IMPDH activity assay

NADH production was measured in real time in a 100-µl cuvette (1-cm path length) in a Cary Eclipse fluorometer (excitation: 340 nm; emission: 440 nm) equipped with a temperature-controlled multicuvette holder maintained at 37°C. Fluorescence was converted to µmoles NADH produced by comparison with an NADH standard curve. In the standard reaction, 50 mM Tris pH, 7.4, 100 mM KCl, 1 mM DTT, 5 mM NAD⁺, and 3 mM IMP. Reactions were initiated by the addition of 13.8 µg of IMPDH2 per 100-µl reaction. Preliminary studies showed these substrate concentrations to be saturating. IMPDH concentration and buffer conditions were chosen to precisely mimic those used for negative-stain electron microscopy analysis below. In reactions containing ATP, IMPDH2 was preincubated for 10 min at room temperature with the indicated concentration of ATP prior to reaction initiation with substrate. In substrate titration experiments, the constant substrate was included in the preincubation and the reaction was initiated by the addition of the variable substrate. In GTP-containing reactions, IMPDH2 was first preincubated with 1 mM ATP and 3 mM IMP to induce assembly, and then GTP was added to the indicated concentration for an additional 10 min before the reaction was initiated by the addition of 5 mM NAD⁺. Specific activity was calculated by linear interpolation of the reaction slope within the initial 3 min (approximately nine measurements per minute).