Globin Synthesis In Reticulocytes Research Articles

The effect of various chelating agents on the mobilization of iron from reticulocytes in the presence and absence of pyridoxal isonicotinoyl hydrazone

The chelating agent pyridoxal isonicotinoyl hydrazone (PIH) has recently been shown to mobilize 59Fe from reticulocytes loaded with non-heme 59Fe. In this study, various chelating agents were tested for their ability to effect the mobilization of iron from reticulocytes by PIH. They fall into several groups. The largest group includes chelators such as citrate, ethylenediaminetetracetic acid and desferrioxamine, which fail to affect PIH-induced iron mobilization and do not mobilize iron per se. Either these chelators do not enter reticulocytes or they do not take up iron from PIH-Fe complexes. The second group includes chelators such as 2,2′-bipyridine, 1,10-phenanthroline, bathophenanthroline sulfonate and N, N′-ethylenebis( o-hydroxyphenylglycine) which inhibit PIH-induced iron mobilization from reticulocytes and, when added together with PIH, induce radioiron accumulation in an alcohol-soluble fraction of reticulocytes. It appears that these chelators enter the cell and compete with PIH for 59Fe(II), but having bound iron are unable to cross the cell membrane. Spectral analysis suggests that Fe(II) chelators such as 2,2′-bipyridine and 1,10-phenanthroline remove iron from Fe(II)PIH but are not able to do so from Fe(III)PIH. Then there are compounds such as 2,3-dihydroxybenzoic acid and catechol which potentiate PIH-induced iron mobilization although they are unable to mobilize iron from reticulocytes by themselves. Lastly, there is a group of miscellaneous compounds which include chelators that either potentiate the iron-mobilizing effect of PIH as well as mobilizing iron from reticulocytes by themselves (tropolone), or that reduce PIH-induced iron mobilization while themselves having an iron-mobilizing effect ( N, N′-bis(2,3-dihydroxybenzoyl)-1,6-diaminohexane). In further experiments, heme was found to stimulate globin synthesis in reticulocytes, the heme synthesis of which was inhibited by PIH, suggesting that PIH is probably not toxic to the cells.

The stimulation of globin synthesis by cobalt in reticulocytes with inhibited heme synthesis

Cobalt was found to stimulate globin synthesis in reticulocytes the heme synthesis of which was inhibited by isonicotinic acid hydrazide or 4,6-dioxoheptanoic acid (succinyl acetone). These results suggest that in reticulocytes cobalt does not stimulate globin synthesis through the formation of cobalt protoporphyrin. They do, how-ver, support the hypothesis that cobalt causes the formation of a pool of free heme from preformed hemoglobin which stimulates the synthesis of new globin chains.

Cross-linking of hemoglobin and inhibition of globin synthesis in reticulocytes induced by photoactivated protoporphyrin.

The photoactivated protoporphyrin effect on reticulocyte globin synthesizing capability, cross-linking of intracellular hemoglobin and the cell ultrastructural alterations was studied. Low doses up to 15 microM of protoporphyrin markedly inhibited the globin synthesis and decreased amino acid uptake while no lytic effect or ultrastructural deformations were detected by scanning electron microscopy and transmission electron microscopy. On the other hand, at high doses of protoporphyrin, up to 60 microM, the globin synthesis was totally stopped, and the intracellular hemoglobin leaked out. The photodynamic effect induced cross-linking of hemoglobin into condensed spheres which captured numerous polyribosomes. The end-product of protoporphyrin activity was a closed ghost with a rigid membrane containing only cross-linked hemoglobin spheres closely related to the membrane.

Ratios of α-to β-globin mRNA and regulation of globin synthesis in reticulocytes

THE genomes of several mammalian species contain at least two α globin loci and a single β globin locus so that the ratio of α- to β-globin genes is 2 : 1 or more1–3. Despite this difference in number of genes, reticulocytes from these species synthesise equal numbers of α- and β-globin chains indicating that compensatory regulation must occur. Cytoplasmic mRNA is an intermediate between the gene and globin protein, and it has been suggested that the cellular α/β mRNA ratio is an important factor in achieving balanced globin synthesis4–6. We report here an excess of 30% α mRNA on reticulocyte polysomes of rabbits, mice, and sheep. Our data also illustrate the potential for errors in studies in which only poly(A)+ RNA is assayed.

Evidence supporting a physiological role for the hemin-controlled translational repressor of globin synthesis in reticulocytes

The synthesis of heme and globin in rabbit reticulocytes was compared at 35 and 25°C. The lower temperature decreased heme synthesis significantly more than globin synthesis and resulted in a much greater accumulation of globin dimers. After 16 h of incubation in the absence of iron, globin synthesis in reticulocytes which were at 35°C could not be stimulated by iron, whereas cells which were at 25°C responded with nearly control levels of globin synthesis. Since the formation of the hemin-controlled translational repressor in reticulocyte lysates is also decreased much more than protein synthesis at reduced temperature the results provide evidence for a physiological role for the translational repressor in controlling globin synthesis in reticulocytes.

Disturbed haem and globin synthesis in reticulocytes of prenatal flexed-tailed (f-f) anaemic mice.

SUMMARYHaem synthetase activity and co-ordination of α- and β-globin chain synthesis have been investigated in prenatal reticulocytes of congenic FL/4Re +/+Lvb/Lvband FL/1 Ref/f Lvb/Lvb, mice, which have a marked hypochromic, microcytic, siderocytic anaemia, with reduced erythrocyte numbers at birth, and also in other stocks bearing theflesion. Haem synthetase activity inf/freticulocyte homogenates was similar to that in normal cells but was markedly dependent on protoporphyrin added to the homogenate, while activity in normal cell homogenates was relatively independent of added precursor. In cultured normal prenatal reticulocytes α- and β-globin was synthesized in approximately equal amounts during a 4 h labelling period, but inf/freticulocytes there was an approximate 50% deficiency in β-globin chain synthesis. This deficiency could be repaired by added haem but not by protoporphyrin. Such a lesion is quantitatively consistent with the observed hypochromia of neonatalf/ferythrocytes. The relationship of this abnormality to effects of theflocus on early erythropoietic precursor cells is discussed.

The labilization of hemoglobin by cobalt. Its effect on globin synthesis in reticulocytes.

To elucidate a mechanism for cobalt‐stimulated globin synthesis in rabbit reticulocytes, the cells were incubated with 1 mM CoCl2 containing 60Co. Although the cells incorporated cobalt irreversibly, evidence for the synthesis of cobalt‐protoporphyrin was not obtained. More than 90% of the cobalt recovered in the cell cytoplasma was bound to the globin moiety of hemoglobin. Similar results were obtained with erythrocytes and purified hemoglobin incubated with 60CoCl2.By CM‐Sephadex chromatography and isoelectric focusing it was found that cobalt‐labelled hemoglobin differs slightly in charge from native hemoglobin, possessing a lower isoelectric point.It was also demonstrated that the heme in cobalt‐labelled hemoglobin is less firmly bound to globin than the heme in the native protein, and that the purified cobalt‐treated protein can stimulate globin synthesis in rabbit reticulocytes.It is postulated that in reticulocytes cobalt stimulates globin synthesis indirectly by labilizing intracellular hemoglobin.

The ability of exogenous heme to restore globin synthesis in reticulocytes with impaired heme formation

The addition of isonicotinic acid hydrazide (INH) or cycloheximide decreases the incorporation of 75Se-methionine and 14C-labeled aminoacids into globin. 10 −4M hemin added to the incubation mixture removes the inhibition of globin synthesis caused by INH but is without effect on the synthesis of globin inhibited by cycloheximide.