Identification of glutamine synthetase as a contryphan-Bt binding protein by His-tag pull-down

2021 ◽  
Vol 28 ◽  
Author(s):  
Penggang Han ◽  
Shangyi Liu ◽  
Xiandong Dai ◽  
Chongxu Fan ◽  
Ying Cao ◽  
...  
Keyword(s):  
Glutamine Synthetase ◽  
Binding Protein ◽  
Synthetase Activity ◽  
Glutamine Synthetase Activity ◽  
Intracellular Protein ◽  
Catalytic Center ◽  
His Tag ◽  
Hexahistidine Tag ◽  
Intracellular Binding ◽  
Conus Betulinus

Background: Contryphan-Bt is a D-tryptophan-containing disulfide-constrained decapeptide recently isolated from the venom of Conus betulinus. The molecular targets of contryphans are controversial, and the identification of its interacting proteins may be of great importance. Methods: His-tag pull-down assays were performed to investigate intracellular binding proteins of contryphan-Bt from rat brain lysate. Bt-Acp-[His]6, a contryphan-Bt derivative containing hexahistidine tag, was synthesized and used as the bait. As a control, Acp-[His]6 was used to exclude nonspecific bindings. Results: Glutamine synthetase was identified as a potential contryphan-Bt binding protein by pull-down assays and subsequent LC-MS/MS. The binding of contryphan-Bt to glutamine synthetase was confirmed and determined using microscale thermophoresis, with a Kd of 74.02 ± 2.8 μM. The binding did not affect glutamine synthetase activity, suggesting that the interaction site was distinct from the catalytic center. Conclusions: Glutamine synthetase was identified as a novel contryphan-Bt binding protein. This is the first report in which the conopeptide binds to an intracellular protein.

scholarly journals Identification of glutamine synthetase as a novel contryphan-Bt binding protein by his-tag pull down

2020 ◽  
Author(s):  
Penggang Han ◽  
Ying Cao ◽  
Xiandong Dai ◽  
Shangyi Liu ◽  
Chongxu Fan ◽  
...  
Keyword(s):  
Glutamine Synthetase ◽  
Binding Protein ◽  
Synthetase Activity ◽  
Glutamine Synthetase Activity ◽  
Intracellular Protein ◽  
Catalytic Center ◽  
First Report ◽  
His Tag ◽  
Peptide Toxins ◽  
Conus Betulinus

Abstract(1)BackgroundContryphan-Bt is a D-tryptophan-containing disulfide-constrained decapeptide recently isolated from the venom of Conus betulinus. The molecular targets of contryphans are controversial, and the identification of its interacting proteins may be of great importance.(2)MethodsHis-tag pull down assays were performed to investigate binding proteins of contryphan-Bt from rat brain lysate. Bt-Acp-[His]6, a contryphan-Bt derivative containing hexahistidine tag, was synthesized and used as the bait. As a control, Acp-[His]6 was used to exclude nonspecific bindings.(3)ResultsGlutamine synthetase was identified as a potential contryphan-Bt binding protein by pull down assays and subsequent LC-MS/MS. The binding of contryphan-B to glutamine synthetase was confirmed and determined using microscale thermophoresis, with a Kd of 74.02 ± 2.8 μM. The binding did not affect glutamine synthetase activity, suggesting that the interaction site was distinct from the catalytic center.(4)ConclusionsGlutamine synthetase was identified as a novel contryphan-Bt binding protein. This is the first report that the conopeptide binds to an intracellular protein, therefore offering a new concept and methodology for developing peptide toxins.Key ContributionThis is the first report that the conopeptide binds to an intracellular protein, therefore offering a new concept and methodology for developing peptide toxins.

Respective effects of glucose and glutamine on the glutamine synthetase activity of human skin fibroblasts

1991 ◽  
Vol 102 (2) ◽  
Author(s):  
Th�ophile Soni ◽  
Claire Wolfrom ◽  
Samia Guerroui ◽  
Nicole Raynaud ◽  
Jos�phine Poggi ◽  
...  
Keyword(s):  
Glutamine Synthetase ◽  
Human Skin ◽  
Skin Fibroblasts ◽  
Synthetase Activity ◽  
Glutamine Synthetase Activity ◽  
Human Skin Fibroblasts

Control of nitrogen assimilation in Stichococcus bacillaris by growth conditions

1987 ◽  
Vol 65 (3) ◽  
pp. 432-437 ◽  
Author(s):  
Iftikhar Ahmad ◽  
Johan A. Hellebust
Keyword(s):  
Glutamine Synthetase ◽  
Nitrogen Assimilation ◽  
Continuous Light ◽  
Synthetase Activity ◽  
Glutamine Synthetase Activity ◽  
Dark Cycle ◽  
Continuous Darkness ◽  
Cell Carbon ◽  
Stichococcus Bacillaris ◽  
Mixotrophic Conditions

Stichococcus bacillaris Naeg. (Chlorophyceae) grown on a 12 h light: 12 h dark cycle divides synchronously under photoautotrophic conditions and essentially nonsynchronously under mixotrophic conditions. Photoassimilation of carbon under photoautotrophic conditions was followed by a decline in cell carbon content during the dark period, whereas under mixotrophic conditions cell carbon increased throughout the light–dark cycle. The rates of nitrogen assimilation by cultures grown on either nitrate or ammonium declined sharply during the dark, and these declines were most pronounced under photoautotrophic conditions. Photoautotrophic cells synthesized glutamine synthetase and NADPH – glutamate dehydrogenase (GDH) exclusively in the light, whereas in mixotrophic cells about 20% of the total synthesis of these enzymes during one light–dark cycle occurred in the dark. NADH–GDH was synthesized almost continuously over the entire light–dark cycle. In the dark, both under photoautotrophic and mixotrophic conditions, the alga contained more than 50% of glutamine synthetase in an inactive form, which was reactivated in vitro in the presence of mercaptoethanol and in vivo after returning the cultures to the light. The thermal stability of glutamine synthetase activity was less in light-harvested cells than in dark-harvested cells. The inactivation of glutamine synthetase did not occur in cultures growing either heterotrophically in continuous darkness or photoautotrophically in continuous light. This enzyme appears to be under thiol control only in cells grown under alternating light–dark conditions, irrespective of whether this light regime results in synchronous cell division or not.

scholarly journals Hydrocortisone requirement for the induction of glutamine synthetase in chick-embryo retinas

1968 ◽  
Vol 106 (2) ◽  
pp. 425-430 ◽  
Author(s):  
Liane Reif-Lehrer ◽  
Harold Amos
Keyword(s):  
Chick Embryo ◽  
Glutamine Synthetase ◽  
Messenger Rna ◽  
Active Component ◽  
Calf Serum ◽  
Synthetase Activity ◽  
Glutamine Synthetase Activity

Hydrocortisone has been found to induce glutamine synthetase activity in chick-embryo retinas in culture. Evidence is presented to show that the hydrocortisone is definitely required for transcription; its requirement for translation has not been ruled out. The possible identity of hydrocortisone with an active component of calf-serum diffusate reported earlier is discussed. The data also indicate that the glutamine synthetase messenger RNA is stable for at least several hours.

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