Fig. 4: Active transaminase futile cycling in dried and resuspended metabolite extracts.
From: A diverse proteome is present and enzymatically active in metabolite extracts
a Schematic showing deuterium labeling patterns for a transaminase reaction converting glutamate to α-ketoglutarate and back to glutamate. b Mass spectra of glutamate in the murine liver following D5-glutamate addition at resuspension. Representative AMW20 and AMW50 sample spectra are shown. Representative MS1 spectral peaks corresponding to unlabeled glutamate ([M-H] =146.0460 m/z), D4-glutamate ([M-H] = 150.0711 m/z), and D5-glutamate ([M-H] =151.0773 m/z) are shown. c Relative abundance of D5-glutamate-derived metabolites in AMW50 (-), AMW50 +filter ( + F), AMW50 + aminooxyacetic acid ( + AOA) murine liver samples. D5-glutamate was added at resuspension in all samples. AOA was added at resuspension in AMW50 + AOA samples. Significance calculated by One-way Welch’s ANOVA (t-stat, two-sided) with a Dunnett’s T3 multiple comparisons (mean ± SD, n = 3 technical replicates per group). d Schematic showing 13C and 15N labeling patterns for a cyclical transaminase reaction following [U13C,15N]-glutamate input. e MS1 mass spectra of glutamate in the murine liver following [13C5,15N]-glutamate addition at resuspension. AOA was added at resuspension in AMW50 + AOA samples. Representative AMW50 and AMW50 + AOA sample spectra are shown. Peaks corresponding to unlabeled glutamate ([M-H] = 146.0460 m/z), [15N]-glutamate ([M-H] = 147.0430 m/z), [13C5]-glutamate ([M-H] = 151.0627 m/z), and [13C5,15N]-glutamate ([M-H] = 152.0598 m/z) and are shown. (f) Absolute concentrations of [13C5,15N]-glutamate-derived metabolites in AMW50 (-), AMW50 +filter ( + F), AMW50 + AOA ( + AOA) murine liver samples. [13C5,15N]-glutamate was added at resuspension. AOA was added at resuspension in +AOA samples. Significance calculated by One-way Welch’s ANOVA (t-stat, two-sided) with a Dunnett’s T3 multiple comparisons (mean ± SD, n = 3 technical replicates per group). g Quantitative accounting of the distribution of 13C5 (left) or 15N (right) across metabolic neighbors arising from [13C5,15N]-glutamate added at resuspension. Each bar is an individual replicate arranged in groups of technical triplicates AMW50 (-), AMW50 +filter ( + F), AMW50 + AOA ( + AOA) murine liver samples. The measured concentration of each indicated metabolite isotopologue is stacked to demonstrate conserved molar accounting after metabolic interconversions.
