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. 2011 Oct 18;286(50):42808–42817. doi: 10.1074/jbc.M111.310169

FIGURE 4.

FIGURE 4.

Ceramide 1-phosphate is a direct and specific inhibitor of TACE. A, TACE demonstrates the presence of highly conserved sequences homologous to the C1P-binding site of cytosolic phospholipase A2. The primary structure of human TACE was scrutinized for a potential C1P-binding site in the form of three to four consecutive basic amino acids. Six such sequences were observed from 53 to 58 (N-terminal prodomain), 210 to 215 (mature TACE), 233 to 237 (mature TACE), 625 to 628 (mature TACE), 640 to 645 (mature TACE), and 753 to 758 (mature TACE). A multiple alignment of the corresponding region revealed several highly conserved motifs. B, effect of phospholipids on in vitro TACE activity. TACE activity was measured in vitro using human recombinant TACE enzyme and fluorogenic substrate as described using a Triton X-100 mixed micelle approach to deliver the individual lipids (26). C18:1/18:1 C1P, dioleoyl PA, S1P, and C18:1/18:1 ceramide was used at the designated mol % in regard to Triton X-100. The data are plotted as the mol % of the specific lipid versus TACE specific activity (picomoles of cleaved fluorogenic substrate/min/μg of recombinant TACE protein). The data are representative of the mean ± S.E. of an n = 7 repeated on three separate occasions. C, acyl chain length requirement of C1P for inhibition of TACE activity. TACE activity was measured in vitro using human recombinant TACE enzyme and fluorogenic substrate as described using a Triton X-100 mixed micelle approach to deliver the individual lipids (26). The fluorescence was monitored over time on a fluorescence plate reader. C18:1/2:0 (C2:0 C1P), C18:1/8:0 (C8:0 C1P), C18:1/12:0 (C12:0 C1P), C18:1/16:0 (C16:0 C1P), C18:1/18:1 (C18:1 C1P), and C18:1/24:0 (C24:0 C1P)C1P was used at 0.6 mol % with respect to Triton X-100. The data are plotted as the treatment of the specific lipid and controls versus TACE specific activity (picomoles of cleaved fluorogenic substrate/min/μg of recombinant TACE protein). The data are representative of the mean ± S.E. of an n = 4 repeated on two separate occasions.