Temperature-dependent αCaMKII activity and Ca2+ sequestration-induced inactivation of Ca2+/CaM·phospho-Thr286-αCaMKII. A, steady-state activity measured by an NADH-coupled assay as described under “Materials and Methods.” 50 nm αCaMKII was added (indicated by the asterisk) to start the reaction in the presence of 50 μm syntide 2 as peptide substrate at 10 μm [Ca2+] concentration at 21 °C. Temperature-dependent steady-state activity and inactivation induced by the addition of 4.2 mm EGTA at 21 °C (B), 30 °C (C), and 37 °C (D) were measured. The reaction was started by the addition of the enzyme, marked by an asterisk. A double asterisk denotes where 4.2 mm EGTA was added. As a base-line control, NADH fluorescence was monitored prior to starting the reaction by the addition of the enzyme (the section before that marked with an asterisk). In B and C, which had the longest time courses, additionally independent photobleaching controls were also run for the same duration as the experiment (top lines). The fluorescence decay rates due to enzyme activity or inactivation by EGTA were corrected for the appropriate base line.