GCP activity measurements were carried out following published procedures [
3,
25]. Briefly, the reaction mixture contained [
3H]-NAAG (70 nM, 50 Ci/mmol) and reconstituted pellet (human skin, paw pad, or sciatic nerve) in Tris-HCl containing 1 mM CoCl
2 in a total volume of 90 μL. The reaction was carried out at 37°C at different times as indicated, and stopped with ice-cold sodium phosphate buffer (pH 7.4, 0.1 M, 90 μL). When human skin was used as GCP source, the reaction was carried out in the presence and absence of the selective GCP inhibitor 2-PMPA (1 μM). When rat tissue was used from the
ex vivo study, 2-PMPA was administered i.p. and the animals were sacrificed and their paw pads removed for GCP enzymatic determinations. In both cases, blanks were obtained by incubating the reaction mixture without pellet. Duplicate aliquots of 90 μL from each terminated reaction was transferred to a well in a 96-well spin column containing AG1X8 ion- exchange resin; the plate was centrifuged at 1000 rpm for 5 minutes using a Beckman GS-6R centrifuge equipped with a PTS-2000 rotor. [
3H]-NAAG bound to the resin and [
3H]-glutamate eluted in the flow through. Columns were then washed twice with formate (1 M, 90 μL) to ensure complete elution of [
3H]-glutamate. The flow through and the washes were collected in a deep 96-well block; from each well with a total volume of 270 μL, a 200 μL aliquot was transferred to a glass scintillation vial, to which 10 ml of Ultima-Gold (Perkin Elmer) was added. The radioactivity in each vial corresponding to [
3H]-glutamate was determined via a Beckman LS-6000IC scintillation counter. Radioactivity values in dpm were converted to fmoles of glutamate using the relation 1 pCi/2.2 dpm and the specific activity of [
3H]-glutamate (same as that of [
3H]-NAAG: 1 fmole/50 pCi). As a result, if 16711 dpm [
3H]-glutamate were measured after incubating 10 mg tissue for 1 h, the normalized activity would be: 16711 dpm × (1 pCi/2.2 dpm) × (1 fmole/50 pCi)/10 mg tissue = 15 fmole/h/mg tissue.