Sodium Chloride → Citrulline

Beteiligte Stoffe

Reaktionsschritte

IC50's for compounds 2 to 11 were determined analogously to the methods used to determine the IC50 for F3-amidine, 1. Briefly, inhibitors were pre-incubated with PAD4 (0.2 μM) for 15 min at 37° C. in a buffer containing 100 mM HEPES, pH 7.6, 50 mM NaCl, 10 mM CaCl2, and 0.25 mM TCEP. BAEE was then added to a final concentration of 10 mM to initiate the reaction. Reactions were then quenched after a further 15 min by flash freezing. The amount of Cit produced in the reaction was quantified using our assay for Cit production. IC50 values were determined by fitting the concentration-response data to Equation 3, Fractional activity of PAD4=1/(1+([I]/IC50))  (Eq. 3), using the Grafit™ version 5.0.11 software package. The concentration of an inhibitor that corresponds to the midpoint (fractional activity=0.5) is referred to as the IC50. The calcium dependence of the IC50 was determined identically, except that CaCl2 was omitted during the initial pre-incubation step and then added (10 mM final) with BAEE to initiate the reaction.

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