Figure 4. Analysis of CLS activity of the recombinant hCLS1 enzyme.

The recombinant hCLS1 protein transiently expressed in COS-7 cells was analysed for CLS activity using two different assay conditions. In the first assay condition (A), FLAG–hCLS1 was co-expressed with the human LPGAT1, which was used to synthesize [¹⁴C]PG as a substrate for the CLS assay. CLS activity was assayed in a mixture of 200 μl containing 50 mM Tris/HCl (pH 8.0), 4.0 mM MgCl₂, 20 μM [¹⁴C]oleoyl-CoA, 2.0 mM LPG and 2.0 mM CDP-DAG. As a positive control for the detection of radiolabelled cardiolipin, recombinant ACLAT1 was used to catalyse the synthesis of [¹⁴C]cardiolipin with monolysocardiolipin and [¹⁴C]acyl-CoA as substrates. In the second assay condition (B), the recombinant hCLS1 expressed in COS-7 cells was analysed for CLS enzyme activity with purified [¹⁴C]PG in the presence/absence of CDP-DAG as a substrate. The recombinant hCLS1 catalysed the synthesis of cardiolipin only in the presence of both CDG-DAG and [¹⁴C]PG. The radiolabelled PG and cardiolipin (CL) are indicated by arrowheads. FFA, non-esterified fatty acid.