Please help. From fig 1a how was the level of enzyme activity determined?
The authors used a radioactively labelled preparation of cell membranes as a substrate for the gpi-pld. The activity of purified and exogenously added gpi-pld was assayed by measuring the release of the radioactively labelled PA (phosphatidic acid)
Answer:
They have a lipid membrane made of phosphatidylinositol. The acid residues come from tirtium-labelled myristic acid.
And the phosphatidylinositol molecules are bound to a surface glycoprotein via glycosidic bonds.
They start every reaction with 10000 cpm's worth of membrane, and as the enzyme works its magic, it releases the phosphatidylinositol from the protein. This phospholipid is then extracted and radioactively assayed.
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