By Mark Doolittle

A different number of step by step equipment for the characterization, purification, and expression of mammalian lipases and phospholipases. The equipment comprise the training of numerous certain substrates for the decision of lipolytic job, the isolation of purified lipase arrangements, and the advance of excessive expressing recombinant structures. There also are equipment for the immunodectection and construction of immunological reagents and the research of biochemical homes, corresponding to subunit dimension, lipase kinetics, enzyme immobilization, and ligand interactions. The protocols covers greater than a dozen particular enzymes, and supply well-established equipment that may be tailored to the invention of recent lipase enzymes.

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L , Krueger, E T , and Ketm, P S (1977) Ammo acid sequenceof phospholtpaseA2 from the venom of Crotalus adamanteus J Blol Chem 252, 4913-4921 2 Davidson, F F and Dennis, E A (1990) Evoluttonary Relatronshtpsand Imphcations for the Regulation of phospholtpasesA2 from snake venom to human secretedforms J Mel Evol 31,228-238 Wane, M (1987) The Phosphollpases,Plenum, New York Tohkm, M , Ktshmo,J , Ishtzakt, J andArtta, H (1993)J Bzol Chem268,286>287 1 Kudo, I , Murakamt, M , Hara, S , and moue, K. (I 993) Mammaltan non-pancreatic phosphohpaseA, Blochlm Brophys Acta 1170, 2 17-23 1 de Geus, P , van den Bergh, C.

Owing to tts htghly cattonic nature (isoelectric point > lo), hIIa-PLA2 can be easily enriched from the growth medium using a cation exchange column. 1 Prepare the pYS-bv plasmtd by a standard protocol In order to improve transfecnon efficiency, purify the DNA using a lipopolysaccharide extraction ktt 2 Grow Sf9 cells as monolayer cultures m serum-free TNM-FH medium (see Note 3) 3 Co-transfect viral DNA with the pYS-bv vector and incubate cells for 4 d at 27°C 4 Collect the supematant and use tt to amplify the vuus with multiplicity of mfecnon of 0 5-l After three cycles of amphficatton use the high-titer virus stock solutton for the protein expression 5 Seed I x 1O7St-9 cells onto twenty 10 cm-culture plates (total volume of medium, 200 mL) and infect them with the high-titer viral solution with a multtphctty of infection of 10 6 Incubate the cells for 3 d at 27°C.

The vmyl ether lmkage of plasmalogen may also play an important role m protectmg cellular membranes against oxtdattve stresses (2,3). The breakdown of plasmalogen may be a receptor-mediated process related to stgnal transductron (4,s) Heart (6,7), brain (5,8), and kidney (9) contam a novel phosphohpase A, (PLA,) activity that hydrolyzes arachidonic acid from the SM-2position of plasmalogen. Thrs novel PLA2 has been purified from canme myocardmm (6,7), bovine brain (8), and renal proximal tubule (9).

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