Phospholipase D assay

[acdumaua at indyvax.iupui.edu]

Hello Everyone,

        I am looking for some help.  My current project involves measuring
lipid distributions in bilayers (SUVs/LUVs) using different enzymes.  One
Enzyme Assay involves Phospholipase D (i.e., converts PC's to PA's) on
18:0, 18:1 PC LUVs.  I am having trouble getting any PA product.  I incubate
the LUVs with the enzyme in buffer for various times and then I stop the
reaction by precipitating the enzyme.  After extracting and separating my
lipids on TLC, I see no PA in my samples.  Does anyone have any ideas??
NOTE: I do not want to disrupt the bilayer during the reaction because keeping
the bilyar intact is essential to my project.  I believe that my problem has
to do with the ratio of enzyme-to-lipid that I am using and appreciate any
suggestions.

Thanks,

A. Dumaual  (acdumaua at indyvax.iupui.edu)
J. Cannon