Interfacial Binding of cPLA2 to Intracellular Membranes and the Role of Calcium:

This section of the mini-review will discuss the interaction of the C2 domain binding to the intracelluar membranes represented by types of cellular vessicles. There are several propsed interactions, all dealing with the makeup of LUV's (large unilamellar vesicles). Research indicates that cPLA2 and especially the C2 domain differs in how well it binds to LUV's made up of different lipids.

The first study looked at how well cPLA2 bound to Phophatidylcholine LUV's. Observations indicate that in the presence of 10 micromolar calcium, cPLA2 binds with a Kd (enzyme-vesicle dissociation equlibrium constant) = 90 +/- 18 micromolar (Hixon). Subsequently, binding without calcium present is much weaker, with a Kd=700 +/- 200 micromolar(Hixon).

The next study examined how well cPLA2 bound to Phosphatidylmethanol LUV's. Data for this LUV is significantly different from the previous LUV studied. In this case, the Kd = much less than 10 micromolar either with or without calcium present (Hixon). However, this evidence did not suggest that cPLA2 was not catalytically active on this particular LUV. Actually, cPLA2 is highly active on this LUV, even without calcium present.

The third study looked at the binding action of cPLA2 to Phosphatidylserine LUV's. In this case, cPLA2 was shown to have a Kd = 60+/-10 micromolar when calcium was present(Hixon). While this is lower than the Phosphatidylcholine, it is significantly higher than that of Phosphatidylmethanol. No data was present at this time for the Kd without calcium present.

The next study explored how well the C2 domain bound to the same LUV's as above, starting with Phosphatidylcholine LUV's. With calcium present, the C2 domain showed a Kd = 11 +/- 3 micromolar(Hixon). When no calcium was present, the C2 domain showed no binding to the LUV(Hixon).

The fifth set of data indicates that the C2 domain has a Kd = 10 +/- 2 micromolar when binding to Phosphatidylmethanol LUV's (Hixon). Once agian, when no calcium was present no binding took place (Hixon). These results are very similar to that of the immediately precceding research.

The sixth and final set of data collected examined how well C2 bound to Phosphatidylserine. In this case, either with or without calcium, no binding took place (Hixon).

Several explanations exist for the observations that have been seen in these studies. A simple one is that both domains, the C2 and the catalytic domain both contact the the membrane. Another possibility is that while the catalytic domain does not contact the membrane, it somehow influences the binding properties of the C2 domain.

However, this data should not be interpreted as indicating that the binding of cPLA2 is driven only by the C2 domain (Hixon). Several other studies on other substrate-ezyme complexes show that while the enzyme as a whole may bind very tightly, the two seperate domains may either bind weakly or not at all.

Further, it seems very likely that the catalytic domain would have to be in close proximity to its ligand, even to the point of actual bondage with the membrane. This is likely so that the phospholipid can actually access the active site of the catalytic domain. This is true for all known lipases with known three-dimensional structure. Also, some X-ray structure shows that the C2 and catalytic domains are in position to bind simultaneously to the same interface (Hixon).

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W. D. Keeton, III

University of Wisconsin -- Eau Claire

Biochemistry/Molecular Biology