van Heeswijck R, Ristevski S, Hynes M, Hoogenraad N
Department of Biochemistry, La Trobe University, Bundoora, Victoria, Australia.
Biochem Biophys Res Commun. 1990 May 16;168(3):1280-4. doi: 10.1016/0006-291x(90)91167-q.
An Aspergillus nidulans strain which is deficient in ornithine transcarbamylase due to the arg B1 mutation was transformed with a plasmid containing the ornithine transcarbamylase cDNA from rat liver under the control of the amd S promoter. Stable transformants were obtained by selection on arginine free medium indicating complementation of the arg B mutation. Proof of expression of the rat enzyme in transformants was obtained by immunoprecipitation of all ornithine transcarbamylase activity from cell extracts with antibodies specific for the rat enzyme. The presence of catalytically active rat ornithine transcarbamylase in the transformants indicated that it is capable of being imported into mitochondria in A. nidulans, proteolytically processed and assembled into its homotrimeric form. In vitro uptake experiments using isolated A. nidulans mitochondria demonstrate that processing of the precursor of rat ornithine transcarbamylase occurs in two temporally separated steps as it does in rat liver mitochondria suggesting evolutionary conservation of the processing machinery. Up to 560 ng of active rat enzyme was produced per gm wet weight mycelia. Use of beta-D-alanine, an inducer of amd S, as sole N-source resulted in increased levels of active rat ornithine transcarbamylase relative to uninduced cultures.
一株由于arg B1突变而缺乏鸟氨酸转氨甲酰酶的构巢曲霉菌株，用一个含有来自大鼠肝脏的鸟氨酸转氨甲酰酶cDNA且受amd S启动子控制的质粒进行转化。通过在无精氨酸培养基上筛选获得稳定的转化体，这表明arg B突变得到了互补。通过用针对大鼠酶的特异性抗体从细胞提取物中免疫沉淀所有鸟氨酸转氨甲酰酶活性，获得了转化体中大鼠酶表达的证据。转化体中具有催化活性的大鼠鸟氨酸转氨甲酰酶的存在表明，它能够被导入构巢曲霉的线粒体中，经过蛋白水解加工并组装成其同三聚体形式。使用分离的构巢曲霉线粒体进行的体外摄取实验表明，大鼠鸟氨酸转氨甲酰酶前体的加工过程与在大鼠肝脏线粒体中一样，发生在两个时间上分开的步骤，这表明加工机制在进化上是保守的。每克湿重菌丝体可产生高达560纳克的活性大鼠酶。使用β-D-丙氨酸（amd S的诱导剂）作为唯一氮源，相对于未诱导的培养物，活性大鼠鸟氨酸转氨甲酰酶的水平有所增加。
