NADH-Coupled Steady-State ATPase Assay.

ATPase assays were carried out using time-course fluorescence measurements in a Synergy Neo Microplate reader (340 nm excitation, 440 nm emission) (9). All assays were carried out in Mdn1 assay buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 1 mM MgCl₂, 0.5 mM EGTA, 1 mM DTT) supplemented with 200 μM NADH (Sigma N7410), 1 mM phosphoenolpyruvic acid (Sigma P7127), 30 U/mL D-lactic dehydrogenase, and 30 U/mL pyruvate kinase (Sigma P1506). Unless otherwise stated, 1 mM of Mg-ATP (Sigma A2383) was present. In experiments with Rbin-1, Rbin-XL, or additional MIDAS protein present, 2% DMSO and 2 mM sodium sulfate were added to the assay buffer. Reaction velocities were determined by linear fitting to fluorescence time course data and dividing by the total Mdn1 concentration (25 nM). ATPase rates (V) as a function of ATP concentration (S) were fitted to the Michaelis–Menten equation:

Or the Hill equation:

where h reports the Hill coefficient. For Rbin and WT-MIDAS titrations, ATPase rates (V) were fitted to a dose–response equation:

Where A reports the rate with no Rbin present and b reports the maximal inhibition (11). Fits were weighted by the inverse SEM. All data analysis and fitting were performed in MATLAB.