Kim M K, Lee I Y, Ko J H, Rhee Y H, Park Y H
Bioprocess Technology Research Division, Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yusong, Taejon 305-600, Korea.
Biotechnol Bioeng. 1999 Feb 5;62(3):317-23. doi: 10.1002/(sici)1097-0290(19990205)62:3<317::aid-bit8>3.0.co;2-7.
Changes of intracellular nucleotide levels and their stimulatory effects on curdlan synthesis in Agrobacterium species were investigated under different culture conditions. Under nitrogen-limited conditions where curdlan synthesis was stimulated, intracellular levels of UMP were as high as 87 and those of AMP were 78 nmol/mg of cellular protein, while those under nitrogen-sufficient conditions were lower than 45 nmol/mg-protein. The levels of other nucleotides such as UDP, UTP, UDP-glucose, ADP, ATP, and ADP-glucose were lower than 30 nmol/mg-protein under both nitrogen-limited and sufficient conditions. The time profiles of curdlan synthesis and cellular nucleotide levels showed that curdlan synthesis had a positive relationship with intracellular levels of UMP and AMP. After the ammonium concentration in the medium fell below 0.1 g/L, intracellular levels of UMP and AMP increased, followed by curdlan synthesis. However, no significant changes in the specific activities of UMP kinase, UDP kinase, and UDP-glucose pyrophosphorylase were observed during cultivation. In vitro enzyme reactions for the synthesis of UDP-glucose, which serve as a precursor for curdlan synthesis, demonstrated that the synthesis of UDP-glucose increased with the increase of UMP concentration. In contrast, AMP had no effect on UDP-glucose synthesis at all. Addition of UMP in the medium increased the curdlan synthesis, whereas curdlan synthesis was inhibited in the presence of AMP. From these results, we concluded that only the higher intracellular UMP levels caused by nitrogen limitation in the medium enhance the metabolic flux of curdlan synthesis by promoting cellular UDP-glucose synthesis.
在不同培养条件下，研究了农杆菌属中细胞内核苷酸水平的变化及其对凝胶多糖合成的刺激作用。在刺激凝胶多糖合成的氮限制条件下，UMP的细胞内水平高达87 nmol/mg细胞蛋白，AMP的细胞内水平为78 nmol/mg细胞蛋白，而在氮充足条件下，它们低于45 nmol/mg蛋白。在氮限制和充足条件下，其他核苷酸如UDP、UTP、UDP-葡萄糖、ADP、ATP和ADP-葡萄糖的水平均低于30 nmol/mg蛋白。凝胶多糖合成和细胞内核苷酸水平的时间变化曲线表明，凝胶多糖合成与UMP和AMP的细胞内水平呈正相关。培养基中铵浓度降至0.1 g/L以下后，UMP和AMP的细胞内水平升高，随后开始合成凝胶多糖。然而，在培养过程中未观察到UMP激酶、UDP激酶和UDP-葡萄糖焦磷酸化酶的比活性有显著变化。作为凝胶多糖合成前体的UDP-葡萄糖合成的体外酶反应表明，UDP-葡萄糖的合成随着UMP浓度的增加而增加。相反，AMP对UDP-葡萄糖合成完全没有影响。在培养基中添加UMP可增加凝胶多糖的合成，而在AMP存在下凝胶多糖合成受到抑制。从这些结果中，我们得出结论，只有培养基中氮限制导致的较高细胞内UMP水平通过促进细胞UDP-葡萄糖合成来增强凝胶多糖合成的代谢通量。
