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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.
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1Phospholipase A2 and PhosphatidylinositolSpecific Phospholipase C Assays by HPLC and TLC with Fluorescent Substrate H. Stewart Hendrickson 1. Introduction Llpolytic enzymes have traditlonally been assayed by radlometrlc and t&-lmetric methods (I). RadIometrIc methods are quite sensitive but require expensive radlolabeled substrates and tedious separation of labeled substrate and products. In addition, the safe use of radioactive materials 1sof mcreasmg concern. Tltrlmetrtc assaysare contmuous and quite straightforward and use natural substrates but suffer from low sensltlvlty and are subject to condltlons that may alter the amount of free hydrogen Ions released Fluorescence-based assays have sensltlvltles that approach those of radtometrlc methods; although they require synthetic fluorescent-labeled substrates, they are often more convenient and rapid. For a recent review, see Hendrickson (2). We first used dansyl-labeled glycerol ether analogs of phosphatidylcholme as substrates for the assay of enzymes of the platelet-actlvatmg factor (PAF) cycle m peritoneal polymorphonuclear leukocytes (3). This became a general method for the assay