Multiple forms of rat-liver dihydropteridine reductase identified by their differing isoelectric points.
Multiple forms of rat-liver dihydropteridine reductase identified by their differing isoelectric points.
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通过不同的等电点鉴定多种形式的大鼠肝脏二氢蝶啶还原酶。
DOI：
10.1016/0003-9861(86)90432-7
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发表时间：
1986
影响因子：
3.9
通讯作者：
Whiteley,JM
中科院分区：
文献类型：
--
作者：
Webber,S;Hural,JA;Whiteley,JM
纯化的大鼠肝脏二氢蝶呤还原酶经凝胶过滤(Mr~51,000)、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(MR~25,500)和天然聚丙烯酰胺凝胶电泳均一，表明该酶由两个相同的亚基组成。然而，等电聚焦分析揭示了三种酶的近似等电点为6.5、5.9和5.7(分别指定为I、II和III)。通过制备层析聚焦分离得到三种形式，产率为65%，在含有1 HIM-β-巯基乙醇的0.05M磷酸盐缓冲液(pH 6.8)中是稳定的，并且以二氢生物蝶呤为底物比较其催化活性时，表现出相似的动力学常数。所有形式都与酶辅助因子NADH产生复合体，IEF也可以检测到这些复合体。当在6M尿素中变性条件下用IEF进一步检测时，该酶显示其三种形式的不同亚基组成。可以鉴定出两个不同的亚基，命名为α和β，另外的证据表明，天然酶形式I、II和III代表三种不同的二聚体组合αα(形式I)、αβ(形式II)和αβ(形式III)。
Purified rat-liver dihydropteridine reductase is homogeneous by gel filtration (Mr~ 51,000), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr~ 25,500), and native polyacrylamide gel electrophoresis, suggesting that the enzyme is composed of two identical subunits. However, analysis by isoelectric focusing has revealed three enzyme forms with approximate isoelectric points of 6.5, 5.9, and 5.7 (designated forms, I, II, and III, respectively). The three forms, isolated in 65% yield by preparative chromatofocusing, are stable in 0.05mphosphate buffer, pH 6.8, containing 1 Him β-mercaptoethanol and exhibit similar kinetic constants when the catalytic activities of the isolated forms are compared with quinonoid dihydrobiopterin as substrate. All forms generate complexes with the enzymatic cofactor NADH which are also detectable by IEF. When examined further by IEF under denaturing conditions in 6murea the enzyme demonstrates a differing subunit composition for its three forms. Two distinct subunits, designated α and β, can be identified, and additional evidence suggests that the native enzyme forms I, II, and III represent the three differing dimeric combinations αα (form I), αβ (form II), and αβ (form III).