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Purification of the trypanosome phospholipase C which cleaves the variant surface glycoprotein

Conference · · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
OSTI ID:6527393
; ;
The surface coat of Trypanosoma brucei is composed of many copies of the Variant Surface Glycoprotein (VSG). This protein is tethered to the cell membrane by a glycolipid moiety which contains dimyristylphosphatidylinositol. Following cell lysis, an endogenous, membrane-bound phospholipase C cleaves the glycolipid and releases the VSG in soluble form. The authors have purified a lipase which they believe is responsible for VSG release. This enzyme, designated VSG lipase, is assayed by measuring release of butanol-soluble /sup 3/H from VSG labeled with (/sup 3/H)myristate. The purification involves detergent extraction of trypanosome membranes, ammonium sulfate fractionation, hydrophobic chromatography, and cation exchange chromatography. The enzyme is purified roughly 2500 fold and is nearly homogeneous. Based on SDS-PAGE, it has an apparent subunit molecular weight of 37,000 daltons. This polypeptide co-fractionates with the activity during several fractionation procedures. The enzyme has an apparent s/sub 20,w/ of 3.8 S. The purified VSG lipase is active in the presence of EDTA; its activity is inhibited by organomercurials and stimulated by dithiothreitol. The purified enzyme releases dimyristylglycerol from VSG.
Research Organization:
Johns Hoplins School of Medicine, Baltimore, MD
OSTI ID:
6527393
Report Number(s):
CONF-8606151-
Conference Information:
Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:6
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALCOHOLS
CARBOHYDRATES
CARBOXYLESTERASES
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CELL MEMBRANES
CHELATING AGENTS
CHROMATOGRAPHY
CLEAVAGE
CRYSTAL STRUCTURE
EGTA
ELECTROPHORESIS
ENZYME ACTIVITY
ENZYMES
ESTERASES
GLUCOPROTEINS
GLYCOLS
HYDROLASES
HYDROXY COMPOUNDS
ION EXCHANGE CHROMATOGRAPHY
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LIPASE
MASTIGOPHORA
MEMBRANES
MICROSTRUCTURE
MOLECULAR WEIGHT
MONOCARBOXYLIC ACIDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
PARASITES
PROTEINS
PURIFICATION
SACCHARIDES
SEPARATION PROCESSES
TETRADECANOIC ACID
TRACER TECHNIQUES
TRITIUM COMPOUNDS
TRYPANOSOMA