Measuring the Binding of Protein Kinase C to Sucrose-Loaded Vesicles

Abstract

The binding of protein kinase C (PKC) to model membranes provides useful information about the mechanisms dictating the translocation of PKC to biological membranes. The sucrose-loaded vesicle (SLV) assay is useful for this purpose. It was originally designed to measure the binding of phospholipase C to vesicles (1) but later modified to be useful for the study of PKC (2–4). Large unilamellar vesicles filled with sucrose are used as model membranes that can be readily pelleted by low-speed centrifugation (see Subheading 3.1.). PKC is incubated with the vesicles and other cofactors required for binding, and the mixture is centrifuged to pellet the vesicles along with any PKC bound to them. The supernatant containing the unbound PKC is separated from the vesicle pellet containing bound PKC (see Subheadings 3.2. and 3.3.). The relative amounts of enzyme in the supernatant and pellet are determined by a kinase assay (see Subheading 3.4.). The percentage of PKC bound can be calculated from these values (see Subheadings 3.5. and 3.6.). This assay is useful for determining various properties of PKC. For example, different compositions of lipids can be used to make the SLVs and the binding of PKC to each of them can be compared, to determine which lipids or combinations of lipids are preferred by the enzyme as well as optimal conditions for binding. Also, the binding of mutant PKCs can be compared to that of wild-type to determine the differences created by mutating specific residues.