Lipase and phospholipase, Edition: 1st Edition. by Mark Doolittle

By Mark Doolittle

A different choice of step by step equipment for the characterization, purification, and expression of mammalian lipases and phospholipases. The equipment comprise the guidance of quite a few distinctive substrates for the choice of lipolytic task, the isolation of purified lipase arrangements, and the improvement of excessive expressing recombinant structures. There also are equipment for the immunodectection and construction of immunological reagents and the research of biochemical houses, akin to subunit measurement, 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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1996) Plasmalogen-selecttve phosphohpase A2 and its role m stgnal transduction J LzpzdMedlat Cell Szgnall4,9-13 6 Hazen, S. L , Loeb, L. , and Gross, R W (1991) Purtficatton and charactertzatron of cytosoltc phospholtpase A, acttvmes from canme myocardmm and sheep platelets Methods Enzymol 197,400-411. 7 Hazen, S L and Gross, R W (1992) Identtficatlon and characterization of human myocardtal phosphohpase A, from transplant rectptents suffering from end-stage tschemtc heart dtsease C~rc Res 70,486-495.

Farooqui, A A , and Horrocks, L A (1994) Effects of glycosamtnoglycans and glycosphmgohptds on cytosoltc phosphohpases A2 from bovme brain Blochem J 299,91-95 Plasmalogen-Selective PLA, 47 13 Farooqur, A A and Horrocks, L A (1988) Methods for the determmatton of phosphohpases, hpases and lysophospholtpases m Neuromethods, vol. 7 (Boulton, G B and Horrocks, L A , eds ), Humana, Totowa, NJ, pp 179-209 14 Bhgh, E G and Dyer, W J (1959) A raped method of total hptd extraction and purtficatton Can J Blochem Physzol 37,911-917 15.

Elute the bound protem with 1 A4 NaCl m the same buffer 11 Dialyze the eluted protein solution against 25 mA4 HEPES buffer, pH 8 0 12. Equthbrate a Mono S 5/5 column with 25 mM HEPES buffer, pH 8 0 13 Load the protein solutton onto the column, and elute with a linear gradient of NaCl from 0 to 2 A4 m the same buffer 14. Collect the major protein peak eluted with approx 0 8 M NaCI, dialyze against water, and lyophilize The lyophtltzed hs-PLA, can be stored indefinitely at -2O’C without any detectable loss of acttvtty 36 Cho et al.

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