A re-examination of the interaction of N-acetyl-L-aspartyl-L-glutamate with a subpopulation of rat brain membrane L-[3H]glutamate binding sites.
The neuropeptide, N-acetyl-L-aspartyl-L-glutamate (NAAG) has been reported to act at a subpopulation of putative quisqualate receptors on the basis of its competitive inhibition of specific binding of L-[3H]glutamate and on the basis of quisqualate-sensitive binding of [3H]NAAG radiolabeled on the glutamate moiety. Recently, a membrane-bound metallopeptidase, N-acetylated alpha-linked acidic dipeptidase (NAALADase), which cleaves NAAG to N-acetyl-aspartate (NAA) and glutamate, has been characterized and has been shown to exhibit optimal activity under incubation conditions used to measure NAAG binding sites. Accordingly, we have examined whether NAALADase mediated release of glutamate from NAAG might account for the receptor binding results. Insertion of empirically derived kinetic constants for NAALADase hydrolysis of NAAG into a theoretical model for peptide-derived glutamate inhibition of [3H]glutamate binding reveals that NAAG can appear to displace, with high affinity, a subpopulation of [3H]glutamate labeled sites, and yet have little or no intrinsic activity for these sites. Furthermore, empirical data relating time and protein concentration to NAAG displacement of [3H]glutamate binding are more consistent with a proteolytic mechanism rather than an equilibrium interaction of the peptide with membrane receptors. Coupled with recent findings attributing the Cl- -dependent glutamate binding to a sequestration phenomenon, these results demonstrate that the inferred action of NAAG at glutamate synaptic receptors through previous radioligand binding studies is probably incorrect. Furthermore, these studies offer a general caution regarding the conclusions about subpopulations of receptors drawn from receptor binding assays conducted with ligands, which may be structurally modified by enzymatic processes.
Blakely
R D
RD
Department of Psychiatry, Johns Hopkins School of Medicine, Baltimore, MD 21205.
Robinson
M B
MB
Guarda
A S
AS
Coyle
J T
JT
D000818:Animals; D004151:Dipeptides / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter / Q000187:drug effects* / Q000378:metabolism
D004151:Dipeptides; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter; C027172:isospaglumic acid