Stimulation of AtAPY1 activity by CaM.
(A) Similar amounts (~ 150 ng) of the specified proteins were loaded per lane of a 12% SDS gel, separated by PAGE and subsequently either silver-stained or blotted onto a membrane for a CaM overlay in the presence of either calcium or EGTA. For each of the three applications, all samples were processed in parallel and identical volumes of the same protein sample were loaded. Biotinylated CaM bound by the proteins was detected via chemiluminescence after incubation with alkaline phosphatase coupled to streptavidin. The exposure time for the detection of CaM binding on the Ca2+-treated membrane was 1 s versus 60 s for the EGTA-treated membrane based on normalization via a positive control shown in S10 Fig. The results are representative of three independent CaM overlays. (B) The specific activities were determined by the discontinuous assay in the presence of 1 mM CaCl2 and 3 mM ADP (LpNTPDase1) or 3 mM UDP (AtAPY1-GFP, AtAPY1, AtAPY1-δTM) and compared with the activities under the same conditions, except for the addition of 0.8 μM CaM (LpNTPDase1) or 1.0 μM CaM (AtAPY1-GFP, AtAPY1, AtAPY1-δTM). Error bars represent the standard deviations of the means in fold activity change by the presence of CaM from two independent activity assays. (C) The specific activity of AtAPY1-δTM was determined by the discontinuous assay in the presence of 3 mM UDP, 1 mM CaCl2 and rising concentrations of CaM as indicated. The data were fit to the Hill equation. The means + SD of duplicates from one assay are shown. The result is representative of two independent assays. 1 U = 1 μmol Pi /min.