Figure 2.

Transfection of R72 confers NAALADase activity and immunoreactivity. (Upper) Samples of R72-transfected and pcDNA3CAT-transfected (control) cell lysates were assayed for NAALADase activity; activity is expressed as total [³H]Glu liberated from N-Ac-Asp-[³H]Glu ([³H]NAAG) after subtraction of protein-free blank. SEM is contained within the outlines of the bars in the graph. NAALADase inhibitors quisqualate (1, 20) and 2-(phosphonomethyl)glutaric acid (PMG) (16) were included at concentrations of 10 and 100 times their reported Kis. Expressed in terms of percent inhibition, the data translate as follows: 20 μM quisqualate, 91.5%; 200 μM quisqualate, 98.5%; 2.75 nM PMG, 80%; 27.5 nM PMG, 97.4%. Control cell lysates demonstrated no NAAG hydrolytic activity. (Lower) Twenty micrograms of total protein from control or R72-transfected cells was immunoblotted with anti-NAALADase antiserum. A specific immunoreactive band with an apparent molecular mass of approximately 100 kDa is observed.