Active Glutamine synthetase (GS)

Glutamine synthetase (GS) is an evolutionarily conserved, ubiquitous enzyme. It catalyzes ATP-dependent glutamate-ammonia condensation to form glutamine, a key step in nitrogen metabolism, ammonia detoxification, and nitrogen storage. With oligomeric structures and species-specific isoforms, it’s vital for animal liver/brain function, plant nitrogen assimilation, and microbial survival in nitrogen-poor environments.GS catalyzes the reaction of L-glutamate, NH₄⁺, and ATP to produce L-glutamine, ADP, and inorganic phosphate (Pi).The activity of recombinant human GS is measured by detecting the concentration of ADP generated in the reaction system using an ADP detection kit.GS perform gradient dilution to obtain concentrations of 100ug/ml, 50ug/ml, 25ug/ml, 12.5ug/ml, 6.25ug/ml, 3.13ug/ml, 1.56ug/ml, 0ug/ml.Prepare a 200 uL enzymatic reaction system according to the following components:20uL 10mM MgCl₂,40uL 20mM L-glutamate,20uL 10mM NH₄Cl, 50uL Enzyme sample. Add ATP to the system at a final concentration of 200uM ( 4uL of 100mM stock solution), mix rapidly, immediately place in a 37°C incubator, and react precisely for 30 minutes. Detect the fluorescence intensity of each well.Take the ADP standard from the ADP detection kit and perform gradient dilution to obtain concentrations of 200uM, 100uM, 50uM, 20uM, 10uM, 0uM. Plot a standard curve with the concentration of ADP standard (mM) as the x-axis and the corresponding detection value (e.g., absorbance or fluorescence intensity) as the y-axis.Using this standard curve, we calculate the corresponding concentration and subsequently determine the specific enzyme activity. The specific activity of recombinant human GS is > 100 pmol/min/ug.