Purification and characterization of a low molecular weight endogenous glutamate binding inhibitor (LGBI) in porcine brain.
One of the endogenous substances which modulate glutamate receptor binding was isolated and highly purified from porcine brain. The purification involved extraction of brain tissue with doubled distilled water, followed by gel filtration, anion exchange, cation exchange, and several steps of C18 reverse-phase high performance liquid chromatography (HPLC). A low molecular weight glutamate binding inhibitor (LGBI) was purified to apparent homogeneity as judged from the elution profile of an HPLC column, in which a symmetrical peak was obtained when the eluate was monitored at 220 nm. The LGBI appears to be a small molecule (< 2 kD) that is heat- and acid/base-stable. The highly purified LGBI has no effect on GABAA and benzodiazepine receptor binding. The LGBI is not L-glutamate, L-aspartate or other negatively charged endogenous substances, since they are clearly separated from the LGBI in anion exchange chromatography. The inhibitory effect of the LGBI on [3H]L-glutamate binding is reversible, and it only changes the Bmax while the Kd remains the same. Since the membrane preparations used for [3H]L-glutamate binding assays for the detection of LGBI activity were enriched with quisqualate (QA)-sensitive subtypes, it was suggested that the LGBI could be a modulator of the QA receptor. Some amino acids which produce significant inhibition of glutamate binding activity were also compared with the LGBI, and they all showed no resemblance to the LGBI. The chemical structure of the LGBI remains to be determined.
Lee
Y H
YH
Department of Physiology and Cell Biology, University of Kansas, Lawrence 66045-2106, USA.
Bhattacharyya
A
A
Tang
X W
XW
Seah
E C
EC
Schmidt
K
K
Deupree
D L
DL
Wu
J Y
JY
D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D001685:Biological Factors / Q000737:chemistry / Q000302:isolation & purification*; D001921:Brain / Q000378:metabolism*; D003545:Cysteine / Q000494:pharmacology; D004355:Drug Stability; D018691:Excitatory Amino Acid Antagonists / Q000302:isolation & purification; D018698:Glutamic Acid / Q000378:metabolism; D005973:Glutamine / Q000494:pharmacology; D008566:Membranes / Q000378:metabolism; D008970:Molecular Weight; D017470:Receptors, Glutamate / Q000187:drug effects / Q000378:metabolism; D013552:Swine
D000596:Amino Acids; D001685:Biological Factors; D018691:Excitatory Amino Acid Antagonists; D017470:Receptors, Glutamate; D005973:Glutamine; D018698:Glutamic Acid; D003545:Cysteine