A bioluminescence method for the measurement of L-glutamate: applications to the study of changes in the release of L-glutamate from lateral geniculate nucleus and superior colliculus after visual cortex ablation in rats.
We have developed a rapid, simple, specific, and very sensitive bioluminescence method for the measurement of L-glutamate (L-Glu). Oxidation of L-Glu by glutamate dehydrogenase has been coupled with bacterial FMN reductase and luciferase. Light production (i.e., peak height or integral) was linear from less than 0.5 to 500 pmol of L-Glu. Potential interfering substances that may be encountered in brain tissue have been identified. The most potent inhibitors were ascorbate and the biogenic amines. Procedures that conferred long-term stability of the reagent mixture (greater than 8 h) were established. Bioluminescence analysis of L-Glu content in brain tissue extracts, fractions from release experiments, and human CSF corroborated respective results obtained by HPLC analysis. In this study, we have applied the method to monitor changes in the KCl-evoked release of endogenous L-Glu from milligram amounts of brain tissue, i.e., from lateral geniculate nucleus and superior colliculus after visual cortex ablation.
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D038181:FMN Reductase; D005829:Geniculate Bodies / Q000378:metabolism*; D005969:Glutamate Dehydrogenase; D005971:Glutamates / Q000032:analysis* / Q000378:metabolism; D018698:Glutamic Acid; D007202:Indicators and Reagents; D007700:Kinetics; D008156:Luciferases; D008163:Luminescent Measurements; D008297:Male; D009247:NADH, NADPH Oxidoreductases; D010783:Photometry; D051381:Rats; D011919:Rats, Inbred Strains; D013477:Superior Colliculi / Q000378:metabolism*; D014793:Visual Cortex / Q000502:physiology*
D005971:Glutamates; D007202:Indicators and Reagents; D018698:Glutamic Acid; D008156:Luciferases; D005969:Glutamate Dehydrogenase; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases