Ultracentrifugation Technique for Measuring the Binding of Peptides and Proteins to Sucrose-Loaded Phospholipid Vesicles

The binding of extrinsic proteins to membranes is important for their biological activity in many different systems. Consider a few examples from the calcium/phospholipid second-messenger system (reviewed in refs. 1 and 2 ). First, membrane binding facilitates interaction of the G_q protein with both its activated receptor and its membrane-bound effector, phosphoinositide-specific phospholipase C (PLC-β). Second, it enhances the ability of PLC to hydrolyze its membrane-bound substrate phosphatidylmositol 4,5bis-phosphate (PIP₂ ) in a precessive manner (i.e., the enzyme can “scoot” over the surface, hydrolyzing many PIP₂ molecules before desorbing from the membrane). Third, membrane binding increases the probability that protein kinase C (PKC) will phosphorylate its membrane-bound substrates, which include pp60^src (Src) and MARCKS (mynstoylated alanine-rich C kinase substrate). Fourth, membrane binding is required for the biological activity of proteins such as Src and Ras. All these proteins bind to the membrane by a combination of electrostatic and hydrophobic interactions (see refs. 3 and 4 and references therem). This chapter describes a simple ultracentrifugation technique for measuring the binding of extrinsic proteins and peptides to membranes.