Pyruvate kinase

Steady-state ATP hydrolysis was measured using an NADH-coupled assay (63 (link)). The assay buffer contained 50 mM HEPES (pH 8.0 with KOH), 150 mM KCl, 2 mM MgCl₂, 2 mM DTT, 0.06% (w/v) digitonin, 60 µg/mL pyruvate kinase (Roche), 32 µg/mL lactate dehydrogenase (Roche), 9 mM phosphoenolpyruvate, 150 µM NADH, and 200 nM CFTR. Aliquots of 27 µL were distributed into a Corning 384-well Black/Clear Flat Bottom Polystyrene NBS Microplate. The reactions were initiated by the addition of ATP. The rate of fluorescence depletion was monitored at λ_ex = 340 nm and λ_em = 445 nm at 28 °C with an Infinite M1000 microplate reader (Tecan). ATP turnover was then determined with an NADH standard curve.

DLS experiments were performed at protein concentrations of 1–30 μm (with respect to monomeric units) in buffer A, which was either nucleotide-free or supplemented with 2 mm ADP or 2 mm ATP, respectively. In the case of ATP, 2 mm phosphoenolpyruvate and 0.01 mg/ml pyruvate kinase (Roche Applied Science) were present as an ATP-regenerating system. The measurements were performed with a Viscotek 802DAT DLS instrument (Viscotek, Waghäusel, Germany). 40 scans with a measuring time of 5 s/scan were recorded. Hydrodynamic radii and molecular mass values were extracted from the DLS data using the OmniSIZE version 3.0 software package.

ATPase activity was measured at 37°C coupled to NADH oxidation, which was followed as a decrease in absorption at 340 nm using an ATP regenerative assay similar to Tamura and Gellert, 1990 (link): 0.2 mM NADH Di-Na (Roche, Basel, CH); 2 mM phosphoenol pyruvate K-salt (Bachem, Bubendorf, CH); 2 U/ml pyruvate kinase, Roche; 10 U/ml lactate dehydrogenase, Roche; in 40 mM Hepes, 150 mM KCl, 10 mM MgCl₂, pH 7.5.

Lactate dehydrogenase (rabbit muscle), pyruvate kinase (rabbit muscle), and inorganic pyrophosphatase (Baker's yeast) were purchased from Roche Applied Science. (R, S)-[²H₃]methyl-mevalonolactone, (R, S)-mevalonolactone, acetyl-CoA, glutamate dehydrogenase (bovine liver), acetyl-CoA synthetase (Baker's yeast), myokinase (rabbit muscle) and lysozyme (bovine) were purchased from Sigma. Sodium acetate (¹³C, 99%), sodium acetate (²H, 99%) and D₂O (99%) were purchased from Cambridge Isotope Laboratories, Inc. All other chemical reagents were of the highest grades available. Plasmids pET28efTR (encodes a bi-functional enzyme, Enterococcus faecalis acetoacetyl-CoA thiolase/HMG-CoA reductase), pET28efS2 A100G (encodes Enterococcus faecalis HMG-CoA synthase), and pET28-efR (encodes Enterococcus faecalis HMG-CoA reductase) were generous provided by Prof. V. W. Rodwell [43] (link). Mevalonate kinase (Staphylococcus aureus), phosphomevalonate kinase (Streptococcus. pneumoniae), and diphosphomevalonate decarboxylase (Streptococcus. pneumoniae) were expressed and purified as described previously [46] (link), [49] (link).

The ATPase activities of proteins obtained after various purification procedures were determined by NADH-coupled spectrophotometric assays^{69 (link)}. 10 μg of DM-purified or nanodisc-reconstituted protein was added to 140 μL of reaction buffer containing 50 mM HEPES–KOH pH 8.0, 60 μg/mL pyruvate kinase (Roche), 16 μg/mL lactate dehydrogenase (Roche), 10 mM phosphoenolpyruvate (Roche), 0.3 mM NADH, 3 mM ATP, 10 mM MgCl₂, and 0.17% (w/v) DM in the presence or absence of 250 μM peptide. For proteins reconstituted into nanodiscs, DM was not added to the reaction buffer. For lipid-stimulated ATPase assays, the thin film of lipids was hydrated in 100 μL of buffer containing 50 mM HEPES–KOH pH 8.0 and 10 mM MgCl₂ to yield a 10 mM solution. After sonication of the lipid solution for 10 min to decrease its turbidity, the reaction was initiated by adding varying concentrations of lipids to the mixture. ATP hydrolysis activity was monitored at 37 °C for 3 min by measuring the rate of decline of NADH absorbance at 340 nm. To inhibit ATP hydrolysis, the reaction mixture was incubated with 3 mM AMP-PNP or 8 mM NaF/2 mM BeSO₄ for 5 min at RT. The kinetic parameters of ATP hydrolysis were calculated by fitting the initial rates of ATP hydrolysis to the Michaelis–Menten equation using PRISM 4.0 (GraphPad software).

Dioleoyl phosphatidyl choline was purchased from Avanti Polar Lipids, Alabaster, Alabama. Phosphoenolpyruvate, pyruvate kinase, lactate dehydrogenase, NADH, and ATP (disodium salt, special quality) were supplied from Roche Life Science, and apyrase VI and ouabain from Sigma-Aldrich. Oxonol VI, 5-iodoacetamidofluorescein (5-IAF), RH237, RH421, and P³-1-(2-nitro)phenylethyladenosine-5′-triphosphate (caged ATP) were purchased from Molecular Probes (Eugene, Oregon). Na⁺ and K⁺ salts were used in Suprapur quality (Merck). All other reagents were obtained at the highest quality available.