Purification of P(II) and P(II)-UMP and In Vitro Studies of Regulation of Glutamine Synthetase in Rhodospirillum rubrum

The P(II) protein from Rhodospirillum rubrum was fused with a histidine tag, overexpressed in Escherichia coli, and purified by Ni(2+)-chelating chromatography. The uridylylated form of the P(II) protein could be generated in E. coli. The effects on the regulation of glutamine synthetase by P(II), P(II)-UMP, glutamine, and α-ketoglutarate were studied in extracts from R. rubrum grown under different conditions. P(II) and glutamine were shown to stimulate the ATP-dependent inactivation (adenylylation) of glutamine synthetase, which could be totally inhibited by α-ketoglutarate. Deadenylylation (activation) of glutamine synthetase required phosphate, but none of the effectors studied had any major effect, which is different from their role in the E. coli system. In addition, deadenylylation was found to be much slower than adenylylation under the conditions investigated.

深红螺菌（Rhodospirillum rubrum）来源的P(II)蛋白与组氨酸标签（histidine tag）融合后，在大肠杆菌（Escherichia coli）中过表达，并通过Ni²⁺螯合层析（Ni(2+)-chelating chromatography）完成纯化。本研究可在大肠杆菌中制备得到P(II)蛋白的尿苷酰化形式。以不同培养条件下生长的深红螺菌菌体提取物为实验材料，研究了P(II)、尿苷酰化P(II)（P(II)-UMP）、谷氨酰胺及α-酮戊二酸（α-ketoglutarate）对谷氨酰胺合成酶（glutamine synthetase）的调控作用。实验结果显示，P(II)与谷氨酰胺可促进谷氨酰胺合成酶的ATP依赖性腺苷酰化（adenylylation，即酶的失活过程），而该反应可被α-酮戊二酸完全抑制。谷氨酰胺合成酶的去腺苷酰化（deadenylylation，即酶的激活过程）需要磷酸参与，但本研究所考察的各类效应物均未对其产生显著调控效果，这与它们在大肠杆菌系统中的调控作用存在显著差异。此外，在所考察的实验条件下，谷氨酰胺合成酶的去腺苷酰化反应速率远慢于腺苷酰化反应。