Membrane binding affinity of phospholipases β1 and β2 and g proteins subunit dependence

We have measured the binding affinities of mositol-phopholipid -specific phospholipases βl and β2 using fluorescence spectroscopy and sedimentation. We have also measured the binding of β1 and β2 mutants that are lacking the carboxy tail extension and it is this extension that distinguishes the βfrom the δ isoforms. Unlike the δ isoform of PLC, βl and β2 bind with high affinity (10^-5M) to membranes regardless of the presence of negatively charged phospholipids or PIP² substrate. This lack of PIP₂ dependence of binding was unexpected since both β1 and β2 have domains (i.e. pleckstrin horoology or PH domains) which could bind specifically to PIP₂ Even though β1 and β2 are activated by Ca²⁺ and contain possible CALB domain (calcium assisted binding domains), we find that binding is unaffected by Ca²⁺. The two truncated mutants showed binding properties similar to the whole proteins indicating that the carboxy extension of the β isoforms does not play a role in membrane association. We also characterized the binding of PLCβ2 in the presence of varying amounts of the βγ subunits of G proteins, and found that these subunits do not alter PLCβ2 membrane affinity. This finding indicates that activation of PLC by βγ subunits does not involve recruitment of PLC to the membrane surface by the G protein. These studies have also allowed us to estimate the PLCβ2-Gβγ affinity.