Figure 5.

BzATP is hydrolyzed in vitro by E-NTPDase and E-5NTase. A, Incubation of ATP, BzATP, α,β-MeATP, or α,β-Me-ADP with the enzymes in vitro resulted in accumulation of P_i. Note that an identical amount of P_i was released from ATP and BzATP. Without enzymes (E), only negligible amounts of P_i were detected. Incubation of α,β-MeATP yielded only a small amount of P_i. α,β-MeADP was resistant to hydrolysis. B, Time course of P_i release from ATP (▪) and BzATP (□) during the incubation of these adenine nucleotides with ecto-nucleotidases. The reactions were started in parallel and stopped successively after 1, 3, and 6 min. Enzymatic activity and Ca²⁺ concentration were strongly decreased to reduce the reaction rate. After 1 min, only a small portion of substrates has been hydrolyzed, and >6 min would be required under those conditions to achieve equilibrium. Note that the initial accumulation of P_i, being proportional to the initial reaction velocity, is similar for BzATP and ATP. Absorption values were normalized in each run on the absorption value of the ATP vial that was incubated for 6 min (norm.Δabs.).