Kawai T, Nishikimi M, Ozawa T, Yagi K
Institute of Applied Biochemistry, Yagi Memorial Park, Gifu, Japan.
J Biol Chem. 1992 Oct 25;267(30):21973-6.
The osteogenic disorder Shionogi (ODS) rat is a mutant Wistar rat that is subject to scurvy, because it lacks L-gulono-gamma-lactone oxidase, a key enzyme in L-ascorbic acid biosynthesis. Sequencing of polymerase chain reaction-amplified cDNAs for mutant and normal rat L-gulono-gamma-lactone oxidases demonstrated that the mutant cDNA has a single base mutation from G to A at nucleotide 182, which mutation alters the 61st amino acid residue from Cys to Tyr. To test the effect of this mutation on the expression of L-gulono-gamma-lactone oxidase, we inserted a region of the cDNAs coding for normal and mutant L-gulono-gamma-lactone oxidases into an expression vector, pSVL, and transfected COS-1 cells with such vectors. The result indicated that the defined amino acid substitution does decrease both the amount of immunologically detectable protein and the level of enzyme activity to about one-tenth of their normal values, while it does not affect the amount of the mRNA produced in the transfected cells. This situation is similar to our previous observation that L-gulono-gamma-lactone oxidase is expressed in the liver of the ODS rat at a very low level irrespective of the presence of a normal amount of L-gulono-gamma-lactone oxidase-specific mRNA of a normal size (Nishikimi, M., Koshizaka, T., Kondo, K., and Yagi, K. (1989) Experientia (Basel) 45, 126-129). Thus it became clear that the Cys-->Tyr substitution is responsible for the L-gulono-gamma-lactone oxidase deficiency in the ODS rat.
成骨障碍Shionogi（ODS）大鼠是一种突变的Wistar大鼠，因缺乏L-抗坏血酸生物合成中的关键酶L-古洛糖酸-γ-内酯氧化酶而患坏血病。对突变型和正常大鼠L-古洛糖酸-γ-内酯氧化酶的聚合酶链反应扩增cDNA进行测序表明，突变型cDNA在核苷酸182处有一个从G到A的单碱基突变，该突变将第61个氨基酸残基从半胱氨酸改变为酪氨酸。为了测试这种突变对L-古洛糖酸-γ-内酯氧化酶表达的影响，我们将编码正常和突变型L-古洛糖酸-γ-内酯氧化酶的cDNA区域插入表达载体pSVL中，并用这些载体转染COS-1细胞。结果表明，确定的氨基酸取代确实将免疫可检测蛋白的量和酶活性水平降低到正常值的约十分之一，而不影响转染细胞中产生的mRNA的量。这种情况与我们之前的观察结果相似，即无论是否存在正常量的正常大小的L-古洛糖酸-γ-内酯氧化酶特异性mRNA，ODS大鼠肝脏中L-古洛糖酸-γ-内酯氧化酶的表达水平都非常低（Nishikimi，M.，Koshizaka，T.，Kondo，K.，和Yagi，K.（1989）实验（巴塞尔）45，126 - 129）。因此，很明显，半胱氨酸到酪氨酸的取代是ODS大鼠L-古洛糖酸-γ-内酯氧化酶缺乏的原因。
