Inhibition Of Globin Synthesis Research Articles

2,3-Bisphosphoglycerate inhibits hemoglobin synthesis and phosphorylation of initiation factor 2 by casein kinase II in reticulocyte lysates

2,3-Bisphosphoglycerate inhibited protein synthesis in reticulocyte lysates with 50% inhibition at 2 mM. Glycerate 2,3-P2 increased the Mg2+ optimum for protein synthesis by chelation of Mg2+, but Mg2+ addition did not completely reverse the inhibition, suggesting an additional site of action. eIF-2 has been used to examine the activity of casein kinase II in reticulocyte lysates in response to glycerate 2,3-P2. When glycerate 2,3-P2 was increased to 4mM, phosphorylation of eIF-2 beta was increasingly inhibited. Thus inhibition of phosphorylation of translational components by casein kinase II can be correlated with inhibition of globin synthesis at physiological concentrations of glycerate 2,3-P2.

Mechanism of Stimulation of Globin Synthesis by Adenosine-3',5'-Monophosphate and Guanosine 5'-Triphosphate in a Rabbit Reticulocyte Lysate System

The inhibition of globin synthesis in hemin-deficient rabbit reticulocyte lysates is due to the activation of a hemin-controlled translational inhibitor (HCI) that specifically phosphorylates eIF-2 alpha. High concentrations of cAMP (5-10 mM) and GTP (1-2 mM) stimulated the globin synthesis in hemin-deficient lysates when these compounds were added at the initial stage of incubation. The mechanism of the stimulation by cAMP and GTP was studied using hemin-deficient lysates, the N-ethylmaleimide (NEM)-treated HCI-supplemented lysates and a partially purified initiation factor, eIF-2. As the stimulation of globin synthesis by these compounds must be due to the prevention of the inhibition of globin synthesis, or due to the restoration of globin synthesis, or both, the preventive and restorative effects of these compounds were examined. As for the preventive effect, it was observed that a) the activation of HCI in the postribosomal supernatant of reticulocytes was prevented by GTP, but not by cAMP, and b) cAMP and GTP inhibited the phosphorylation of eIF-2 alpha in hemin-deficient lysates. As for the restorative effect of cAMP and GTP, it was observed that c) these compounds restored the globin synthesis and the binding of [35S]Met-tRNAf to the 40S ribosomal subunits, and promoted the dephosphorylation of eIF-2(alpha P), d) the rates of the restored synthesis of globin were lower than the control, and e) cAMP promoted the release of [3H]GDP from the eIF-2(alpha P) X [3H]GDP complex and the formation of eIF-2(alpha P) X eIF-2B complex. Finding (d) indicates that steps involved in the restorative effect of these compounds may not contribute to the stimulation of the globin synthesis in hemin-deficient lysates. The data on the preventive and restorative effects of cAMP and GTP showed that these compounds affected multiple steps. That is, cAMP inhibited the phosphorylation of eIF-2 alpha and promoted both the release of GDP from eIF-2 and the formation of eIF-2(alpha P) X eIF-2B complex, and GTP prevented both the activation of HCI and the phosphorylation of eIF-2 alpha. Though cAMP and GTP affected multiple steps, it is suggested that cAMP stimulates the globin synthesis by inhibiting the phosphorylation of eIF-2 alpha and that GTP stimulates the globin synthesis chiefly by preventing the activation of HCI in hemin-deficient lysates.

Activation and partial characterization of a human reticulocyte heme-dependent eIF-Z alpha kinase.

An inhibitor of globin translation initiation that is activated under heme-deficient conditions and whose activity is associated with phosphorylation of the 38,000 dalton (alpha) subunit of the initiation factor eIF-2 has been previously described in rabbit reticulocytes. We describe here an analogous enzyme in human reticulocytes. The human enzyme can be activated in intact reticulocytes under conditions of heme synthesis inhibition or by incubation of lysates in the absence of hemin. Addition of hemin to lysates prevents activation of the eIF-2 alpha kinase. The partially purified kinase is associated with inhibition of globin synthesis in a rabbit cell-free system and phosphorylates the small subunit of highly purified rabbit eIF-2. We conclude that human reticulocytes contain a protein kinase that is analogous to the rabbit HCR and that may play a role in clinical heme deficiency states. l

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.

Inhibition of globin synthesis by rabbit reticulocyte rRNAs.

The effect of concentration of rabbit reticulocyte rRNAs on the translation of rabbit globin mRNAs was studied by using a rabbit reticulocyte lysate system. Globin synthesis was studied using L-[U-14C]leucine. Both the 18S and 28S rRNA inhibited globin synthesis. The 18S rRNA inhibited the synthesis of alpha-globin chain more than that of beta chain, but the 28S rRNA inhibited the synthesis of alpha- and beta-chains almost equally. Nascent chains prelabelled with [14C]Leu or f[35S]Met were released at various concentrations of rRNAs. Release of the nascent chains was not inhibited at various concentrations of rRNAs. The ratios [14C]alpha/[14C]beta and [35S]alpha/[35S]beta in the released chains were almost constant at various concentrations of rRNAs. It therefore appears that the inhibition of globin synthesis by these rRNAs was at the step of initiation.

Effect of lead on globin synthesis in vitro.

A defect in heme synthesis is well documented to occur in lead intoxication. Globin synthesis and the alpha/beta globin chain synthesis ratio have been shown to be disturbed in lead poisoning. To elucidate further the nature of the inhibitory effect of lead on hemoglobin synthesis, reticulocyte-rich peripheral blood samples have been incubated with lead at concentrations of 10(-6), 10(-5), and 10(-4) M, equivalent to 20, 200, and 2,000 mug/dl (.96, 9.6 and 96.0 mumol/l), respectively. The incorporation of tritiated leucine into globin was determined. Globin chain synthesis decreased to 85, 49 and 15% of the control value with increasing lead concentrations. The effect of heme on inhibition of globin synthesis by lead was studied by incubating reticulocyte-enriched blood with lead 10(-5) M (200 mug/dl; 9.6 mumol/l) in the presence or absence of heme at a concentration of 10(-3) M. Lead alone depressed the incorporation of tritiated leucine to 45% of control, while globin synthesis with heme alone was 109% of control. Although in the presence of both heme and lead, the mean incorporation of leucine into globin was 69.2% of control, the increase was not significantly different from that obtained with lead alone. It is concluded that (1) lead has an inhibitory effect on globin synthesis at a concentration considered to be within the acceptable range encountered in environmental lead pollution, and the extent of inhibition is dose-dependent; (2) the effect of lead on globin synthesis is not significantly prevented by heme and, therefore, does not appear to be mediated through the well-documented inhibitory effect of lead on heme synthesis.

Involvement of hemin, a stimulatory fraction from ribosomes and a protein synthesis inhibitor in the regulation of hemoglobin synthesis

For maximum globin synthesis in lysate cell-free systems prepared from rabbit reticulocytes, there is a requirement for both hemin and a fraction derived from the ribosome by salt treatment (I fraction). A previously described inhibitor of protein synthesis (Q fraction), which accumulates during incubation of the lysate system, is shown to be without effect on completion and release of nascent chains, suggesting that it is acting at the site of chain initiation. Inhibition of globin synthesis by Q fraction is not reversed by addition of salt-treated ribosomes but is blocked and reversed by the addition of the I fraction, suggesting that an I fraction component is limiting in systems containing Q fraction. Several modes of regulation of protein synthesis involving hemin, I fraction and Q fraction are discussed in the light of kinetic data obtained with these systems, and in the light of the finding that intact cells incubated in the absence of added iron accumulate an inhibitor with properties similar to Q fraction desscribed above.

Regulation of iron entry into reticulocytes: II. Relationship between hemoglobin synthesis and entry or iron into reticuloyctes

1. 1. All of the tested inhibitors of globin synthesis (puromycin, cycloheximide, emetine, chloramphenicol, tryptamine, NaF, amino acid deficit) decrease reticulocyte radio-iron uptake. Experimental evidence showed that the mechanism of inhibited incorporation of iron into reticulocytes differed according to the inhibitor used. 2. 2. The direct inhibitors of globin synthesis inhibit the membrane transport of iron by a mechanism which depends on the ability of cells to form heme. This is exemplified by estimating the rate of iron uptake by reticulocytes with inhited proteosynthesis which were incubated under conditions of inhibited or restored heme synthesis. Inhibitors of heme synthesis (isonicotinic acid hydrazide or penicillamine) decrease the inhibition of iron uptake caused by cycloheximide, puromycin or emetine. Pyridoxal phosphate, added to reticulocytes incubated with inhibitors of both globin and heme synthesis, was shown to restore heme synthesis which is accompanied by inhibited reticulocyte iron uptake. 3. 3. Experimental evidence is presented that chloramphenicol, tryptamine and NaF inhibit reticulocyte iron uptake by a mechanism which is not directly dependent on the inhibition of globin synthesis. These substances either decrease teh production of energy-rich compounds or damage the cell membrane.

Regulation of Iron Entry into Reticulocytes. I. Feedback Inhibitory Effect of Heme on Iron Entry into Reticulocytes and on Heme Synthesis

Abstract In the presence of hemin 59Fe incorporation into both heme and reticulocytes decreases. Isonicotinic acid hydrazide which inhibits heme synthesis in vitro is shown to increase reticulocyte radioiron uptake. During inhibition of globin synthesis by cycloheximide, reticulocyte radioiron uptake decreases. This decrease is probably caused by free heme which accumulates in reticulocytes after inhibition of globin synthesis. Simultaneous addition of an inhibitor of heme synthesis—isonicotinic acid hydrazide—to reticulocytes treated by cycloheximide decreases the inhibitory effect of this antibiotic on the iron entry into reticulocytes. The authors suppose the feedback linkage by which heme regulates iron entry into reticulocytes.

Hemoglobin synthesis during rabbit reticulocyte maturation in vitro

A defined medium for the study of rabbit reticulocyte maturation in vitro is described; the use of this medium led to the following observations. 1. 1. During maturation, hemoglobin synthesis itself had no effect on the rate of decay of the reticulocyte's ability to synthesize hemoglobin. 2. 2. Iron deprivation during maturation led to an irreversible loss in the reticulocyte's ability to utilize exogenous iron for hemoglobin synthesis. 3. 3. Addition of hemin to the maturation medium resulted in inhibition of globin synthesis in fresh cells, but stimulation in cells which had been incubated for 20 h. 4. 4. Comparison of whole cell and cell-free protein synthesizing rates of fresh cells and cells incubated for 20 h showed a much greater decline in activity of the whole cells than the cell-free systems derived from them. The conclusion drawn from these observations is that heme synthetic capacity declines more rapidly than globin synthetic capacity during maturation in vitro. This suggests that the duration of heme synthetic capacity may be a quantitatively controlling feature in the development of erythroid cells.