Kohno H, Furukawa T, Yoshinaga T, Tokunaga R, Taketani S
Department of Hygiene, Kansai Medical University, Osaka, Japan.
J Biol Chem. 1993 Oct 5;268(28):21359-63.
Coproporphyrinogen oxidase (EC 1.3.3.3), the enzyme involved in the sixth step of heme biosynthesis, was purified to apparent homogeneity from bovine liver; it has a molecular mass of 37,000 daltons. Partial amino acid sequences were determined. Two degenerate oligonucleotides based on the sequences of trypsin-digested peptides were used in a polymerase chain reaction to amplify a 198-base pair fragment of coproporphyrinogen oxidase DNA, using bovine kidney cells cDNA as a starting template. This fragment was used as a hybridization probe to isolate full-length coproporphyrinogen oxidase clones from a mouse erythroleukemia (MEL) cell cDNA library. Sequence analysis revealed that coproporphyrinogen oxidase comprises 354 amino acid residues (M(r) 40,647), with a putative leader sequence of 31 amino acid residues, the result being a mature protein of 323 amino acid residues (M(r) 37,255). RNA blot analysis revealed a 3.0-kilobase coproporphyrinogen oxidase mRNA in mouse liver and in MEL cells. Treatment of MEL cells with dimethyl sulfoxide led to an increase in coproporphyrinogen oxidase mRNA within 10 h, the induction reached a maximum at 24 h, and was in parallel with the induction of ferrochelatase mRNA. The cDNA allows for the expression of active coproporphyrinogen oxidase, the activity of which is mainly present in mitochondria of transfected cultured cells, thereby indicating that mammalian coproporphyrinogen oxidase is mitochondrial enzyme.
粪卟啉原氧化酶（EC 1.3.3.3）是参与血红素生物合成第六步的酶，从牛肝脏中纯化至表观均一；其分子量为37,000道尔顿。测定了部分氨基酸序列。基于胰蛋白酶消化肽段序列的两条简并寡核苷酸用于聚合酶链反应，以牛肾细胞cDNA为起始模板扩增粪卟啉原氧化酶DNA的198碱基对片段。该片段用作杂交探针，从小鼠红白血病（MEL）细胞cDNA文库中分离全长粪卟啉原氧化酶克隆。序列分析表明，粪卟啉原氧化酶由354个氨基酸残基组成（M(r) 40,647），具有31个氨基酸残基的推定前导序列，结果是一个由323个氨基酸残基组成的成熟蛋白（M(r) 37,255）。RNA印迹分析显示在小鼠肝脏和MEL细胞中有一条3.0千碱基的粪卟啉原氧化酶mRNA。用二甲基亚砜处理MEL细胞导致10小时内粪卟啉原氧化酶mRNA增加，诱导在24小时达到最大值，并且与铁螯合酶mRNA的诱导平行。该cDNA允许表达活性粪卟啉原氧化酶，其活性主要存在于转染培养细胞的线粒体中，从而表明哺乳动物粪卟啉原氧化酶是线粒体酶。
