Figure 3. LPAAT Activity of Purified Murine and Human I148M Variants.
(A) Coomassie blue stain showing purified TF and recombinant mAdpn, mI148M, hADPN, and hI148M. I148M mutants were generated by site-directed mutagenesis, purified, and analyzed for LPAAT activity in comparison to wild-type proteins. Purified proteins were detected by western blotting using anti-His monoclonal antibody.
(B and C) (B) LPAAT activity of mAdpn and mI148M and (C) hADPN and hI148M using 1 μg of purified protein in a reaction with 20 μM [1-14C] oleoyl-CoA and 100 μM LPA. Purified TF was used as negative control with negligible LPAAT activity (<0.1 nmol PA/min*mg).
(D) Substrate specificity of recombinant hADPN and hI148M for acyl donors was measured by incubating 2 μg of partially purified proteins with 25 μM [3H] LPA (600,000 cpm/assay) and 20 μM of various acyl-CoAs. Relative LPAAT activity was determined by normalizing the specific activity of hADPN and hI148M (4.2 and 8.1 nmol/min*mg, respectively) in the presence of [3H] LPA and oleoyl-CoA to 100%.
(E) Substrate specificity of recombinant hADPN and hI148M for acyl acceptors was measured by incubating 2 μg of partially purified proteins with 20 μM [1-14C] oleoyl-CoA as acyl donor in the presence of 100 μM of various acyl acceptors (LPA, lysophosphatidyl choline [LPC], lysophosphatidyl ethanolamine [LPE], lysophosphatidyl inositol [LPI], or lysophosphatidyl serine [LPS]). Relative LPAAT activity was determined by normalizing the specific activity of hADPN and hI148M (as shown in Figure 3C) in the presence of LPA and [1-14C] oleoyl-CoA to 100%.
(F) Acylation of glycerol-3-phosphate (G3P) was measured by incubating 2 μg purified TF, hADPN, and hI148M with 300 μM G3P and 20 μM [1-14C] oleoyl-CoA. Data are presented as mean ± SD and are representative of at least two independent experiments with each sample in duplicates. Statistical significance was determined by a two-tailed Student’s t test, **p < 0.01, ***p < 0.001.