TY - JOUR
T1 - Quantitation of N-acetyl-aspartyl-glutamate in microdissected rat brain nuclei and peripheral tissues
T2 - findings with a novel liquid phase radioimmunoassay
AU - Guarda, Angela S.
AU - Robinson, Michael B.
AU - Ory-Lavollée, Laure
AU - Forloni, Gian Luigi
AU - Blakely, Randy D.
AU - Coyle, Joseph T.
PY - 1988/6
Y1 - 1988/6
N2 - Antibodies were raised in rabbits against the neuropeptide N-acetyl-l-aspartyl-l-glutamate (NAAG) coupled to bovine serum albumin via a carbodiimide linkage. One of these rabbit antisera, which preferentially recognizes coupled NAAG-like immunoreactivity (LIR), has been previously used to immunocytochemically localize NAAG-LIR. We have now employed a second of these antisera, which preferentially recognizes free NAAG, to develop a competitive liquid phase radioimmunoassay (RIA). Using this assay, we were able to detect picomole amounts of NAAG in rat tissue extracts. The specificity of the assay revealed a 60-fold greater affinity of the antibody for NAAG over N-acetyl-aspartate (NAA) and greater than one million-fold specificity for NAAG over both aspartate and glutamate. High-pressure liquid chromatographic (HPLC) separation of tissue extracts yielded only two detectable peaks of NAAG-LIR in collected fractions and these co-chromatographed with NAAG and NAA. NAAG levels determined by this liquid phase RIA and by HPLC were essentially identical after correction for the presence of NAA crossreactivity. The antibody that preferentially recognizes coupled NAAG was used to immunocytochemically localize NAAG-LIR to the red nucleus, the facial nucleus, the dorsal raphe, and the locus coeruleus. To further confirm this localization of NAAG, these and other nuclei were microdissected and levels of NAAG were determined by liquid phase RIA. Nuclei which stained intensely were found to contain high levels of NAAG by RIA and between 60 and 100% of this NAAG-LIR co-chromatographed with NAAG. These results support our previous conclusion that NAAG is co-localized in noradrenergic, serotonergic and cholinergic neurons. We have also used this RIA to identify NAAG-LIR in striated muscle and other peripheral tissues.
AB - Antibodies were raised in rabbits against the neuropeptide N-acetyl-l-aspartyl-l-glutamate (NAAG) coupled to bovine serum albumin via a carbodiimide linkage. One of these rabbit antisera, which preferentially recognizes coupled NAAG-like immunoreactivity (LIR), has been previously used to immunocytochemically localize NAAG-LIR. We have now employed a second of these antisera, which preferentially recognizes free NAAG, to develop a competitive liquid phase radioimmunoassay (RIA). Using this assay, we were able to detect picomole amounts of NAAG in rat tissue extracts. The specificity of the assay revealed a 60-fold greater affinity of the antibody for NAAG over N-acetyl-aspartate (NAA) and greater than one million-fold specificity for NAAG over both aspartate and glutamate. High-pressure liquid chromatographic (HPLC) separation of tissue extracts yielded only two detectable peaks of NAAG-LIR in collected fractions and these co-chromatographed with NAAG and NAA. NAAG levels determined by this liquid phase RIA and by HPLC were essentially identical after correction for the presence of NAA crossreactivity. The antibody that preferentially recognizes coupled NAAG was used to immunocytochemically localize NAAG-LIR to the red nucleus, the facial nucleus, the dorsal raphe, and the locus coeruleus. To further confirm this localization of NAAG, these and other nuclei were microdissected and levels of NAAG were determined by liquid phase RIA. Nuclei which stained intensely were found to contain high levels of NAAG by RIA and between 60 and 100% of this NAAG-LIR co-chromatographed with NAAG. These results support our previous conclusion that NAAG is co-localized in noradrenergic, serotonergic and cholinergic neurons. We have also used this RIA to identify NAAG-LIR in striated muscle and other peripheral tissues.
KW - Glutamate
KW - Immunocytochemistry
KW - N-Acetyl-aspartate
KW - N-Acetyl-aspartyl-glutamate
KW - Optic tract
KW - Radioimmunoassay
UR - http://www.scopus.com/inward/record.url?scp=0023940297&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0023940297&partnerID=8YFLogxK
U2 - 10.1016/0169-328X(88)90045-9
DO - 10.1016/0169-328X(88)90045-9
M3 - Article
C2 - 3401700
AN - SCOPUS:0023940297
SN - 0169-328X
VL - 3
SP - 223
EP - 231
JO - Molecular Brain Research
JF - Molecular Brain Research
IS - 3
ER -