PMC

Schematic of vesicle formation, purification and analysis. Initial chloroform dry down and lipid rehydration was done with constant stirring.

TLC of native lens lipids. Native lens lipids were separated by TLC using chloroform:methanol:ammonium hydroxide (65:25:4) as the solvent system. Spots were visualized by H₂SO₄ charring followed by digital image analysis to determine the relative mobilities. The origin is indicated by a solid line and the solvent front is indicated by a dashed line.

Native lens lipid vesicle binding. Human recombinant AlexaFluor350™-conjugated α-crystallins were incubated for 15 h at 37 °C with vesicles composed of either SPH + 40 mol% cholesterol () or native lens lipids (). Most points represent the average of 4 assays. No major differences were seen for human recombinant αA (A), αB (B) or reconstituted recombinant 3:1 heterocomplex (C).

Synthetic lipid vesicle binding. Human recombinant AlexaFluor350™-conjugated α-crystallins were incubated for 15 h at 37 °C with vesicles composed of either PC 16:0 (black), PC 16:1t (), PC 20:0 (), PC 20:4c (), or egg SPH (purple). Most points represent the average of 4 assays. No major differences between vesicle types were seen for human recombinant αA (A), αB (B) or reconstituted recombinant 3:1 heterocomplex (C).

The effect of cholesterol on vesicle binding. Human recombinantAlexaFluor350™-conjugated α-crystallins were incubated for 15 h at 37 °C with vesicles composed of either SPH (black), SPH + 40 mol% cholesterol (), PC 16:0 (), or PC 16:0 + 40 mol% cholesterol (). Most points represent the average of 4 assays. No major differences between vesicle types were seen for human recombinant αA (A), αB (B) or reconstituted recombinant 3:1 heterocomplex (C).

Comparison of native and recombinant α-crystallin PC binding. Vesicle binding of human recombinant αA (), αB (), and 3:1 heterocomplex (black) was compared to the binding of native bovine (purple) and human () α-crystallins. Each α-crystallin complex was incubated for 15 h at 37 °C with vesicles composed of PC 16:0. All points represent the average of 2–4 assays. No major differences were seen between the protein binding capacities.