In erythrocytes gamma glutamyl cysteine ligase represents the most important enzyme involved in GSH synthesis.In fact, a lot of drugs or compounds can modulate its enzyme activity either during oxidative stress or no stress condition.
A simple method to purify and evaluate its activity could be suitable.
I hope that following information will be helpful.
http://gut.bmj.com/content/42/4/485.long
Dear Arbace,
gGLYCys activity determination in red cells is difficult becouse it is very low. So if you must measure it by spectrophotometry it is a problem. In fact Hb which adsorbs at 340 make impossible this determination in this kind of biological matrix becouse you have to use Hb separation procedures and/or enzyme purification.
On my opinion you should follow this procedure but it requires HPLC separation of the products and fluorescent detection. However even if it is time consuming it can be applied in the presence of Hb requiring only the absence of relevant gGT activity (which is exactly the case of red cells by the way). We use this method.
C.C. Yan, R.J. Huxtable, J. Chromatogr, B. Biomed, J. Chromatogr. B Biomed.
Appl. 672 (1995) 217–224.
Briefly, a solution containing 10 mM ATP,
150 mM KCl, 2 mM K3EDTA, 20 mM MgCl2, 10 mM glutamic acid,
2.5 mM cysteine, 0.5 mM DTT in 1 M Tris/Cl, pH 8.1, was let to
incubate at 37 C for 15 min. Thereafter, 10 ll-aliquots of sample
were added to 150 ll of the incubation solution; after 5 min at
37 C, the sample was deproteinized by 1:1 (v/v) addition of
12% (w/v) TCA containing 1 mM K3EDTA. 0.1 ml of the obtained
supernatant was then reacted with 2.5 mM (final concentration)
mBrB in the presence of 2 M Tris (25 ll). After 10 min of incubation
in the dark at room temperature, samples were acidified by
addition of 5 ll of 37% (v/v) HCl and analyzed by HPLC for the
thiol content as above described.
Dear Arbace
we recently determined gGLYCys activity in bovine epithelial mammary cells. Please, find the paper attached. If you need more information please don't esitate to contact me directly [email protected]
Umberto
- Badges
- Science topic
- Similar topics
- Chemistry
- Pharmacology
- Medical Pharmacology
- Clinical Pharmacology