This band was cut out from the gel and digested with trypsin, and peptides from it were separated from one another by reverse phase high performance liquid chromatography.
[5] Three corresponded to vimentin, an intermediate filament protein of 56 kDa believed to be a contaminant, and the other two matched the cDNA clone subsequently identified as NAPE-PLD.
When recombinant NAPE-PLD was tested in COS cells in vitro it had similar activity toward several radiolabeled substrates: N-palmitoylphosphatidylethanolamine, N-arachidonoylphosphatidylethanolamine, N-oleoylphosphatidylethanolamine, and N-stearoylphosphatidylethanolamine all reacted with a Km between 2–4 micromolar and a Vmax between 73 and 101 nanomole per milligram per minute as calculated by Lineweaver–Burk plot.
[2] (These generate N-palmitoylethanolamine, anandamide, N-oleoylethanolamine, and N-stearoylethanolamine, respectively) The enzyme also reacted N-palmitoyl-lyso-phosphatidylethanolamine and N-arachidonoyl-lyso-phosphatidylethanolamine with similar Km but at one-third to one-fourth the Vmax.
It also lacks the transphosphatidylation activity of phospholipase D that allows the creation of phosphatidyl alcohols rather than phosphatidic acid in the presence of ethanol or butanol.
Bile acids bind with high affinity to selective pockets in this cavity, enhancing dimer assembly and enabling catalysis.