反应详情

Initial rates were determined in the absence and presence of various amounts of H3-amidine (0, 5, 10 mM), using BAEE as the substrate. BAEE and H3-amidine were pre-incubated in assay buffer containing 100 mM HEPES, pH 7.6, 50 mM NaCl, 0.5 mM TCEP, and 10 mM CaCl2 for 10 min at 37° C. Reactions were initiated by the addition of PAD4 to a final concentration of 0.2 μM. After incubation at 37° C. for 15 min, the reactions were quenched and the amount of Cit produced quantified. The initial rates derived from these experiments were fit by non-linear least fit squares to equations representing linear competitive inhibition (Equation 4) and linear noncompetitive inhibition (Equation 5), using the using the GraFit™ version 5.0.11 software package. v=Vm[S]/(Km(1+[I]/Kis)+[S])  (Eq. 4) v=Vm[S]I(Km(1+[I]/Kis)+[S](1+[I]/Kii))  (Eq. 5)

1. additionReactions were initiated by the addition of PAD4 to a final concentration of 0.2 μM
2. customthe reactions were quenched