Hsp82 ATPase Kinetics Assay

ATP hydrolysis rates were measured in 100 μL reactions containing 2 μM (monomer) of the indicated Hsp82 proteins and 25 mM HEPES pH 7.4, 7 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 0.6 mM NADH⁺, 10 mM phospho-enol-pyruvate, and 2.5% pyruvate kinase/lactate dehydrogenase (Sigma P0294)^{33 (link)}. Where included, Aha1 was at 4 μM. Reactions were initiated by the addition of ATP to 2 mM. Background ATPase activity was controlled for by measuring ATPase of matched samples containing radicicol (200 μM) and subtracting these values from the non-inhibited rates. Data were collected in a BMG Omega plate reader using BMG software version 5.5.