Fig. 4.

Factors affecting lysophosphatidic acid acyltransferase activity of recombinant CGI-58. Recombinant His₁₂-CGI-58 (1 μg) was added to reaction mixtures containing 10 μM [¹⁴C]oleoyl-CoA and 50 μM 1-oleoyl-lysophosphatidic acid in 50 mM Tris-HCl, pH 7.5, unless otherwise noted. Reaction mixtures were extracted with solvents and phosphatidic acid was separated from other lipids by TLC. Phosphorimaging and scintillation counting of scrapings from silica gel plates were used to quantify phosphatidic acid formation. A: Time dependence of phosphatidic acid formation. His₁₂-CGI-58 was added to the reaction mixture and samples were incubated at 30°C for the indicated times. Data represent means ± SD for three samples in one representative experiment out of four. Error bars that are not visible are contained within the symbol. B: Enzyme concentration dependence of phosphatidic acid formation: various amounts of His₁₂-CGI-58 were added to reaction mixtures, which were then incubated at 30°C for 10 min. Data represent means ± SD for duplicate samples for one out of two experiments. C: Recombinant His₁₂-CGI-58 (0.5 μg) was incubated at various temperatures for 10 min prior to addition of [¹⁴C]oleoyl CoA (10 μM) and lysophosphatidic acid (50 μM) in 50 mM Tris-HCl, pH 7.5 and further incubation at 30°C for 10 min. Data represent mean ± SD for duplicate samples from one of two experiments; error bars that are not visible are contained within the symbol. D: Determination of optimal pH for phosphatidic acid formation. His₁₂-CGI-58 was added to the reaction mixture in solutions buffered with N-(2-acetamido)-2-aminoethanesulfonic acid, Tris, or ethanolamine between pH 5.0 and 9.5. Reactions were incubated at 30°C for 10 min. Data represent means ± SD for triplicate samples for one out of four experiments. E: Detergent inhibition of phosphatidic acid formation. His₁₂-CGI-58 was added to reaction mixtures containing no additions (none), 0.4 mM Triton X-100, or 6 mM CHAPS, and samples were incubated at 30°C for 10 min. Data represent means ± SD for triplicate samples for one out of three experiments. F: Metal ion inhibition of phosphatidic acid formation: 0.5 μg His₁₂-CGI-58 was added to reaction mixtures containing various concentrations of magnesium chloride (•), or calcium chloride (○) up to 1 μM. Acyltransferase activity is expressed as percent of maximal activity in the absence of added metal ions. Data represent means ± SD for duplicate samples from one out of two experiments.