Regulation of phosphate-activated glutaminase (PAG) by glutamate analogues

The ability of structural analogues of glutamate (GLU) to modulate phosphate activated glutaminase (PAG) was assessed in the present series of studies. A number of GLU receptor agonists and antagonists were tested for their ability to inhibit synaptosomal PAG activity. PAG activity was determined by measuring GLU formation from 0.5mM glutamine (GLN) in the presence of 10 mM phosphate. GLU analogues at 5–10 mM were found to significantly inhibit PAG activity. It was determined that PAG inhibition occurred regardless of whether the GLU analogues were receptor agonists or antagonists, however, PAG inhibition was influenced by analogue chain length, isomeric form and substituent substitution. The glutamate uptake blockers, dihydrokainic acid and DL-threo--hydroxyaspartic acid were relatively weak inhibitors of PAG (<25% inhibition) as were the receptor agonists, ibotenic acid and (±)cis-2,3-piperidine-dicarboxylic acid. Other GLU analogues produced inhibition of PAG in the range of 40–70%. PAG inhibition by GLU analogues did not appear to differ substantially among the brain regions evaluated (cortex, striatum and hippocampus). The endogenous amino acids, glycine, taurine and N-acetylaspartic acid, also significantly inhibited PAG activity in the 5–10 mM range. The noncompetitive NMDA antagonists, (+)MK801 and ketamine, at a concentration of 5 mM, significantly stimulated PAG activity 1.5–2 fold over control values. The activation of PAG by (+)MK801 was dose-related, stereoselective and appeared to result from a synergistic interaction with phosphate to enhance substrate (GLN) binding to PAG. The results of these studies suggest that GLU analogues could potentially alter neurotransmitter GLU synthesis if sufficient concentrations of these drugs are used in in vitro or in vivo studies. Furthermore, preliminary evidence suggests that other endogenous amino acids (glycine, taurine, N-acetylaspartic acid) may modulate PAG activity. These studies have further characterized the structural requirements for the allosteric regulation of PAG by glutamate and its analogues.