Silman N J, Carr N G, Mann N H
Department of Biological Sciences, University of Warwick, Coventry, United Kingdom.
J Bacteriol. 1995 Jun;177(12):3527-33. doi: 10.1128/jb.177.12.3527-3533.1995.
Glutamine synthetase (GS) inactivation was observed in crude cell extracts and in the high-speed supernatant fraction from the cyanobacterium Synechocystis sp. strain PCC 6803 following the addition of ammonium ions, glutamine, or glutamate. Dialysis of the high-speed supernatant resulted in loss of inactivation activity, but this could be restored by the addition of NADH, NADPH, or NADP+ and, to a lesser extent, NAD+, suggesting that inactivation of GS involved ADP-ribosylation. This form of modification was confirmed both by labelling experiments using [32P]NAD+ and by chemical analysis of the hydrolyzed enzyme. Three different forms of GS, exhibiting no activity, biosynthetic activity only, or transferase activity only, could be resolved by chromatography, and the differences in activity were correlated with the extent of the modification. Both biosynthetic and transferase activities were restored to the completely inactive form of GS by treatment with phosphodiesterase.
在添加铵离子、谷氨酰胺或谷氨酸后,在蓝藻聚球藻属PCC 6803菌株的粗细胞提取物和高速上清液组分中观察到谷氨酰胺合成酶(GS)失活。对高速上清液进行透析导致失活活性丧失,但通过添加NADH、NADPH或NADP +,以及在较小程度上添加NAD +,可以恢复这种活性,这表明GS的失活涉及ADP-核糖基化。通过使用[32P]NAD +的标记实验和对水解酶的化学分析,均证实了这种修饰形式。通过色谱法可以分离出三种不同形式的GS,分别表现出无活性、仅具有生物合成活性或仅具有转移酶活性,并且活性差异与修饰程度相关。通过用磷酸二酯酶处理,生物合成活性和转移酶活性均恢复到完全无活性的GS形式。