Glutamate dehydrogenase

Terephthalic acid (99%), trimesic acid (99%), cystamine hydrochloride, and 2,3,4,5,6-pentafluorobenzyl bromide (99%) were purchased from J&K Scientific Ltd. (Beijing, China). Chromium nitrate, copper(II) acetate, and L-glutamic acid were bought from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Carbonic anhydrase (CA, bovine red blood cell), formate dehydrogenase (FateDH, lyophilized), and glutamate dehydrogenase (GDH, bovine liver) were provided by Sigma-Aldrich (St. Louis, MO, USA). Nicotinamide adenine quinone dinucleotide (NADH, 98%) was obtained from Aladdin Biotechnology Co., Ltd. (Shanghai, China). CO₂ (>99%) and ¹³CO₂ (>99%) were purchased from Beijing Ruyuan Ruquan Technology Co., Ltd. (Beijing, China). All other chemicals were obtained from Beijing Chemical Factory (Beijing, China). Double-distilled water was used in all experiments.

Glutamate release from synaptosomes was detected through a glutamate dehydrogenase reaction, as described previously [48 (link),49 (link)]. Briefly, synaptosomes (0.5 mg/mL of final protein concentration) were incubated in HEPES-buffered medium containing glutamate dehydrogenase (20 U/mL) (Sigma, St. Louis, MO, USA), β-nicotinamide adenine dinucleotide (NAD⁺, 1 mM), and CaCl₂ (1.2 mM), at 37 °C for 5 min. In the presence of glutamate, glutamate dehydrogenase reduced NAD⁺ to NADH, a product that fluoresces (excitation and emission wavelengths of 340 and 460 nm, respectively). Fluorescence intensity of NADH was measured in a stirred thermostated cuvette (37 °C) using a PerkinElmer LS55 spectrofluorimeter. Endogenous glutamate released from the synaptosomes to the incubation medium was detected as an increase in NADH fluorescence. Released glutamate was calibrated by a standard of exogenous glutamate (5 nmol) and expressed as nanomoles of glutamate per milligram of synaptosomal protein (nmol/mg protein). Values quoted in the text and depicted in bar graphs represent the levels of glutamate that were cumulatively released after 5 min of depolarization, and are expressed as nmol/mg protein/5 min.

Determination of metabolites in the extracellular medium was performed as in Díaz-Troya et al. (2020) (link) with minor modifications. A 6 ml aliquot of culture samples was centrifuged (15 000 g for 10 min at 4 °C) and supernatants were quick-frozen in liquid nitrogen, lyophilized (VirTis BenchTop Pro Freeze dryer, SP Scientific), and stored at –20 °C until analyzed. Samples were resuspended in 600 µl of H₂O, and 10–100 µl aliquots were analyzed by enzyme-coupled assays in 375 mM Tris–HCl (pH 7.5) with 0.11 mM NADH (and 50 mM NH₄Cl for 2-oxoglutarate quantification). The reaction was triggered by the addition of 5 mU of lactate dehydrogenase (Sigma) or 12 mU of glutamate dehydrogenase (Sigma) for the determination of pyruvate or 2-oxoglutarate, respectively. When NADH oxidation was completed, the remaining NADH was measured spectrophotometrically at 340 nm to calculate the concentration of the metabolite compared with standard curves of known amounts of pyruvate (Sigma) and 2-oxoglutarate (Sigma).

Lysogeny broth (LB) medium, isopropyl-β-d-thiogalactopyranoside
(IPTG), and NADPH were purchased from
Research Products International. The protease inhibitor cocktail,
lysozyme, DNase I, glutamate dehydrogenase, acetaldehyde dehydrogenase,
acetaldehyde, kanamycin, imidazole, Tris, HEPES, PLP, and l-glutamate were obtained from Sigma-Aldrich. Ammonium bicarbonate,
potassium phosphate, 2-mercaptoethanol, KCl, and MgCl₂ were
also acquired from Sigma-Aldrich. DNase I was purchased from Roche.
Vivaspin 20 spin filters and HisTrap and HiTrap Q columns were obtained
from Cytiva. The 10 kDa Nanosep spin filters were purchased from Pall
Corporation (Port Washington, NY). Deuterium oxide was acquired from
Cambridge Isotope Laboratories Inc., and oxygen-18 labeled water (98%)
was obtained from Medical Isotopes Inc.

Glutamate release was indirectly quantified by a fluorescent method^{44 (link)}. Fluorescence quantification was measured using a fluorimeter (Synergy TM2, Biotek^®). Excitation and emission wavelengths were recorded at 360 nm and 450 nm, respectively. Isolated nerve terminals were incubated with CaCl2 (1 mM), and NADP+ (1 mM) in KRH medium (2 min). Glutamate dehydrogenase (Sigma Aldrich^®; 50 units per well) was added to each well, and readings were performed until the fluorescence reached balance (5 min). KCL 33 mM was used as a depolarizing stimulus (10 min). Finally, calibration curves were obtained after standard amount of glutamate (Sigma Aldrich^®; 5 nM/μL) was added to the medium (5 min). Glutamate levels were normalized to the total amount of synaptosomal protein that was obtained through Bradford assay.