Mixed Micellar Assay For Phorbol Ester Binding

This chapter provides an overview of mixed micellar assay for phorbol ester binding. Phorbol esters, the most potent tumor promoters known, elicit complex biology in various cells, including differentiation, altered cell proliferation, intracellular communication, and biochemical changes. Understanding of the molecular mechanisms of phorbol esters action advanced with the demonstration of a specific and high affinity receptor for biologically active phorbol esters using [³H]phorbol dibutyrate (PDBu). This receptor was associated with the particulate fraction of cells and was found to be widely distributed in tissues. The binding of phorbol esters to cytosolic protein kinase C is dependent on Ca²⁺ and phospholipids and is displaced by sn-1,2-diacylglycerols. The mixed micellar assay for phorbol dibutyrate binding described in this chapter provides a convenient and reproducible method for studying the phospholipid requirement for phorbol binding and for evaluating the stoichiometry of this binding.