Figure 6.

NPC4 Activity toward Different Phospholipids.

(A) Immunoblotting of NPC4 protein produced in E. coli. NPC4 from total E. coli lysate was purified and separated on 10% SDS-PAGE and transferred onto a filter and blotted with anti-His antibody. M, marker; 1, total lysate from empty vector; 2 and 3, total lysate harboring NPC4; 4, empty vector extract bound to His-agarose beads; 5, bound protein eluted from empty vector extract; 6, NPC4 extract bound to His-agarose beads; 7, NPC4 eluted from beads. Arrow indicates the 60-kD NPC4 protein.

(B) Lipid hydrolysis activity assayed in the presence of different phospholipids using crude E. coli lysate. DAG was quantified indirectly by measuring the release of the phosphorylated head group using a colorimetric phosphate assay. Values are means ± sd (n = 3). Vector control refers to the lysate from E. coli harboring the empty vector. Lipid abbreviations are described in the legend to Figure 5.

(C) NPC4 activity on PA using purified NPC4. DAG was quantified using gas chromatography (GC) analysis of fatty acid methyl esters derived from the DAG. Vector control refers to the lysate from E. coli harboring the empty vector that was purified in the same manner as NPC4. Values are means ± sd (n = 3).