Sakakibara H, Shimizu H, Hase T, Yamazaki Y, Takao T, Shimonishi Y, Sugiyama T
Department of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University, Nagoya 464-01, Japan.
J Biol Chem. 1996 Nov 22;271(47):29561-8. doi: 10.1074/jbc.271.47.29561.
In maize, a small multigene family encodes the cytosolic isoforms of glutamine synthetase (GS), and five cDNAs, designated pGS1a, pGS1b, pGS1c, pGS1d, and pGS1e, have been cloned (Sakakibara, H., Kawabata, S., Takahashi, H., Hase, T., and Sugiyama, T. (1992) Plant Cell Physiol. 33, 49-58; Li, M., Villemur, R., Hussey, P. J., Silflow, C. D., Gantt, J. S., and Snustad, D. P. (1993) Plant Mol. Biol. 23, 401-407). This report describes the identification and enzymatic characterization of the cytosolic isoforms of GS in maize roots, namely GS1 and GSr. The purified isoforms, as well as recombinant enzymes that had been overexpressed in Escherichia coli, were analyzed by capillary liquid chromatography/electrospray ionization-mass spectrometry, and GS1 and GSr were identified as the products of the GS1a/GS1b and GS1c/GS1d genes, respectively. Upon the addition of ammonia to the culture medium, significant amounts of GSr accumulated and a preferential increase in GS synthetase activity, as compared to GS transferase activity, was found in the root extract. Assays with the purified recombinant enzymes confirmed that the specific biosynthetic and synthetase activities of GSr were 1.6-fold higher than those of GS1. Marked differences in stability were also found between the two isoforms: GSr was more sensitive to heat than GS1 and octameric aggregates of the subunits of GSr were easily dissociated to monomers than those of GS1 at low concentrations of Mn2+ and Mg2+ ions. These characteristics of the ammonia-induced isoform of GS seem to be physiologically important for the primary assimilation of external ammonia by roots.
在玉米中，一个小的多基因家族编码谷氨酰胺合成酶（GS）的胞质同工型，并且已经克隆了五个cDNA，分别命名为pGS1a、pGS1b、pGS1c、pGS1d和pGS1e（Sakakibara, H., Kawabata, S., Takahashi, H., Hase, T., and Sugiyama, T. (1992) Plant Cell Physiol. 33, 49 - 58; Li, M., Villemur, R., Hussey, P. J., Silflow, C. D., Gantt, J. S., and Snustad, D. P. (1993) Plant Mol. Biol. 23, 401 - 407）。本报告描述了玉米根中GS胞质同工型即GS1和GSr的鉴定及酶学特性。通过毛细管液相色谱/电喷雾电离质谱对纯化的同工型以及在大肠杆菌中过表达的重组酶进行了分析，结果表明GS1和GSr分别是GS1a/GS1b和GS1c/GS1d基因的产物。向培养基中添加氨后，根提取物中积累了大量的GSr，并且与GS转移酶活性相比，GS合成酶活性优先增加。对纯化的重组酶进行的测定证实，GSr的比生物合成活性和合成酶活性比GS1高1.6倍。两种同工型在稳定性上也存在显著差异：GSr比GS1对热更敏感，并且在低浓度的Mn2+和Mg2+离子条件下，GSr亚基的八聚体聚集体比GS1的更容易解离为单体。GS的氨诱导同工型的这些特性似乎对于根对外部氨的初级同化在生理上具有重要意义。
