The enzyme phsopholipase C cleaves off the phosphate and hea

The enzyme phsopholipase C cleaves off the phosphate and head group from phospholipids. Suppose you add the enzyme to the outside of intact HeLa cells, incubate for 30 minutes, and then extract all of the phospholipids in organic solvents and measure the amounts of intact phosphatidyl choline and phosphatidyl ethanolamine before and after treatment. In a second experiment, you first break open the same number of cells and add Phospholipase C to the broken membranes. The following results are obtained:

Experiment 1: intact cells Before                         After

phosphatidyl choline               100 umoles                  10 umoles

phosphatidyl ethanolamine     90 umoles                 88 umoles

Experiment 2: broken membranes

phosphatidyl choline               98 umoles                    2 umoles

phosphatidyl ethanolamine     95 umoles                 0.5 umoles

What can you conclude about the asymmetric placement of these phospholipids in the membrane? Why was Experiment 2 carried out?

Solution

A common feature of all eukaryotic membranes is the non-random distribution of different lipid species in the lipid bilayer (lipid asymmetry). Lipid asymmetry provides the two sides of the plasma membrane with different biophysical properties and influences numerous cellular functions.

In the plasma membrane of normal eukaryotic cells,phosphatidylcholine (PC)is present predominantly in the outer leaflet whereas Phosphatidylethanolamine (PE) is present in the inner leaflet of the plasma membrane.

In our Experiment 2, since Phosphatidylethanolamine (PE) is present in the inner leaflet of the plasma membrane in the broken membranesenzyme phospholipase C interacts with PE and cleaves the phosphate and head group from phospholipids. Thus resulting the low amounts of PE in the broken cells than in the intact cells (i.e 88 umoles to 0.5 umoles)

* The second experiment is carried out inorder to compare the action of the enzyme phospholipase C in both intact cells and broken membranes before and after the treatment with it. The intearction of the enzyme depends on the arrangement of the phospholips in the membrane also.