Phospholipase overview

Prior to being able to study the function and mechanism of an enzyme, it is essential that suitable assays be available to monitor enzyme activity toward different substrates and to determine the kinetic parameters kcat and Km for the reactions. A brief overview of the known assays for the evaluation of PLCB(. activity is thus appropriate. The ideal assay for a phospholipase C would utilize a phospholipid substrate, not an analogue with a modified headgroup or side chains. Such an assay should be sensitive to minimize the quantities of enzyme and substrates that would be required, and it should be convenient to implement so that analyses may be readily performed. 

Figure 1.9. Overview of the biosynthesis of ecosanoids. The 20 carbon fatty acid arachidonic acid is released from cell membrane phospholipids by the actions of phospholipase A2. Free arachidonic acid forms the precursor of prostaglandins and thromboxanes via the multi-enz5une cyclooxygenase pathway, while leukotrienes are formed via the lipoxygenase pathway...

During recent years, groups interested in the role of Ca in secretion and in the control of membrane cytoskeleton have identified some intracellular phospholipid-binding proteins that appear to be distinct from the calmodulin superfamily these include lipocortin, endonexin, calelectrin, p36, and calpac-tin These membrane-binding proteins are collectively called annexins, and contain repeated domains distinct from EF-hands. The Ca sites are very similar to that observed in phospholipase A2, as shown by the recently determined x-ray structure of annexin A condensed overview of the interaction of Ca + with intracellular proteins is shown in Figure 3.16. We will now go on to discuss the molecular properties of some of the proteins mentioned above, starting with calmodulin. 

Reactions of heterolipids are generally similar to those of other lipids, but reactions of bound phosphoric acid are specific to heterolipids. Phospholipids are hydrolysed by various phospholipases an overview of which is given in Table 3.47 and the mechanism of their action is shown in Figure 3.89. Phosphatidylcholine is a demanded product and its concentration in lecithin can be increased by enzymatically controlled interesterification after adding choline. Phosphatidylcholine can also be produced directly from diacylglycerols or phosphatidic acids using immobilised enzymes. 

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