytes are iron deficient. We show that a chromatographic method is able to measure the amount of 'free' heme in reticulocytes. Most of the 'free' heme is the result of biosynthesis. Succinylacetone, an inhibitor of heme synthesis, decreases the level of 'free' heme and cycloheximide, an inhibitor of globin synthesis, increases the 'free' heme level. In a pulse-chase experiment with 59Fe-transferrin, the 'free' heme pool behaves as an intermediate, with a half-life of just over 2 h. Belgrade reticulocytes contain about 40% as much 'free' heme as do heterozygous or homozygous reticulocytes. This deficiency of 'free' heme slows initiation of translation in Belgrade reticulocytes by increasing the level of an inhibitor of initiation. Thus the Belgrade rat makes a whole animal model available with chronic heme deficiency.