Hemmens B, Carrey E A
Department of Biochemistry, University of Dundee, Scotland.
Eur J Biochem. 1995 Jul 1;231(1):220-5.
We have purified mammalian dihydroorotase as a polypeptide fragment of 46 kDa from an elastase digest of CAD, the 240-kDa multienzyme that catalyses the first three reactions of pyrimidine biosynthesis. The thermal unfolding of the domain was analysed through the change in circular dichroism, indicating a sharp transition at 45 degrees C in which most of the native alpha-helix is lost. Although there is good evidence that the fragments associate as dimers in solution, chemical cross-linking was only possible when the dihydroorotase domain was included in a larger proteolytic fragment of 190-195 kDa. Cross-linking of the isolated domain yielded a species that appeared to result from links between two or more sub-domains, and did not yield the expected 90-kDa dimer of dihydroorotase. We speculate that the presence of other folded regions of CAD stabilises the interactions between dihydroorotase domains.
我们从CAD（一种催化嘧啶生物合成前三个反应的240 kDa多酶）的弹性蛋白酶消化物中纯化出了46 kDa的哺乳动物二氢乳清酸酶多肽片段。通过圆二色性的变化分析了该结构域的热解折叠，结果表明在45℃时发生了急剧转变，此时大部分天然α-螺旋结构丧失。尽管有充分证据表明这些片段在溶液中以二聚体形式缔合，但只有当二氢乳清酸酶结构域包含在190 - 195 kDa的较大蛋白水解片段中时，化学交联才有可能实现。分离结构域的交联产生了一种似乎是由两个或更多亚结构域之间的连接导致的产物，并未产生预期的90 kDa二氢乳清酸酶二聚体。我们推测CAD其他折叠区域的存在稳定了二氢乳清酸酶结构域之间的相互作用。
