Phospholipid Signaling Protocols (Methods in Molecular Biology, 105)

Cell membranes are not, as once believed, inert structures designed to contain the cell contents, but are in fact dynamic structures that are as me- bolically active as the cytosol and other cellular compartments they surround. Thus membranes not only contain mixtures of lipid and phospholipids, but also many proteins both embedded deeply within the membrane structure itself and also more loosely attached on the membrane surfaces. Though many such proteins have long been known to act as transport proteins, ion channels, hormone receptors, G proteins, cytoskeletal anchorage points, and so on, the major advance of recent years is the increasing understanding that the lipids and phospholipids in the membrane bilayer itself are also metabolized to b- logically active products that can diffuse either in the cytosol or in the m- brane bilayer to control the function of other proteins. Thus the concept of lipid-derived second messengers is now firmly established.

In Phospholipid Signaling Protocols, state-of-the-art techniques for monitoring the major lipid and phospholipid-derived second messenger pathways to identify and quantify pathway activation are detailed by experts intimately experienced in their use. The assays described cover all the major phospholipases (C, D, A2), as well as sphingomyelinase and its associated metabolites. Additional protocols are provided for the assay of phosphoinositide 3-, 4-, and 5-kinase and sphingosine kinase activity, and for the quantification, separation, and rigorous identification of phospholipids, diacylglycerol, and sphingolipids, as well as their metabolites, including phosphoinositols, choline metabolites, and fatty acid metabolites. In addition, there is extensive information on the extraction, size separation, detection, and quantification of cellular signaling proteins and corresponding mRNA, as well as a description of their localization by immunohistochemistry and immunocytochemistry.

Phospholipid Signaling Protocols offers a wide-ranging collection of cutting-edge techniques for the study of signal transduction through phospholipid intermediates and their metabolites. The book is an indispensable reference for both the newcomer and the experienced researcher seeking to expand knowledge of these critical pathways, and strongly complements its companion volumes-R.A.J. Challiss' Receptor Signal Transduction Protocols, D. Bar-Sagi's, Transmembrane Signaling Protocols, and D. A. Kendall and S.J. Hill's Signal Transduction Protocols-in building a unique library of tried-and-tested protocols relating to the ever expanding signal transduction field.