Pyruvate Kinase

Based on the metabolomics results, three key enzymes involved in essential pathways, including xanthine oxidase (XOD), pyruvate kinase (PK), and glucose 6-phosphate dehydrogenase (G6PDH), were examined for changes of their activities under desiccation stress. In brief, Salmonella cell suspensions at different sampling points were diluted with sodium phosphate buffer (0.5 M, pH 7.0) to obtain a final concentration of 10⁷ CFU/mL. The extraction solution was added according to the manufacturer’s instructions (Comin Biotechnology Co. Ltd., Suzhou, China). The mixture was ultrasonically broken with an ultrasonic ice bath (200 W, ultrasonic time 3 s at intervals of 10 s, and 30 times of ultrasound treatment). The supernatant was obtained by centrifugation at 8,000 × g and 4°C for 10 min and then incubated at room temperature for 30 min after adding the reaction reagent. UV spectrophotometric (UV–VIS, Thermo Fisher Scientific, United States) analysis was used to measure the concentration change of the reaction products catalyzed by specific enzymes. The characteristic absorption wavelength was 290 nm for XOD, and 340 nm for PK and G6PDH, respectively. The enzyme activity was determined in units of nmol/min/10⁴ cells.
In addition, the effect of continuous stress on the intracellular ATP content was also determined to further verify the regulation of metabolic pathways. Salmonella cell suspensions at different sampling points were centrifuged at 5,000 rpm for 5 min at 4°C, and then the supernatant was removed. Five milliliter of phosphate buffer saline solution (PBS, pH7.2) were added for resuspension. Cell suspensions were broken with ultrasound in an ice bath for 5 min. The supernatants were collected after centrifugation at 12,000 rpm for 20 min at 4°C. The ATP levels were assayed using an ATP assay kit (Jiancheng Bioengineering Institute, Nanjing, China). The results were analyzed by UV–VIS with an absorption wavelength of 636 nm. All assays were performed in biological triplicates.
The results were expressed as mean ± standard deviation. ANOVA was performed in SPSS 22.0 to determine significant differences between groups (p < 0.05).

Actin-activated ATPase rates were measured across a range of Blebbistatin concentrations using the NADH-coupled assay in a 96-well plate (De La Cruz and Ostap, 2009 (link)) with a 0.1 μM myosin S1 concentration and 10 μM actin concentration. Before the experiment, actin was polymerized by dialysis in ATPase buffer containing 20 mM Imidazole, 10 mM KCl, 2 mM MgCl₂, and 1 mM DTT followed by 1.1 x molar ratio phalloidin stabilization. Experiments were conducted in ATPase buffer with the addition of the NADH-coupled enzymes (0.5 mM phospho(enol)pyruvate [Sigma, P0564], 0.47 mM NADH [Sigma, N7410], 100 U/mL pyruvate kinase [Sigma, P9136], and 20 U/mL lactate dehydrogenase [Sigma, L1254]). Blebbistatin (Selleckchem, S7099) was dissolved in DMSO. The Blebbistatin concentration was varied using serial dilutions. Before gathering data, 2 mM ATP was added to each well. Experiments were performed at 25 ° C using a BioTek Syngergy H1 microplate reader. Absorbance was monitored at 340 nm and it decreased linearly with time. Rates for each well were determined based on the linear fitting of the absorbance as a function of time. A control well containing actin, no myosin, and 20 μM Blebbistatin was used as a baseline. Finally, a Hill equation was fit to the data to determine an IC50 for each experiment. Each data point consists of five technical replicates.

Enzymatic activity was evaluated using retinal samples from six eyes per animal group. Glutamine synthetase (GS) activity was evaluated using the method employed in a previous study [29 (link)]. Briefly, the reaction mixture was prepared with 50 mM Imidazole-HCl buffer (pH 7.1), 7.6 mM ATP, 1.0 mM phosphoenolpyruvate, 50 mM MgCl₂, 10 mM KCl, 40 mM NH₄Cl₂, 0.35 mM NADH, 0.1 M monosodium glutamate, 25 μg of pyruvate kinase, and 50 μg of lactate dehydrogenase in a volume of 1.0 ml. To eliminate traces of ADP and pyruvate, the reaction mixture was equilibrated at 30°C for 10 minutes. The retinal samples were added to the reaction mixture in a 1 : 1 ratio and the rate of change in NADH absorbance was measured at 340 nm, 37°C using the EnSpire® Multimode Plate Reader for 10 minutes. The specific enzyme activity of GS was normalised to total retinal protein concentration and expressed as µmoles per minute per milligram protein.
The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) activity Assay kit (#ab204732, Abcam, Australia) was used to evaluate GAPDH enzyme activity. In this assay, GAPDH catalyses the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphate glycerate resulting in a stoichiometric NADH generation, which further reacts with the developer to form a coloured product with an absorbance maximum at 450 nm. The retina was homogenised in GAPDH assay buffer and centrifuged at 10,000 × g for 5 minutes at 4°C to remove any cellular debris. The retinal supernatant was added to the reaction mix containing GAPDH assay buffer, GAPDH developer, and GAPDH substrate in a 1 : 1 ratio and the colorimetric change was measured kinetically every 5 minutes, for a total of 20 minutes at 450 nm using a plate reader. A NADH standard curve was generated to calculate the specific GAPDH activity. GAPDH activity was expressed as nmoles per minute per milligram of protein.