Podschun B, Wahler G, Schnackerz K D
Institute of Physiological Chemistry, University of Würzburg, Federal Republic of Germany.
Eur J Biochem. 1989 Oct 20;185(1):219-24. doi: 10.1111/j.1432-1033.1989.tb15105.x.
Dihydropyrimidine dehydrogenase was isolated from cytosolic pig liver extracts and purified 3100-fold to apparent homogeneity. Purification made use of ammonium sulfate fractionation, precipitation with acetic acid and chromatography on DEAE-cellulose and 2',5'-ADP-Sepharose with 28% recovery of total activity. The native enzyme has a molecular mass of 206 kDa and is apparently composed of two similar, if not identical, subunits. Proteolytic cleavage reveals two fragments with apparent molecular masses of 92 kDa and 12 kDa. The C-terminal 12-kDa fragment seems to be extremely hydrophobic. The enzyme contains tightly associated compounds including four flavin nucleotide molecules and 32 iron atoms/206-kDa molecule. The iron atoms are probably present in iron-sulfur centers. The flavins released from the enzyme were identified as FAD and FMN in equal amounts. An isoelectric point of 4.65 was determined for the dehydrogenase. Apparent kinetic parameters were obtained for the substrates thymine, uracil, 5-aminouracil, 5-fluorouracil and NADPH.
二氢嘧啶脱氢酶从猪肝脏胞质提取物中分离出来，并纯化了3100倍，达到表观均一性。纯化过程采用硫酸铵分级沉淀、乙酸沉淀以及在DEAE-纤维素和2',5'-ADP-琼脂糖上进行层析，总活性回收率为28%。天然酶的分子量为206 kDa，显然由两个相似（即便不是完全相同）的亚基组成。蛋白水解裂解产生两个表观分子量分别为92 kDa和12 kDa的片段。C末端的12 kDa片段似乎极具疏水性。该酶含有紧密结合的化合物，包括四个黄素核苷酸分子和32个铁原子/206 kDa分子。铁原子可能存在于铁硫中心。从该酶释放的黄素被鉴定为等量的FAD和FMN。测定该脱氢酶的等电点为4.65。获得了底物胸腺嘧啶、尿嘧啶、5-氨基尿嘧啶、5-氟尿嘧啶和NADPH的表观动力学参数。
