Smith S J, Cox T M
Department of Medicine, University of Cambridge, Addenbrooke's Hospital, U.K.
Cell Mol Biol (Noisy-le-grand). 1997 Feb;43(1):103-14.
Heme formation in immature erythroid cells is subject to end-product negative feedback control. Although studies with immature erythroid cells obtained from animals have shown that increased intracellular hemin inhibits the acquisition of iron from transferrin, our experiments with human reticulocytes indicate that feedback inhibition of heme biosynthesis is primarily regulated at one or more steps that lead to formation of the first committed precursor, delta-aminolevulinate (ALA). To identify the site of control of heme biosynthesis in the human erythron further, region-specific antibodies to human erythroid delta-ALA synthase (e-ALA synthase) were used to immunoprecipitate newly-synthesised enzyme from human reticulocytes after biosynthetic labelling. Low concentrations of exogenous hemin (30-35 microM) inhibited the biosynthetic labelling of mature erythroid ALA synthase that was detected by exon 4 peptide-specific antibodies and antibodies raised against the entire recombinant human erythroid ALA synthase molecule. Pulse-chase experiments after biosynthetic labelling indicated no differences in the effect of hemin on the turnover of the radiolabelled enzyme and hemin did not influence the distribution of precursor froms of the ALA synthase molecule. Parallel experiments using antibodies directed against human H-chain ferritin confirmed the specificity of the effects of hemin on translation of the e-ALA synthase mRNA. At the concentrations of hemin used to inhibit heme formation from 14C-glycine, no significant effects on the rate of overall protein synthesis were observed. We conclude that heme regulates synthesis of the first committed precursor of the porphyrin biosynthetic pathway in immature human erythroid cells by effects on the synthesis of the e-ALA synthase molecule. Although the mechanism of hemin action is unknown, it is apparently independent of 5'-iron-response elements and influences the translational activity of erythroid ALA synthase mRNA.
未成熟红细胞中的血红素形成受到终产物负反馈控制。尽管对从动物获得的未成熟红细胞的研究表明，细胞内血红素增加会抑制从转铁蛋白获取铁，但我们用人网织红细胞进行的实验表明，血红素生物合成的反馈抑制主要在导致第一个定向前体δ-氨基乙酰丙酸（ALA）形成的一个或多个步骤中受到调节。为了进一步确定人类红细胞生成中血红素生物合成的控制位点，使用针对人类红细胞δ-ALA合酶（e-ALA合酶）的区域特异性抗体，在生物合成标记后从人网织红细胞中免疫沉淀新合成的酶。低浓度的外源性血红素（30 - 35 microM）抑制了成熟红细胞ALA合酶的生物合成标记，这是通过外显子4肽特异性抗体和针对整个重组人类红细胞ALA合酶分子产生的抗体检测到的。生物合成标记后的脉冲追踪实验表明，血红素对放射性标记酶的周转没有影响，并且血红素不影响ALA合酶分子前体形式的分布。使用针对人类H链铁蛋白的抗体进行的平行实验证实了血红素对e-ALA合酶mRNA翻译作用的特异性。在用于抑制从14C-甘氨酸形成血红素的血红素浓度下，未观察到对总体蛋白质合成速率有显著影响。我们得出结论，血红素通过影响e-ALA合酶分子的合成来调节未成熟人类红细胞中卟啉生物合成途径的第一个定向前体的合成。尽管血红素作用的机制尚不清楚，但它显然独立于5'-铁反应元件，并影响红细胞ALA合酶mRNA的翻译活性。
