Fig. 4.
Helix-dependent recruitment of Pah1p onto liposomes. (A) SDS-PAGE of the purified Pah1p and Pah1p-AH3A from yeast, stained with Coomassie blue. (B and C) The helix mediates the binding of Pah1p onto PA/Triton X-100 micelles and is required for its PAP activity. The PAP activities of Pah1p or Pah1p-AH3A were measured as a function of the indicated molar (B) or surface (C) concentrations of PA. For the experiment shown in B, the molar ratio of Triton X-100 to PA was maintained at 10∶1 (9.1 mol % PA). For the experiment shown in C, the molar concentration of PA was held constant at 0.2 mM, and the Triton X-100 concentration was varied to obtain the indicated surface concentrations. The kinetic parameters for Pah1p or Pah1p-AH3A for each of the two assays are shown. Kinetic data were analyzed according to the Michaelis–Menten and Hill equations using the enzyme kinetics module of SigmaPlot software. The data shown are means ± SD from triplicate enzyme determinations. (D) Effect of dephosphorylation on the liposome-dependent increase in fluorescence of Pah1p or Pah1p-AH3A. Native and dephosphorylated forms of Pah1p were incubated with increasing concentrations of liposomes that were made of phosphatidylcholine and PA at a molar ratio of 10∶1. Following 10 min incubation, the increase in fluorescence was measured. The data are plotted with respect to total phospholipid concentration in the liposomes and are means ± SD from triplicate determinations. Open symbols: native proteins, closed symbols: dephosphorylated proteins. The dissociation constants (Kd) shown were determined as previously described (32).