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

The effect of INH-inhibited heme synthesis on globin synthesis.

In the rabbit reticulocyte cell-free system, optimal globin synthesis is dependent upon an adequate level of heme. The effect of heme is mediated by an inhibitor of globin synthesis initiation termed HCR. In addition to marked inhibition of total globin synthesis, HCR results in a decreased alpha/beta globulin synthesis ratio. We describe here the use of INH as a relatively nontoxic inhibitor of heme synthesis in intact rabbit reticulocytes with a resultant inhibition of globin synthesis. In parallel with the inhibition of globin synthesis in reticulocytes, an inhibitor of globin synthesis in the hemin-supplemented cell-free system is generated. No INH-induced alterations in alpha/beta synthesis ratio could be found in "stress" reticulocytes from phenylhydrazine-treated rabbits, but "normal" reticulocytes from untreated rabbits showed a decreased alpha/beta ratio. Inhibition of heme synthesis and the resulting decrease in globin synthesis and intracellular hemoglobin concentration may have application as a potential treatment of homozygous sickle cell disease.

Asynchronous globin chain synthesis in rabbit reticulocyte lystates induced by hemin-controlled repressor.

To define further the role of hemin-controlled repressor (HCR) in globin synthesis, we studied its effect on the synthesis of individual globin chains in a rabbit reticulocyte lysate cell-free system. In the presence of HCR there was a marked globin chain imbalance, resulting in a lowered alpha/beta ratio. These findings in vitro may have relevance to certain clinical heme deficiency states in which a similar globin chain imbalance has been observed.