Abstract
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.
Highlights
From the Department of Hygiene, Kansai Medical University, Moriguchi, Osaka570, Japan andthe $Department of Public Health, Faculty of Medicine, Kyoto University, Kyoto 606, Japan
PorphyrinogenoxidaseDNA, using bovine kidnceyells cDNA as a starting template. This fragmentwas used as a hybridization probe to isolate full-length coproporphyrinogen oxidase clones from a mouse erythroleukemia (MEL) cell cDNA library
The gene for yeast coprogen oxidase was isolated and was found to encode a protein of 328 amino acids; it has a calculated molecular mass of 37,673 Da [8].Expression of the gene proved to be negatively regulated by heme and oxygen
Summary
In contrast to its location in mitochondria in liver cells, liver; it has a molecular mass 3o7f,000 daltons.Partial yeast coprogen oxidase is located inthe cytosol [7]. PorphyrinogenoxidaseDNA, using bovine kidnceyells cDNA as a starting template This fragmentwas used as a hybridization probe to isolate full-length coproporphyrinogen oxidase clones from a mouse erythroleukemia (MEL) cell cDNA library. The gene for yeast coprogen oxidase was isolated and was found to encode a protein of 328 amino acids; it has a calculated molecular mass of 37,673 Da [8].Expression of the gene proved to be negatively regulated by heme and oxygen
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