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NSF ATPase activity was quantified using the Malachite Green Assay by measuring the release of inorganic phosphate (Pi) due to the ATP hydrolysis with spectrophotometer. The assay was adapted from the method of Lanzetta et al. [50 (link)]. The Malachite Green Stock solution used for the assay was a mixture of two different solutions (one with 34 mg Malachite Green oxalate salt (Sigma-Aldrich. St. Louis, MO, USA) into 40 ml HCl 1 M and the other with 1 g (NH₄)₂MoO₄ (Sigma-Aldrich, St. Louis, MO, USA) into 14 ml HCl 4 M to a final volume of 100 ml with distilled water and then filtered through 0,45 nm. The concentration of human NSF used for the ATPase assay was 216 nM (36 nM hexameric concentration) with different ATP concentration. Reaction was performed at 37 °C and followed for 120 min. The time point aliquots collected (20 μl) were mixed with 150 μl of Malachite Green stock solution until the solution became homogenous and the absorbance measured at 640 nm using a corresponding Malachite Green solution as blank. The values of absorbance were then converted into μmol of free Pi in solution using a standard curve. To reported values for the kinetic constants (K_m, k_cat and V_max) were obtained by data fitting with the Michaelis-Menten kinetic model (Y = Vmax*S/(Km + [S])).