Abstract
This chapter discusses the analysis of the binding of several isoforms of profilin with phospholipids. Human platelet profilin binds to clusters of phosphatidylinositol 4,5-bisphosphate (PIP2) and in doing so inhibits the hydrolysis of the PIP2, by a soluble phosphoinositide-specific phospholipase C (PLC). So profilin might participate in the regulation of both actin polymerization and the phosphoinositide (PI) signaling pathway. The chapter discusses binding of profilin to phospholipids, how profilin inhibits soluble phospholipase C, interaction of profilin with actin, and what profilin actually does in a cell. The binding of profilin isoforms to various phospholipids is specific for both the protein and the phospholipid. Human platelet profilin binds to PIP2 and PIP, with dissociation constant, in the range of 1μM. Acanthamoeba profilin II binds to PIP, with a similar affinity and stoichiometry. The acidic isoform of Acantharnoeba profilin, profilin I, only binds weakly to PIP2, with a dissociation constant greater than 50 to 100 μM. This remarkable difference between profilin I and profilin II provides evidence for the specificity of binding, because these two profilins have similar amino acid sequences. Platelet profilin is the strongest inhibitor of the enzyme phospholipase C, followed by Acanthamoeba profilin II and Acanthamoeba profilin I that is a weak inhibitor of the enzyme. These observations have been confirmed, with purified brain PLC-γ, a soluble phospholipase C. Biochemical experiments, combined with the knowledge about the concentrations of profilin and PIP2 in cells, suggest that the profilins can influence the PI signaling pathway by binding to PIP, the substrate for PIC, which gives rise to the second messengers IP and diacylglycerol (DAG). Thus, profilin is a candidate for the mysterious inhibitor of PLC in the resting cells.