N-acyl Phosphatidylethanolamine-specific Phospholipase D - Identification and Purification

Identification and Purification

NAPE-PLD is an enzyme activity - a phospholipase, acting on phospholipids found in the cell membrane. It is not homology but the chemical outcome of its activity that classes it as phospholipase D. The enzymatic activity was discovered and characterized in a series of experiments culminating in the 2004 publication of a biochemical purification scheme from which peptide sequencing could be accomplished. Researchers homogenized (finely ground) hearts from 150 rats and subjected the resulting crude lysate to sucrose sedimentation at 105,000 x g to separate out the cell membranes from the remainder of the cell. The integral membrane proteins were then solubilized using octyl glucoside and subjected to four column chromatography steps (HiTrap SP HP cation-exchange column, HiTrap Q anion-exchange column, HiTrap Blue affinity column, Bio-Gel HTP hydroxyapatite column). Each of these separates the different types of membrane proteins into different sample containers when the proteins are eluted from the column over time, and by measuring the activity of samples in each container it was possible to track which ones received the active enzyme. Measurement of the enzyme activity was done by thin layer chromatography of a radioactive substrate sensitive to the NAPE-PLD enzymatic activity: Cleavage of the substrate affected where it appeared on the plate when the radiation was detected on a bioimaging analyzer.

The result of this extensive procedure was still not a pure protein, but it produced a limited number of bands by SDS-PAGE, and one band of 46 kilodaltons was found to correlate in intensity with the enzymatic activity. 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. The resulting fragments were then microsequenced by an automated Edman degradation. 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.

Once this clue had been obtained, the identification could be confirmed by a less onerous procedure: Overexpression of the putative NAPE-PLD cDNA in COS-7 cells yielded a strong NAPE-PLD enzymatic activity, whose characteristics were shown to be similar to those of the original heart extract.