Vilchis F, Ramos L, Timossi C, Chávez B
Department of Reproductive Biology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga no. 15, Del. Tlalpan C.P. 14000, México DF, Mexico.
J Endocrinol. 2006 Apr;189(1):103-12. doi: 10.1677/joe.1.06300.
Ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), the terminal enzyme of the haem biosynthetic pathway, catalyses the insertion of ferrous iron into protoporphyrin IX to form protohaem. The Syrian hamster Harderian gland (HG) is known for its ability to produce and accumulate large amounts of protoporphyrins. In this species, the female gland contains up to 120 times more porphyrin than the male gland. Data from biochemical studies suggest that this gland possesses the enzymatic complex for haem biosynthesis but lacks ferrochelatase activity. The abundance of intraglandular haem proteins does not support this idea. To gain more insight into this process, we isolated cDNA for ferrochelatase from hamster liver, using the 5'- and 3'- rapid amplification of complementary DNA ends (RACE), and investigated its expression in HG from males and females. The full-length cDNA comprises an open reading frame of 1269 bp encoding a polypeptide of 422 amino-acid residues. Hamster DNA sequence exhibits 92% identity to mouse and 87% identity to human sequences. The predicted hamster enzyme was shown to have structural features of mammalian ferrochelatase, including a putative NH2- terminal presequence, a central core of about 330 amino-acid residues and an extra 30-50-amino-acid stretch at the carboxyl-terminus. RNA blotting experiments indicated that this cDNA hybridized to a liver mRNA of about 2.1 kb, while a weak hybridization signal was observed with mRNA from HG preparations. RT-PCR assays confirmed the expression of specific transcripts in both tissues. Male glands contained approximately twofold more enzyme mRNA than female glands. Likewise, the intraglandular content of mRNA varied during the oestrous cycle, with the highest levels found in the oestrous phase. These cyclic variations were less evident in liver. Ovariectomy plus treatment with progesterone or 17beta-oestradiol plus progesterone increased ferrochelatase mRNA of the gland. In HG of short- or long-term castrated males, the administration of testosterone did not affect the ferrochelatase mRNA concentration. Based on mRNA expression levels, we conclude that Harderian ferrochelatase may play an active role in maintaining the physiological pool of haem required for processing cytochromes and other glandular haem proteins. Likewise, the sex-steroid hormones appear to have only a modest influence upon Harderian ferrochelatase.
亚铁螯合酶（原卟啉亚铁裂解酶，EC 4.99.1.1）是血红素生物合成途径的末端酶，催化亚铁离子插入原卟啉IX中以形成原血红素。叙利亚仓鼠的哈德氏腺（HG）以其产生和积累大量原卟啉的能力而闻名。在该物种中，雌性腺体中的卟啉含量比雄性腺体多120倍。生化研究数据表明，该腺体拥有血红素生物合成的酶复合物，但缺乏亚铁螯合酶活性。腺体内血红素蛋白的丰度并不支持这一观点。为了更深入了解这一过程，我们利用5'-和3'-互补DNA末端快速扩增（RACE）技术从仓鼠肝脏中分离出亚铁螯合酶的cDNA，并研究了其在雄性和雌性HG中的表达。全长cDNA包含一个1269 bp的开放阅读框，编码一个422个氨基酸残基的多肽。仓鼠DNA序列与小鼠序列的同一性为92%，与人类序列的同一性为87%。预测的仓鼠酶显示具有哺乳动物亚铁螯合酶的结构特征，包括一个假定的NH2-末端前序列、一个约330个氨基酸残基的中央核心以及羧基末端额外的30 - 50个氨基酸延伸。RNA印迹实验表明，该cDNA与约2.1 kb的肝脏mRNA杂交，而在HG制剂的mRNA中观察到较弱的杂交信号。RT-PCR分析证实了两种组织中特异性转录本的表达。雄性腺体中的酶mRNA含量大约是雌性腺体的两倍。同样，腺体内mRNA含量在发情周期中有所变化，在发情期含量最高。这些周期性变化在肝脏中不太明显。卵巢切除加孕酮或17β-雌二醇加孕酮处理可增加腺体中亚铁螯合酶mRNA的含量。在短期或长期阉割的雄性HG中，给予睾酮并不影响亚铁螯合酶mRNA的浓度。基于mRNA表达水平，我们得出结论，哈德氏亚铁螯合酶可能在维持处理细胞色素和其他腺体内血红素蛋白所需的血红素生理库中发挥积极作用。同样，性甾体激素对哈德氏亚铁螯合酶似乎只有适度的影响。
