Ns. Protein concentrations have been determined as described by Bradford (53) or by applying a

Ns. Protein concentrations have been determined as described by Bradford (53) or by applying a NanoDrop 2000 ULK supplier spectrophotometer (Fisher Scientific, Schwerte, Germany) and the calculated extinction coefficient of (51.590 mM 1 cm 1) at 280 nm. IMAC. Immobilized metal-chelate affinity chromatography (IMAC) was performed as follows. To acquire purified histidine-tagged fusion proteins, His Spin Trap affinity columns (GE Healthcare, Uppsala, Sweden) were applied in line with the manufacturer’s directions. Ni-nitrilotriacetic acid (NTA) columns had been equilibrated with 20 mM sodium phosphate buffer (pH 7.four), containing 20 mM imidazole and 500 mM sodium chloride. Exactly the same buffer containing 40 mM imidazole was utilised for the washing step, even though the elution buffer contained 500 mM imidazole. Analytical SEC. The molecular mass of ActTBEA6 was determined by analytical size exclusion chromatography (SEC) applying a Superdex 200 HR column. The column was equilibrated with 50 mM sodium phosphate buffer (pH 7.5), containing 150 mM sodium chloride. NLRP1 Biological Activity Calibration was performed applying chymotrypsinogen A (25 kDa), ovalbumin (44 kDa), conalbumin (75 kDa), aldolase (158 kDa), ferritin (440 kDa), and blue dextran 2000 (GE Healthcare, Uppsala, Sweden) in accordance with the manufacturer’s guidelines. For each and every determination, 300 g of purified heterologous ActTBEA6 was applied towards the column. The column was operated at a flow rate of 0.750 ml/min. Enzyme assays. (i) Initial identification of an acceptable CoA donor for ActTBEA6. The heterologously expressed ActTBEA6 was assayed by incubating 20 g/ml purified enzyme in 50 mM sodium phosphate buffer (pH 7.four) for 1 h at 30 in the presence of 5 mM 3SP and five mM acetylCoA, propionyl-CoA, butyryl-CoA, crotonyl-CoA, or succinyl-CoA, respectively. The reaction was stopped by addition of 50 l (ten [wt/vol]) trifluoroacetic acid (TFA). The samples have been analyzed by high-pressure liquid chromatography (HPLC)-electrospray ionization (ESI) MS for the formation of 3SP-CoA. Samples with heat-inactivated protein (15 min at 95 ) and soluble protein fractions from cells harboring only the expression vector devoid of actTBEA6 (vector manage) served as a handle, or one of the substrates was omitted at a time. (ii) Determination of kinetic parameters. 3SP-CoA formation by ActTBEA6 was measured by applying A. mimigardefordensis strain DPN7T 3SP-CoA desulfinase (AcdDPN7) as a coupling enzyme in an aerobic continuous spectrophotometric assay (51). Kinetic parameters have been determined in cuvettes using a total volume of 1 ml containing 50 mM TrisHCl (pH 7.6), 150 mM NaCl, 0.2 mM five,5=-dithiobis(2-nitrobenzoic acid) (DTNB), and purified AcdDPN7 as an auxiliary enzyme. Different amounts of AcdDPN7 had been tested to make sure that the auxiliary enzyme was not rate limiting. Just after preincubation for 2.five min at 30 , the reaction was began by addition of purified recombinant ActTBEA6 (0.five g). The enhance in absorption was measured at 412 nm ( 14.150 mM 1 cm 1) and corrected for the observed boost in absorbance determined by the nonenzymatic decomposition of succinyl-CoA at pH 7.six. Activity was measured for 10 distinctive concentrations of succinyl-CoA ranging from 0.0 mM to 1.0 mM (with a constant concentration of 10 mM 3SP) or for 12 concentrations of 3SP ranging from 0 to 100 mM (using a continual concentration of 0.2 mM succinyl-CoA). All measurements were carried out in triplicate at 30 . The apparent Vmax and Km have been determined by fitting the obtained data to the Michaelis-Men.