Chefalo P J, Oh J, Rafie-Kolpin M, Kan B, Chen J J
Harvard-MIT Division of Health Sciences and Technology, MIT, Cambridge, Massachusetts 02139, USA.
Eur J Biochem. 1998 Dec 1;258(2):820-30. doi: 10.1046/j.1432-1327.1998.2580820.x.
The regulation of protein synthesis by the availability of heme in reticulocytes is well established. However, the mechanism by which heme regulates translational initiation is not clear. In this study, we have examined the heme regulation directly on the homogeneous heme-regulated eIF-2alpha kinase (HRI), which is activated during heme deficiency. We found that HRI purified as a hemoprotein with the characteristic Soret band of hemoprotein at 424 nm. This HRI was an active autokinase and eIF-2alpha kinase, and its kinase activities were inhibited by submicromolar concentrations of hemin with an apparent Ki of 0.5 microM. Homogeneous HRI was a homodimer, and its activities could not be inhibited by incubation with purified inactive K199R HRI in vitro. Our results suggest that there are two distinct types of heme-binding sites in the HRI homodimer. The binding of heme to the first site is stable, while the binding of heme to the second site is responsible for the rapid downregulation of HRI activity by heme. These results indicate that HRI binds heme and serves as a sensor of the availability of heme to coordinate the balanced synthesis of globins and heme in erythroid cells.
网织红细胞中血红素的可用性对蛋白质合成的调节已得到充分证实。然而，血红素调节翻译起始的机制尚不清楚。在本研究中，我们直接研究了血红素对同源的血红素调节的eIF-2α激酶（HRI）的调节作用，该激酶在血红素缺乏时被激活。我们发现纯化的HRI是一种血红蛋白，在424nm处有血红蛋白特有的Soret带。这种HRI是一种活性自身激酶和eIF-2α激酶，其激酶活性受到亚微摩尔浓度的血红素抑制，表观Ki为0.5μM。同源的HRI是一种同二聚体，在体外与纯化的无活性K199R HRI孵育不能抑制其活性。我们的结果表明，HRI同二聚体中有两种不同类型的血红素结合位点。血红素与第一个位点的结合是稳定的，而血红素与第二个位点的结合则导致HRI活性被血红素快速下调。这些结果表明，HRI结合血红素并作为血红素可用性的传感器，以协调红细胞中珠蛋白和血红素的平衡合成。
