Measurement of glutamine synthetase activity in rat muscle by a colorimetric assay.
Glutamine synthetase catalyses the formation of L-Gln from L-Glu and NH4+. This enzyme also exerts a glutamyl-transferase activity that produces gamma-glutamyl-hydroxamate from Gln and hydroxylamine. This gamma-glutamyl-transfer reaction can be used to determine glutamine synthetase activity by colorimetric assay. This method has never been applied to rat muscle. The aim of this work was to study and optimize the glutamine synthetase assay conditions in rat muscle. Enzyme activity was linear with time of incubation (30 min at 37 degrees C) and linear with enzyme concentration in the incubation medium. The method was specific. In addition, this assay correlated well with a radiometric assay (y = 0.76x + 340, where x and y are the glutamine synthetase activities measured by radiometry and colorimetry respectively; r = 0.94; P = 0.05). Finally, no glutamine synthetase activity was found in muscles of rats treated with methionine sulfoximine, an inhibitor of glutamine synthetase, and activity dramatically rose in muscles from rats treated with dexamethasone, an activator of glutamine synthetase (in extensor digitorum longus: 2717 +/- 54 nmol/min/g protein in dexamethasone-treated rats versus 1228 +/- 114 nmol/min/g protein in control rats, P < 0.0001). In conclusion, the method presented here is accurate and reliable for measurement of glutamine synthetase activity in muscles.
Minet
R
R
Department of Biochemistry, Molecular Biology and Nutrition, Pharmacy School, Clermont-Ferrand, France.
Villie
F
F
Marcollet
M
M
Meynial-Denis
D
D
Cynober
L
L
D000818:Animals; D003124:Colorimetry / Q000379:methods*; D003907:Dexamethasone / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005974:Glutamate-Ammonia Ligase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008297:Male; D008717:Methionine Sulfoximine / Q000494:pharmacology; D018482:Muscle, Skeletal / Q000187:drug effects / Q000201:enzymology*; D011874:Radiometry; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results
D004791:Enzyme Inhibitors; D008717:Methionine Sulfoximine; D003907:Dexamethasone; D005974:Glutamate-Ammonia Ligase