Grace A. Vanderhoff

Control of globin synthesis: The role of heme☆

Abstract The initial rate of globin synthesis in reticulocyte lysates is close to the steady-state rate in intact reticulocytes, but the amount of globin made by these preparations is low because the initiation of new chains of globin becomes the rate-limiting step shortly after the start of cell-free incubation. The amount of globin made by a given lysate is constant and independent of the initial rate of protein synthesis if that rate is altered by making the incubations at low concentrations of cycloheximide or at different temperatures. There are two exceptions to these general observations: (1) below about 25 °C, the capacity to initiate is much more stable and protein synthesis continues at a constant rate to a level which exceeds the amount made at the initial rate for higher temperatures. (2) In the presence of 3 × 10−5 m-hemin, the initial rate of initiation is preserved for much longer than in the absence of added hemin. Lysates whose rate of protein synthesis has markedly declined may be reactivated by the addition of ribosome-free cytoplasm from a fresh lysate. A few lysates can be reactivated by the addition of hemin, but in this case synthesis resumes only after a lag of a few minutes. From these observations we draw the conclusion that initiation in reticulocyte lysates involves one or more “soluble” initiation factors which are inactivated by protein synthesis above 25 °C and which are stabilized by hemin.

Glutathione: VIII. The effects of glutathione disulfide on initiation of protein synthesis

Abstract 1. 1. Glutathione disulfide (GSSG) additions (1 · 10−4–2· 10−4 M or less) to rabbit reticulocyte lysates containing glutathione (GSH) in normal amounts (0.7 · 10−3–3 · 10−3 M) have no immediate effect on the rate of protein synthesis at 33°C but cause a rapid decrease in synthesis rate after 6–9 min (the lag period), followed by complete cessation of protein-synthesizing activity. 2. 2. The lag period varies with temperature; the length of the lag period and the time dependence of the decrease in synthesis provide important information for the interpretation of the GSSG effect on protein synthesis. 3. 3. Reduction of GSSG to GSH before or during the lag period prevents inhibition of protein synthesis. Such reduction has no effect on the behavior of the system if carried out after the lag period. 4. 4. Cessation of protein synthesis is accompanied by a conversion of polysomes to monosomes, with little dissociation into subunits. 5. 5. A ribosome-free supernatant is effective in reversing the inhibition of protein synthesis caused by GSSG. Fractionation of the supernatant leads to a protein (precipitated by 40–70 % (NH4)2SO4) which reverses the GSSG inhibition. Further fractionation yields a high molecular weight fraction with inhibition-reversing activity. 6. 6. Factor Q is the name given to the material with inhibition-reversing activity, the inhibition-reversal factor. A scheme for the behavior of Factor Q in protein synthesis is proposed. 7. 7. The effect of the antibiotic, cycloheximide, on initiation of protein synthesis is interpreted in light of the GSSG effect. 8. 8. A small rise in the usual GSSG concentration of a cell is recognized as a potentially important event, one requiring careful scrutiny.

Iron removal from transferrin. An experimental study.

We have studied the facilitation of iron transfer from transferrin to desferrioxamine by various anions. Most of the anions which can substitute for HCO-3 in the ternary complex of transferrin - Fe - HCO3 do not facilitate iron transfer; anions which do facilitate iron transfer do not necessarily form stable ternary complexes. Combinations of anions effective in transfer have a less-than-additive effect, suggesting a common reaction pathway. We suggest that the transfer of iron from transferrin to desferrioxamine involves a substitution step and a subsequent chelation step, and that the efficiency of the overall reaction is a function of both these attributes of the anion.

Chelate mediated transfer of iron from transferrin to desferrioxamine.

SUMMARY. Desferrioxamine, widely used for the treatment of iron overload in Cooleys anaemia, binds iron so tightly that it should quantitatively remove iron from transferrin. Studies conducted in vivo and in vitro, however, have failed to demonstrate significant depletion of transferrin‐bound iron by a stoichiometric excess of desferrioxamine. However, low molecular weight chelating agents, capable of forming ternary complexes with transferrin and ferric iron, can promote a rapid transfer of iron from transferrin to desferrioxamine. A possible mechanism for this facilitated exchange is offered.

Inhibition of protein synthesis by glutathione disulfide in the presence of glutathione

Abstract As little as 5 × 10−5 M glutathione disulfide (GSSG) in the presence of 80 × 10−5 M glutathione (GSH) in a rabbit reticulocyte lysate caused a profound inhibition of initiation of protein synthesis. A potential physiological regulatory role for GSSG in protein synthesis is thus revealed.

The Effects of Nucleosides On the Resistance of Normal Human Erythrocytes to Osmotic lysis

The investigations reported in this paper proceeded on the hypothesis that maintenance of the structural integrity of the human erythrocyte is dependent on continued production and utilization of energy by the cell. In an attempt to test this hypothesis the susceptibility of fresh human erythrocytes to osmotic lysis was studied in terms of the influence of various compounds that might serve as substrates for energy yielding reactions within the erythrocyte. Particular attention was paid to glucose (3) and purine nucleosides (4, 7) which have been shown to prolong the viability of stored erythrocytes and to retard their progressive lysis and diminished resistance to hypotonic solutions. It may be noted, however, that the effectiveness of the purine nucleosides in the preservation of erythrocytes has recently been questioned (8).

Erythroid Cell Differentiation and the Synthesis and Assembly of Hemoglobin

Publisher Summary This chapter focuses on erythroid cell differentiation and the synthesis and assembly of hemoglobin. The control of hemoglobin synthesis is concerned with the following: (1) the determination of the primary structure of each chain, (2) differentiation of erythroid cells and the induction of hemoglobin synthesis, (3) the switch from the synthesis of embryonic to fetal hemoglobin and of fetal to adult hemoglobin during gestation and early postnatal life, (4) the coordination of synthesis of alpha chains and of their complementary beta, gamma, delta, and epsilon chains, and (5) the regulation and coordination of the syntheses of heme and of globin. The sensitivity of hemoglobin synthesis to 5-fluorouracil and to bromodeoxyuridine only up to the head process stage suggests that DNA synthesis is necessary for hemoglobin synthesis only during these early stages. The differentiation of erythroid cells, the induction of hemoglobin synthesis, and the switch from embryonic and fetal to adult hemoglobin synthesis may be explicable in terms of control at the level of transcription of genes. The mechanism of action of heme in promoting the formation of polyribosomes, accelerating the synthesis of globin, and regulating the assembly of hemoglobin is under study. The synthesis of heme is controlled by feedback inhibition of the formation of ALA. Heme stimulates the synthesis of globin and promotes the coordinate assembly of hemoglobin. This schema of regulation is helpful in understanding the induction and control of hemoglobin synthesis in developing erythroid cells, and it provides a framework for clarifying various disorders of hemoglobin metabolism in man.