Use of an ABP to identify a DPAP1-selective substrate in parasite lysates.

Abstract

<p><b>A.</b> Structure and reaction mechanism of the (Pro-Arg)₂-Rho substrate. <b>B.</b> Measurement of (Pro-Arg)₂-Rho apparent <i>K</i>_m in trophozoite lysates (circles) and with recombinant DPAP1 (triangle). Turnover rates at increasing concentrations of substrate were fitted to a Michaelis-Menten equation as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011985#s4" target="_blank">methods</a> section. <b>C.</b> Labeling of DPAP1 activity in parasite lysates with FY01. Trophozoite lysates were incubated for 1 h with increasing concentrations of FY01. Labeling was stopped by boiling the sample in SDS-PAGE loading buffer. DPAP1 activity was measured using a flatbed fluorescent scanner. <b>D.</b> DPAP1 labeling correlates with substrate turnover inhibition. An aliquot of the samples treated for 1 h with FY01 was diluted in assay buffer containing 10 µM of (Pro-Arg)₂-Rho, and the initial turnover rate was measured in a 96-well plate (circles). This turnover rate is plotted with the labeling quantified in C.</p