Kohashi M, Clement R P, Tse J, Piper W N
Biochem J. 1984 Jun 15;220(3):755-65. doi: 10.1042/bj2200755.
Rat hepatic uroporphyrinogen III co-synthase was isolated and purified 73-fold with a 13% yield by (NH4)2SO4 fractionation and sequential chromatography on DEAE-Sephacel, Sephadex G-100 (superfine grade) and folate-AH-Sepharose 4B. The purified co-synthase has an Mr of approx. 42 000, and is resolved into two bands, each possessing co-synthase activity, by polyacrylamide-gel electrophoresis. A factor was dissociated from the purified co-synthase. Results of both microbiological and competitive protein-binding assays suggest that it is a pteroylpolyglutamate. The isolated pteroylpolyglutamate factor was co-eluted with authentic N5-methyltetrahydropteroylheptaglutamate on DEAE-Sephacel. Uroporphyrinogen III is formed by cosynthase-free preparations of uroporphyrinogen I synthase in the presence of tetrahydropteroylglutamate. Tetrahydropeteroylheptaglutamate is also able to direct the formation of equivalent amounts of uroporphyrinogen III at a concentration approximately one-hundredth that of tetrahydropteroylmonoglutamate. These results suggest that a reduced pteroylpolyglutamate factor is associated with rat hepatic uroporphyrinogen III co-synthase, and that this may function as a coenzyme for the biosynthesis of uroporphyrinogen III.
通过硫酸铵分级分离以及在DEAE-葡聚糖凝胶、葡聚糖凝胶G-100（超细级）和叶酸-AH-琼脂糖4B上的连续层析，大鼠肝脏尿卟啉原III合酶被分离纯化，纯化了73倍，产率为13%。纯化后的合酶的相对分子质量约为42000，在聚丙烯酰胺凝胶电泳中可分离为两条带，每条带都具有合酶活性。从纯化的合酶中分离出一种因子。微生物学和竞争性蛋白结合试验的结果均表明它是一种蝶酰多谷氨酸。分离出的蝶酰多谷氨酸因子在DEAE-葡聚糖凝胶上与标准的N5-甲基四氢蝶酰庚谷氨酸共洗脱。在四氢蝶酰谷氨酸存在的情况下，尿卟啉原I合酶的无合酶制剂可形成尿卟啉原III。四氢蝶酰庚谷氨酸也能够在浓度约为四氢蝶酰单谷氨酸百分之一的情况下指导形成等量的尿卟啉原III。这些结果表明，一种还原型蝶酰多谷氨酸因子与大鼠肝脏尿卟啉原III合酶相关，并且这可能作为尿卟啉原III生物合成的辅酶发挥作用。
