PURIFICATION AND PROPERTIES OF COPROPORPHYRINOGEN OXIDASE FROM THE YEAST SACCHAROMYCES-CEREVISIAE
PURIFICATION AND PROPERTIES OF COPROPORPHYRINOGEN OXIDASE FROM THE YEAST SACCHAROMYCES-CEREVISIAE
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DOI：
10.1111/j.1432-1033.1986.tb09617.x
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发表时间：
1986-05-02
期刊：
影响因子：
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通讯作者：
LABBE, P
中科院分区：
文献类型：
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作者：
CAMADRO, JM;CHAMBON, H;LABBE, P
粪卟啉原氧化酶定位于酵母细胞的胞质溶胶中。从血红素突变株中纯化该酶至同质，该血红素突变株表现出高比活性（15-20酶单位/mg可溶性蛋白，而野生型菌株为1-2酶单位/mg可溶性蛋白）。通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（Mr = 35 000）和等电聚焦（pI = 6.2）判断，最终制备物是均匀的。在AcA 44上的凝胶过滤得到70 000的相对分子质量。N-末端氨基酸序列分析显示一个单一的多肽链。因此，该酶似乎是具有相同亚基的二聚体。两个铁原子/分子的天然蛋白质被检测到，他们不能被删除，通过彻底透析或凝胶过滤Sephadex G-25。然而，参与的铁原子的氧化催化活性的酶是活跃的，只有当分子氧被用作电子受体，没有厌氧活性可以检测到。 巯基导向试剂部分抑制酶，表明活性需要一个SH基团。酵母粪卟啉原氧化酶被中性洗涤剂的磷脂激活，如牛肝酶所述。
Coproporphyrinogen oxidase has been located in the cytosol of yeast cells. The enzyme was purified to homogeneity from a heme mutant strain exhibiting a high specific activity (15-20 enzyme units/mg soluble protein compared to 1-2 enzyme units/mg soluble protein of the wild-type strain). The final preparation was homogeneous as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (Mr = 35 000) and isoelectrofocusing (pI = 6.2). Gel filtration on AcA 44 gave a relative molecular mass of 70 000. N-terminal amino-acid sequence analysis revealed a single polypeptide chain. Thus the enzyme appears to be a dimer with identical subunits. Two iron atoms/molecule of native protein were detected; they could not be removed by exhaustive dialysis or gel filtration on Sephadex G-25. However the involvement of the iron atoms in the oxidative catalytic activity of the enzyme was active only when molecular oxygen was used as electron acceptor; no anaerobic activity could be detected. Thiol-directed reagents partially inhibited the enzyme, indicating that an SH group is required for activity. Yeast coproporphyrinogen oxidase was activated by phospholipids of neutral detergents as described for the bovine liver enzyme.