FIGURE 5.

Activity of human ABHD6 and mutant variants of the enzyme. A, the coding sequence of hABHD6 was cloned into a mammalian expression vector (pcDNA4/HisMaxC), and the protein was expressed in COS-7 cells. Mutations were introduced by site-directed mutagenesis. For enzymatic assays, cell lysates (1000 × g supernatant) were incubated with BMP(S,S), and FFA release was determined. Lysates obtained from cells expressing β-galactosidase (LacZ) were used as a negative control. B, selected mutations of hABHD6 as reported by the NCBI dbSNP database (http://www.ncbi.nlm.nih.gov/projects/SNP). C, BMPH activity of wild-type hABHD6 and mutant variants of the enzyme. Data are expressed as percent activity of wild-type hABHD6 and were normalized to the expression levels of His-tagged proteins. The activity detected in lysates of LacZ-expressing cells was set as blank. Inset, Western blot of His-tagged variants using an anti-His antibody. Data are presented as mean ± S.D. ***, p < 0.001; analysis of variance.