Methods in Molecular Biology [Methods Mol. Biol.], Humana Press Inc., 1998, vol. 105, 380 pp
This book attempts to summarize for the newcomer to this field those methodological protocols that allow monitoring of the major lipid- and phospholipid-derived second messenger pathways, as well as some additional physiologically important enzymes (the phosphoinositide kinases) involved in further regulating pathway activity. The book in particular covers the investigation of the hormone-sensitive phosphoinositidase C pathway, which gives rise to the formation of inositol 1,4,5-trisphosphate and diacylglycerol, each implicated in the release of intracellular Ca super(2+) and in the activation of protein kinase C, respectively. In addition, the assay of other phospholipases C to yield diacylglycerol from such phospholipids as phosphatidylcholine and phosphatidylethanolamine are described, together with assay of phospholipase A sub(2) and diacylglycerol/monoacylglycerol lipase activity to yield free arachidonate and its metabolites. Though the bulk of this book focuses on the hormone-stimulated pathways of phospholipid and lipid metabolism that are often found to mediate acute regulation of cell function, also included are information on the assay of sphingomyelinase activity, on the mass determination and species analysis of ceramides and sphingomyelins, and a protocol for the activity of sphingosine kinase, since there has recently been increasing evidence for a role of these pathways in the control of mitogenesis vs apoptosis. An additional feature is the inclusion of information on extraction, size separation, and quantification of cellular protein and mRNA, as well as a description of their immunohistochemistry and immunocytochemistry. This may seem unusual, but in endocrinology a question often arises that cannot be answered with a metabolic assay alone. For instance, there are many different isoforms of phospholipases as well as associated receptors, G proteins, or protein kinases, and so on. The most definitive method for identification of an isoform in a given cell or tissue is by protein immunodetection or mRNA quantification. All chapters have been contributed by scientists with considerable experience in the areas covered, so the protocols are each thoroughly tried and tested. In each article, after a brief introduction, the materials are listed, and then a detailed step-by-step protocol is given. A final section of notes is then provided, in which many of the tricks of the trade are hidden.