110 Chapter 4 Glycolytic enzyme kinetics Glycolytic enzyme-catalyzed reactions were measured by means of photometric assays. Alveolar macrophages were seeded on plates coated with either fibrous or globular collagen, or left uncoated (control) and incubated for 72 hours. Adherent cells were then detached with 1mM EDTA, centrifuged, and washed twice with icecold PBS. Cells were then resuspended in PBS containing protease inhibitors (SigmaAldrich, cOmplete™ Protease Inhibitor Cocktail) and stored at -80 °C. On the day of analysis, Triton X-100 (Sigma-Aldrich) was added to a final concentration of 0.1% and the suspension was centrifuged at 21000 g for 10 min at 4 °C. The cell-free supernatant was collected and used for the analysis. The enzyme activity, expressed in µmol/min/mg protein was determined from the difference in the slope of NAD(P) H absorbance (340 nm) before and after the addition of a substrate. The activities, unless stated otherwise, were measured in buffers with 5 mM KCl, 1 M Tris-HCl, 0.15 M NaCl, 5 mM CaCl2, and 0.1 M MgSO4, pH 7.4, 37 °C. The contents of the reaction mixtures were as follows: the phosphofructokinase (PFK) reaction mixture contained 0.1 M ATP, 15 mM NADH, 620 U/mL glycerol-3-phosphate dehydrogenase (G3PDH), 330 U/mL aldolase, 1800 U/mL triosephosphate isomerase (TPI), and 0.1 M fructose-6-phosphate. The phosphoglycerate kinase mixture contained 0.1 M ATP, 15 mM NADH, 800 U/mL glyceraldehyde-3P-dehydrogenase (GAPDH), and 0.125 M 3-phosphoglyceric acid. The aldolase mixture contained 15 mM NADH, 620 U/mL G3PDH, 1800 U/mL TPI, and 0.02 M of fructose-1,6-bisphosphate (F-1,6BP). The pyruvate kinase mixture contained 0.1 M ADP, 0.02 M of F-1,6-BP, 15 mM NADH, 9300 U/mL lactate dehydrogenase, and 0.02 M phosphoenolpyruvate. Statistical analysis Differentially expressed proteins by cells growing on collagen were investigated by comparing macrophages grown on plastic with macrophages grown on any type of collagen (globular + fibrous) using an unpaired two-sided two-sample Wilcoxon test with a Benjamini-Hochberg correction for multiple testing. To uncover differentially expressed proteins induced by a specific type of collagen, we compared macrophages grown on globular collagen with macrophages grown on fibrous collagen using a paired two-sided two-sample Wilcoxon test with a Benjamini-Hochberg correction for multiple testing. A corrected p-value <0.05 was considered significant. No proteins were found to be significantly differentially expressed when using this correction. For pathway analyses we therefore used the uncorrected p-values and included all proteins that had an uncorrected p-value <0.025 (collagen versus control) or <0.05 (fibrous versus globular) and a fold change <0.875 or >1.25. Analyses were performed using R (version 4.2.0 (2022-04-22)—“Vigorous Calisthenics”).
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