Journal: Biochemistry

Article Title: Cholesterol exposure at the membrane surface is necessary and sufficient to trigger perfringolysin O binding.

doi: 10.1021/bi9002309

Figure Lengend Snippet: FIGURE 1: Cholesterol dependence of PFO binding to liposomes composed of cholesterol and various phospholipids. Changes in the PFO Trp emission intensity are shown as a function of the cholesterol content of the different liposomes. The net F/F0 was calculated as described in Experimental Procedures. (A) DOPC, (; SOPC, 9; POPC, b. (B) DSPC, 2; DPPC, 9; DMPC, O. The cholesterol dependence curve for POPC (b) is shown for reference. Error bars indicate the standard deviation observed for three independent measurements per data point.

Article Snippet: Cholesterol (5-cholesten-3β-ol) and3-epicholesterol (5-cholesten-3R-ol)wereobtained from Steraloids.

Techniques: Binding Assay, Liposomes, Standard Deviation

Journal: Biochemistry

Article Title: Cholesterol exposure at the membrane surface is necessary and sufficient to trigger perfringolysin O binding.

doi: 10.1021/bi9002309

Figure Lengend Snippet: FIGURE 3: Effect of cholesterol concentration and phospholipid unsaturation on lipid order in liposomes as measured by DPH anisotropy. DPH was incorporated into the PC/cholesterol mem- branes as described in ExperimentalProcedures. DPPC, 2; DSPC, O; POPC, b; DOPC, 9.

Article Snippet: Cholesterol (5-cholesten-3β-ol) and3-epicholesterol (5-cholesten-3R-ol)wereobtained from Steraloids.

Techniques: Concentration Assay, Liposomes

Journal: Biochemistry

Article Title: Cholesterol exposure at the membrane surface is necessary and sufficient to trigger perfringolysin O binding.

doi: 10.1021/bi9002309

Figure Lengend Snippet: FIGURE 2: Cholesterol dependence of PFO oligomerization on POPC- or DOPC-cholesterol liposomes. The amount of cholesterol present in the PC-liposomes is shown below each lane. Lanes A-F show PFO oligomerization with POPC/cholesterol liposomes, while lanes G-L contain DOPC/cholesterol liposomes. The NBD-PFO monomer and oligomer bands were visualized with a BioRad Mole- cular Imager FX as described in the Experimental Procedures.

Article Snippet: Cholesterol (5-cholesten-3β-ol) and3-epicholesterol (5-cholesten-3R-ol)wereobtained from Steraloids.

Techniques: Liposomes

Journal: Biochemistry

Article Title: Cholesterol exposure at the membrane surface is necessary and sufficient to trigger perfringolysin O binding.

doi: 10.1021/bi9002309

Figure Lengend Snippet: FIGURE 4: Detergent insolubility of various PC/cholesterol lipo- somes. Liposomes with trace amounts of [3H]cholesterol and [14C] PC were extracted with TX-100 at 0 C. Radioactivity in the pellet fractions was measured after ultracentrifugation, and the percent of radioactivity in the pellet was plotted as a function of cholesterol concentration. (A) Fraction of [3H]cholesterol found in pellet. (B) Fraction of [14C]PC found in pellet. In both (A) and (B), the PC species are the following: DOPC, O; POPC, b; and DPPC, 9.

Article Snippet: Cholesterol (5-cholesten-3β-ol) and3-epicholesterol (5-cholesten-3R-ol)wereobtained from Steraloids.

Techniques: Liposomes, Radioactivity, Concentration Assay

Journal: Biochemistry

Article Title: Cholesterol exposure at the membrane surface is necessary and sufficient to trigger perfringolysin O binding.

doi: 10.1021/bi9002309

Figure Lengend Snippet: FIGURE 5: PFO binding to DOPC/SM/cholesterol membranes. (A) Changes in the Trp emission intensity of PFO are shown as a function of the percentage of SM in the nonsterol fraction of the membranes. The net F/F0 was calculated as described in Experimen- tal Procedures. Error bars indicate the standard deviation observed for three independent measurements per data point. (B) NBD-PFO oligomerization on DOPC/SM/cholesterol membranes. After incu- bation of PFO with the membranes, samples were solubilized with sample buffer and separated by 1.5% SDS-AGE. The NBD-PFO monomer and oligomer bands were visualized with a BioRad Mole- cular Imager FX as described in the Experimental Procedures. In both (A) and (B), the membranes were composed of 50 mol% cholesterol and various proportions of DOPC and SM comprising the remaining 50 mol% lipid in the nonsterol fraction. For example, when SM is at 50% in the nonsterol fraction, there is an equimolar amount of SM and DOPC (25 mol% each). (C). Formation of detergent-resistant domains in DOPC/SM/cholesterol membranes. The liposomes were composed of 40 mol% cholesterol and various proportions of DOPC and SM comprising the remaining 60 mol% lipid in the nonsterol fraction. Liposomes with trace amounts of [3H] cholesterol were extracted with TX-100 at 0 C. Radioactivity in the pellet fractions was measured after ultracentrifugation, and the percent of radioactivity in the pellet was plotted as a function of the percentage of SM in the nonsterol fraction of the membrane.

Article Snippet: Cholesterol (5-cholesten-3β-ol) and3-epicholesterol (5-cholesten-3R-ol)wereobtained from Steraloids.

Techniques: Binding Assay, Standard Deviation, Liposomes, Radioactivity, Membrane

Journal: Biochemistry

Article Title: Cholesterol exposure at the membrane surface is necessary and sufficient to trigger perfringolysin O binding.

doi: 10.1021/bi9002309

Figure Lengend Snippet: FIGURE 6: NBD-PFO oligomerization on membranes containing cholesterol and epicholesterol. The liposomes contained 52 mol% POPC and 48 mol% sterol. The mol% of the individual sterols comprising the sterol fraction of the liposome is indicated below each lane in the above figure. After incubation of PFO with the mem- branes, samples were solubilized with sample buffer and separated by 1.5% SDS-AGE. The NBD-PFO monomer and oligomer bands were visualized with a BioRad Molecular Imager FX as described in the Experimental Procedures.

Article Snippet: Cholesterol (5-cholesten-3β-ol) and3-epicholesterol (5-cholesten-3R-ol)wereobtained from Steraloids.

Techniques: Liposomes, Incubation