This chapter discusses the methods used for analyzing the involvement of N proteins in the chemoattractant-induced breakdown of phosphatidylinositol 4, 5-bisphosphate (PIP2) in human polymorphonuclear leukocytes (PMNs). The study utilizes three systems: intact cells, detergent-permeabilized cells, and isolated plasma membranes. These approaches provide increasing ability to define the biochemical mechanisms of the coupling of occupied receptors to phospholipase C activation and prove to be generally applicable to other cells and receptor systems. The chapter discusses the quantitation of polyphosphoinositide hydrolysis in intact PMNs. Radiolabeled PMNs are exposed to hormones or to buffer. Hormone-stimulated polyphosphoinositide hydrolysis is measured as the decrease in the amount of radioactivity in phosphatidylinositol 4-phosphate (PIP) and PIP2 or the increase in radioactivity in inositol phosphates. Radioactivity in PIP and PIP2 is measured by liquid-scintillation counting after the isolation of these lipids by affinity chromatography using immobilized neomycin. Inositol phosphates are analyzed by anion-exchange chromatography followed by liquid-scintillation spectrometry.
All Science Journal Classification (ASJC) codes
- Molecular Biology