Figure 1. Calibration for glutamate release. (A) Fluorescence intensity (FI) response at 460 nm (excitation at 340 nm) versus time with the sudden addition of glutamate (1-7 nmol) to the standard medium at 40 s, using an enzyme-coupled assay system using 50 U/ml glutamate dehydrogenase. FI increases because of the production of fluorescent product nicotinamide adenine dinucleotide phosphate (NADPH) from NADP sup + (1 mM) in the medium. According to standard enzyme kinetics, when K^mof the enzyme for the substrate (where K^m= the substrate concentration at which 50% of the maximum velocity is obtained for a constant enzyme concentration) exceeds the substrate concentration by > 20-fold, the initial rate of enzyme reaction will be proportional to the substrate concentration. (B) The initial slope of FI increase at 460 nm for the addition of various amounts of glutamate. Circles = the slopes determined from A; squares = an additional experiment. The response is linear over the range studied.