Purification and properties of mouse liver coproporphyrinogen oxidase

Abstract

Coproporphyrinogen oxidase was purified to homogeneity from mouse liver. The specific activity of the pure enzyme was 3500 nmol · h−1· mg−1; its apparent molecular mass (35 kDa) was confirmed by immunological characterization of the enzyme in a trichloroacetic‐acid‐precipitated total‐liver‐protein extract. The native enzyme appeared to be a dimer of 70 kDa as determined by gel filtration under nondenaturating conditions. The Km value for coproporphyrinogen III was 0.3 μM. The purified enzyme was activated by neutral detergents and phospholipids (affecting both Vmax and Km) but inhibited by ionic detergents. Reactivity toward sulfhydryl agents suggested the possible involvement of (an) SH group(s) for the activity. When compared to the previously purified coproporphyrinogen oxidases (from bovine liver and yeast), the mouse liver coproporphyrinogen oxidase appears to share many common catalytic properties with both enzymes. However, its apparent molecular mass is very different from that of the bovine liver enzyme (71.6 kDa) but identical to that found for the yeast (Saccharomyces cerevisiae) enzyme.

Crystal Structure of the Oxygen-dependant Coproporphyrinogen Oxidase (Hem13p) of Saccharomyces cerevisiae

“…Mature odCPO is an ϳ35-kDa protein that exists as a stable ϳ70-kDa dimer in solution (3,9,11,12,(15)(16)(17). It is located in the mitochondria of higher eukaryotes (1,12,15,18,19), but resides in the cytosol of S. cerevisiae (16).…”

Molecular cloning, sequencing, and functional expression of a cDNA encoding human coproporphyrinogen oxidase.

“…Two different phenotypes of unrelated homozygous patients have been described, both with a reduction of CPX activity to MATERIALS AND METHODS Purification, Activity, and Amino Acid Sequening of CPX from Murine Liver. CPX was purified from 600 g of mouse (Swiss strain) livers according to the method of Bogard et al (5). To remove ampholytes associated with the protein, the enzyme preparations with the highest purity were further applied to a Pharmacia Mono-P FPLC column equilibrated with 50 mM ammonium hydrogen carbonate buffer (pH 7.5) and the enzyme was eluted in a linear gradient ofNaCI (0-500 mM in the equilibration buffer).…”

“…Coproporphyrinogen oxidase (CPX; EC 1.3.3.3) is a soluble mitochondrial protein that is localized in the intermembrane space within mammalian cells (1,2) and catalyzes the sixth step in heme biosynthesis, the conversion of the two propionate groups at positions 2 and 4 of coproporphyrinogen III to two vinyl groups, thus producing protoporphyrinogen IX (3). CPX has been characterized and purified to homogeneity from a variety of sources, including the yeast Saccharomyces cerevisiae (4), mouse liver (5), and bovine liver (6). The CPX genes from S. cerevisiae (7), Salmonella typhimurium (8), and soybean (9) have been cloned and sequenced.…”

In situ conversion of coproporphyrinogen to heme by murine mitochondria: Terminal steps of the heme biosynthetic pathway

“…All three of these enzymes have been purified from mouse liver and characterized to various extents Dailey & Karr, 1987;Bogard et al, 1989;Proulx & Dailey, 1992; see Dailey, 1990). The terminal two enzymes have also been reconstituted into phospholipid vesicles and their membrane-bound versus soluble properties examined .…”