Purification of P(II) and P(II)-UMP and in vitro studies of regulation of glutamine synthetase in Rhodospirillum rubrum

Abstract

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.

FIG. 1

SDS-PAGE analysis of R. rubrum P_II purified from E. coli. Lanes: A1, E. coli extract with induced P_II; A2, purified histidine-tagged P_II; A3, the same as A2, but with P_II after thrombin digestion; B1, histidine-tagged P_II-UMP; B2, mixture of histidine-tagged P_II and P_II-UMP; B3, histidine-tagged P_II. P_II-UMP was purified from E. coli treated with glutamine, methionine sulfoximine, and UTP, as described in the text. M, molecular weight markers. His-tag, histidine-tagged.

FIG. 2

Adenylylation of glutamine synthetase in extracts. Extracts were supplemented with 2 mM ATP, 6 mM MgCl₂, and other effectors (final concentrations as follows). ⊞, no addition; ▵, 5 mM α-ketoglutarate; ■, 5 mM α-ketoglutarate and 0.6 μM P_II; ◊, 0.6 μM P_II; , 10 mM glutamine and incubated at 30°C. Extracts are from an N-starved culture (A), an N₂-grown culture (B), and a switch-off culture (C). The experiments were carried out several times. The data are from one representative experiment with duplicate samples. Glutamine synthetase activity is given in micromoles of γ-glutamyl hydroxamate/minute · milligrams of protein.

FIG. 3

The two forms of glutamine synthetase in extracts, shown by SDS–10% PAGE and Western blotting. (A) N-starved cells; (B) extract from N₂-grown cells; (C) extract from switch-off cells. Lanes: 1, no addition; 2, P_i added and extract incubated at 30°C for 1 h; 3, glutamine, MgCl₂, and ATP incubated at 30°C for 1 h. GS, unmodified glutamine synthetase; GS-AMP, adenylylated glutamine synthetase.

FIG. 4

The effect of α-ketoglutarate on glutamine synthetase activity. The extract from a N₂-grown culture was supplemented with 2 mM ATP, 6 mM MgCl₂, and α-ketoglutarate (final concentrations as follows). ⊞, no addition; , 0.2 mM; □, 0.5 mM; ▵, 1 mM; , 2.5 mM; ▾, 5 mM. Extracts were incubated at 30°C. The experiments were carried out several times. The data are from one representative experiment with duplicate samples. Glutamine synthetase activity is given in micromoles of γ-glutamyl hydroxamate/minute · milligrams of protein.

FIG. 5

The effects of P_II and glutamine on glutamine synthetase activity. The extracts were supplemented by 2 mM ATP and 6 mM MgCl₂. (A) Extract from N₂-grown cells with 1 mM α-ketoglutarate and P_II (final concentrations as follows). ⊞, no addition; , 0.09 μM; ■, 0.17 μM; ▴, 0.43 μM; , 0.85 μM; □, 1.31 μM; ▵, 1.97 μM; ◊, 2.56 μM. The boxed insert shows glutamine synthetase activity after 10 min at different P_II concentrations. (B) Extract from N-starved cells with 2 mM α-ketoglutarate and glutamine (final concentrations as follows). ⊞, no addition; , 1 mM; ■, 5 mM; ▴, 10 mM; , 20 mM. The boxed insert shows glutamine synthetase activity after 10 min at different glutamine concentrations. Experiments were carried out several times. The data are from one representative experiment with duplicate samples. Glutamine synthetase activity is given in micromoles of γ-glutamyl hydroxamate/minute · milligrams of protein.

FIG. 6

Activation of glutamine synthetase in extracts. (A) Extract from switch-off culture supplemented by 10 mM P_i and effectors (final concentrations as follows). ⊞, no addition; ▵, 10 mM α-ketoglutarate; ■, 10 mM α-ketoglutarate and 0.85 μM P_II-UMP; , 0.85 μM P_II-UMP; ◊, 10 mM glutamine. (B) Extract from an N₂-grown culture supplemented by 2 mM ATP and 6 mM MgCl₂. After 20 min, the extract was divided into tubes containing 10 mM P_i and other effectors as in (A). The experiments were carried out several times. The data are from one representative experiment with duplicate samples. Glutamine synthetase activity is given in micromoles of γ-glutamyl hydroxamate/minute · milligrams of protein.