The lipases and phospholipases represent a diverse group of enzymes that are expressed in animals, plants, fungi, and bacteria. Their ubiquitous distribution among all species is a testament to the essential roles played by these enzymes in lipid storage, mobilization and metabolism, membrane homeostasis and remodeling, endocrine and immune functions, and signal tra- duction. In humans, lipases and phospholipases are also thought to contribute to complex diseases, such as atherosclerosis, obesity, arthritis, and cancer, as well as to single gene defects, such as Wolman's disease and Type I hyperlipoproteinemia. Enzymatically, the lipases are unique, since they hydrolyze substrates that are either insoluble, or only partly soluble, in aq- ous solvents; thus, enzyme catalysis takes place at a lipid-water interface. The interface comprises at least two, and often more, discrete bulk and s- face phases, in which the enzyme, substrate, and products oflipolysis disperse among these phases based on their physical properties. Furthermore, the d- tribution of these components changes continuously as lipolysis proceeds. Thus, the lipases and phospholipases are fundamentally different from any other enzyme because of the physical complexity of the environment in which catalysis occurs.

Klappentext

A unique collection of step-by-step methods for the characterization, purification, and expression of mammalian lipases and phospholipases. The methods include the preparation of a variety of unique substrates for the determination of lipolytic activity, the isolation of purified lipase preparations, and the development of high expressing recombinant systems. There are also methods for the immunodectection and production of immunological reagents and the analysis of biochemical properties, such as subunit size, lipase kinetics, enzyme immobilization, and ligand interactions. The protocols covers more than a dozen specific enzymes, and provide well-established methods that can be adapted to the discovery of new lipase enzymes.

Inhalt
Lipase Activity, Purification, and Expression.- Phospholipase A2 and Phosphatidylinositol-Specific Phospholipase C Assays by HPLC and TLC with Fluorescent Substrate.- Fluorometric Phospholipase Assays Based on Polymerized Liposome Substrates.- Triglyceride Lipase Assays Based on a Novel Fluorogenic Alkyldiacyl Glycerol Substrate.- Purification and Assay of Mammalian Group I and Group IIa Secretory Phospholipase A2.- Determination of Plasmalogen-Selective Phospholipase A2 Activity by Radiochemical and Fluorometric Assay Procedures.- Human Plasma Platelet-Activating Factor Acetylhydrolase.- Assays for Pancreatic Triglyceride Lipase and Colipase.- Bile Salt-Activated Lipase.- Determining Lipoprotein Lipase and Hepatic Lipase Activity Using Radiolabeled Substrates.- Lysosomal Acid Lipase.- Hormone-Sensitive Lipase and Neutral Cholesteryl Ester Lipase.- Lecithin-Cholesterol Acyltransferase.- Purification of Lipases and Phospholipases by Heparin-Sepharose Chromatography.- Large-Scale Lipoprotein Lipase Purification from Adipose Tissue.- Purification of Rat Hepatic Lipase Essentially Free of Apolipoprotein E and Apolipoprotein B.- High-Level Expression and Purification of Human Hepatic Lipase from Mammalian Cells.- High-Level Baculoviral Expression of Hormone-Sensitive Lipase.- High-Level Baculoviral Expression of Lysosomal Acid Lipase.- One-Step Purification and Biochemical Characterization of Recombinant Pancreatic Lipases Expressed in Insect Cells.- Modulation of the Expression Level of Human Acidic Lipases by Various Signal Peptides.- Biochemical Characterization.- Immunodetection of Lipoprotein Lipase: Antibody Production, Immunoprecipitation, and Western Blotting Techniques.- Immunological Characterization of Digestive Lipases.- Determining Lipase Subunit Structure by Sucrose Gradient Centrifugation.- Detecting Ligands Interacting with Lipoprotein Lipase.- Monolayer Techniques for Studying Lipase Kinetics.- Efficient Immobilization of Phospholipase A2.- Molecular Genetic Characterization.- An Introduction to Internet Resources for the Molecular and Genetic Analysis of the Lipases.- Techniques for the Measurement of Lipoprotein Lipase Messenger RNA.- In Vitro Transcription and Translation of Lipoprotein Lipase.- Induced Lipase Mutations in the Mouse.