Hasegawa H
J Biochem. 1977 Jan;81(1):169-77. doi: 10.1093/oxfordjournals.jbchem.a131432.
Dihydropteridine reductase [EC 1.6.99.7] was purified from bovine liver in 50% yield and crystallized. The physicochemical properties of the purified enzyme were quite similar to those of sheep liver dihydropteridine reductase. During the course of purification, however, the enzyme was found to be separated into 2 major peaks together with minor peaks by column chromatography on CM-Sephadex, and one of the major peaks was identified as a binary complex of the enzyme with NADH. The reductase-NADH complex was also prepared in vitro and crystallized. Upon addition of quinonoid-dihydropterin to the complex, NADH was oxidized and released from the enzyme. The amount of bound NADH was calculated to be 2 moles per mole of the reductase. The occurrence of the reductase-NADH was calculated to be 2 moles per mole of the reductase. The occurrence of the reductase-NADH complex in bovine liver extract as a predominant form was in accord with the pyridine nucleotide specificity for NADH as a coenzyme. The results further support the view that NADH is the natural coenzyme of this reductase.
二氢蝶啶还原酶[EC 1.6.99.7]从牛肝中纯化出来，产率为50%，并进行了结晶。纯化后的酶的物理化学性质与羊肝二氢蝶啶还原酶非常相似。然而，在纯化过程中，通过CM - 葡聚糖凝胶柱色谱法发现该酶被分离成2个主要峰以及一些小峰，其中一个主要峰被鉴定为该酶与NADH的二元复合物。还原酶 - NADH复合物也在体外制备并结晶。向该复合物中加入醌型二氢蝶呤后，NADH被氧化并从酶中释放出来。计算得出每摩尔还原酶结合的NADH量为2摩尔。还原酶 - NADH复合物在牛肝提取物中以主要形式存在，这与NADH作为辅酶的吡啶核苷酸特异性一致。这些结果进一步支持了NADH是该还原酶天然辅酶的观点。
