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 alpha-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 alpha-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.
Johansson
M
M
Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden.
Nordlund
S
S
D000249:Adenosine Monophosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002474:Cell-Free System; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D051922:PII Nitrogen Regulatory Proteins; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000302:isolation & purification; D012247:Rhodospirillum rubrum / Q000201:enzymology*
D001426:Bacterial Proteins; D007656:Ketoglutaric Acids; D051922:PII Nitrogen Regulatory Proteins; D011994:Recombinant Proteins; D005973:Glutamine; D000249:Adenosine Monophosphate; C404143:PIID regulatory protein, Bacteria; D011494:Protein Kinases; D005974:Glutamate-Ammonia Ligase