Gantt J S, Chiang C S, Hatfield G W, Arfin S M
J Biol Chem. 1980 May 25;255(10):4808-13.
The growth of Chinese hamster ovary cells in a complete medium lacking asparagine is inhibited by beta-aspartylhydroxamate. The inhibition is overcome by the presence of asparagine in the growth medium. beta-Aspartylhydroxamate inhibits the activity of both asparagine synthetase and asparaginyl-tRNA synthetase in vitro. beta-Aspartylhydroxamate-resistant clones of Chinese hamster ovary cells have been isolated and three of these have been characterized. One clone, AH12, is 3-fold more resistant to beta-aspartylhydroxamate than the parental line and has 2 times higher levels of asparagine synthetase activity. Strains AH2 and AH5 are 6- to 7-fold more resistant to beta-aspartylhydroxamate and have 5 times higher levels of asparagine synthetase. The regulation of the expression of asparagine synthetase is altered in all three resistant cell lines. Whereas asparagine synthetase activity varies 2- to 3-fold in response to the asparagine content of the medium or to the extent of aminoacylation of tRNALeu in the parental cells, the activity of asparagine synthetase in the resistant cells is elevated under all growth conditions. No significant changes in the Km for substrates, Ki for beta-aspartylhydroxamate, or thermal stability were found for the asparagine synthetase of the resistant cells. These variants should prove useful in understanding the mechanisms involved in regulating the levels of asparagine synthetase in mammalian cells.
在缺乏天冬酰胺的完全培养基中，中国仓鼠卵巢细胞的生长受到β-天冬氨酰异羟肟酸的抑制。生长培养基中存在天冬酰胺可克服这种抑制作用。β-天冬氨酰异羟肟酸在体外抑制天冬酰胺合成酶和天冬氨酰-tRNA合成酶的活性。已分离出中国仓鼠卵巢细胞的β-天冬氨酰异羟肟酸抗性克隆，并对其中三个进行了表征。一个克隆AH12对β-天冬氨酰异羟肟酸的抗性比亲本细胞系高3倍，天冬酰胺合成酶活性水平高2倍。菌株AH2和AH5对β-天冬氨酰异羟肟酸的抗性高6至7倍，天冬酰胺合成酶水平高5倍。在所有三个抗性细胞系中，天冬酰胺合成酶表达的调节都发生了改变。在亲本细胞中，天冬酰胺合成酶活性随培养基中天冬酰胺含量或tRNALeu氨酰化程度的变化而变化2至3倍，而在抗性细胞中，天冬酰胺合成酶的活性在所有生长条件下均升高。抗性细胞的天冬酰胺合成酶在底物的Km、β-天冬氨酰异羟肟酸的Ki或热稳定性方面没有发现显著变化。这些变体应有助于理解调节哺乳动物细胞中天冬酰胺合成酶水平的机制。
