Adenosine triphosphate (atp)

The phosphorylation reactions were performed at room temperature in buffer containing 50 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM MgCl₂, 20 mM imidazole, 2% glycerol, 0.2 mg/ml BSA, 500 nM Cks1 and 500 µM ATP [(with added [γ-32P]-ATP (Hartmann Analytic)]. PKA (murine cAMP Dependent protein kinase^{53 (link)} and Clb2-Cdk1 were 20 nM and 1 nM, respectively. The concentration of 6xHis-Nth1 was 1 µM. The reactions were stopped by addition of SDS-PAGE sample buffer at indicated time points.

ATPase activity was measured as described by [23 (link)] with 0.5 μM HscA, 10 μM IscU, and HscB at the indicated concentrations or with 0.5 μM Ssq1, 10 μM Isu1, 0.5 μM Mge1, and Hsc20 at the indicated concentrations in buffer A (40 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES–KOH), pH 7.5, 100 mM KCl, 1 mM dithiothreitol, 10 mM MgCl₂, and 10% (v/v) glycerol). Reactions (15 μL) were initiated by the addition of ATP (2 μCi, 2200 Ci/mmol, HARTMANN ANALYTIC GmbH, Braunschweig, Germany) to a final concentration of 120 μM. Incubation was carried out at 25 °C, and the reaction was terminated after 15 min by the addition of 100 μL of 1 M perchloric acid and 1 mM sodium phosphate. After addition of 400 μL of 20 mM ammonium molybdate and 400 μL of isopropyl acetate, samples were mixed and the phases were separated by a short centrifugation. An aliquot of the organic phase (150 μL), containing the radioactive orthophosphate-molybdate complex, was removed and radioactivity was determined by liquid scintillation counting. Control reactions lacking protein were included in all experiments. Values were plotted in GraphPad Prism 7 (version 7.02, GraphPad Software, San Diego, CA, USA) using the Michaelis–Menten hyperbolic equation to fit the data.