FIGURE 4.

PA binding to SPHKs. A, lipid binding specificity of SPHK1 and SPHK2 on filters. Different lipids and PA species (10 μg) were spotted onto a nitrocellulose membrane and incubated with equal amounts of purified SPHK1, SPHK2, or total protein from E. coli transformed with empty vector. EY, egg yolk PA. B, SPHK1 binding to liposomes containing PC only or PC plus different PA species. Purified SPHK1 (20 μg) was incubated with different liposomes for 1 h at room temperature. The vesicles were pelleted by centrifugation. The protein was visualized by immunoblotting with anti-His antibody. C, SPR quantitative analysis of PA binding to SPHK1. Liposomes containing PC only or PC plus 16:0/16:0 PA or 18:1/18:1 PA were used. SPHK1 was first immobilized on the NTA chip followed by injection of liposomes.