Ter Schure Eelko G, Silljé Herman H W, Raeven Leon J R M, Boonstra Johannes, Verkleij Arie J, Verrips C Theo
1Department of Molecular Cell Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
2Unilever Research Laboratorium Vlaardingen, Olivier van Noortlaan 120, 3133 AT Vlaardingen, The Netherlands.
Microbiology (Reading). 1995 May;141 ( Pt 5):1101-1108. doi: 10.1099/13500872-141-5-1101.
Variations in the transcription of nitrogen-regulated genes and in the activities of nitrogen-regulated enzymes of the yeast Saccharomyces cerevisiae were studied by changing the carbon and nitrogen fluxes. S. cerevisiae was grown in continuous culture at various dilution rates (D) under nitrogen limitation with NH4Cl as sole nitrogen source. With an increase in D from 0.05 to 0.29 h-1, both the glucose and the ammonia flux increased sixfold. The activities of the two ammonia-incorporating enzymes, NADPH-dependent glutamate dehydrogenase (NADPH-GDH) and glutamine synthetase (GS), encoded by GDH1 and GLN1, respectively, increased with increasing D, while the activity of the glutamate-degrading enzyme, NAD-dependent glutamate dehydrogenase (NAD-GDH), decreased. Surprisingly, no changes were observed in the transcription of GDH1 and GLN1; however increased D was accompanied by an increase in GAP1 transcription. At the metabolite level, the increase in the glucose and nitrogen flux did not result in changes in the intracellular 2-oxoglutarate, glutamate or glutamine concentrations. It is shown that growth on ammonia alone is not sufficient to cause repression of GAP1 and GLN1 transcription and that the regulation of GAP1 transcription and both NADPH-GDH and GS activity is not an on/off switch, but is gradually modulated in correlation with the ammonia concentration.
通过改变碳通量和氮通量，研究了酿酒酵母氮调节基因转录和氮调节酶活性的变化。酿酒酵母在以氯化铵为唯一氮源的氮限制条件下，以不同稀释率（D）进行连续培养。随着D从0.05 h-1增加到0.29 h-1，葡萄糖通量和氨通量均增加了六倍。分别由GDH1和GLN1编码的两种氨掺入酶，即NADPH依赖型谷氨酸脱氢酶（NADPH-GDH）和谷氨酰胺合成酶（GS）的活性，随着D的增加而增加，而谷氨酸降解酶NAD依赖型谷氨酸脱氢酶（NAD-GDH）的活性则下降。令人惊讶的是，未观察到GDH1和GLN1转录的变化；然而，D的增加伴随着GAP1转录的增加。在代谢物水平上，葡萄糖和氮通量的增加并未导致细胞内2-氧代戊二酸、谷氨酸或谷氨酰胺浓度的变化。结果表明，仅以氨为氮源生长不足以导致GAP1和GLN1转录的抑制，并且GAP1转录以及NADPH-GDH和GS活性的调节不是一个开/关开关，而是与氨浓度相关地逐渐调节。
