A phospholipase C from Trypanosoma brucei which selectively cleaves the glycolipid on the variant surface glycoprotein.
The surface coat of Trypanosoma brucei is composed of 10(7) molecules of the variant surface glycoprotein (VSG). Each VSG molecule is tethered to the cell membrane by a glycolipid moiety which contains 1,2-dimyristoyl-sn-phosphatidylinositol (Ferguson, M. A. J., Low, M. G., and Cross, G. A. M. (1985) J. Biol. Chem. 260, 14547-14555). Following cell lysis, an endogenous phospholipase C cleaves dimyristoyl glycerol from the glycolipid, releasing soluble VSG. We have purified this enzyme, which we designate VSG lipase, by detergent extraction, (NH4)2SO4 fractionation, hydrophobic chromatography, and cation exchange chromatography. It is purified 2600-fold and is virtually homogeneous. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent molecular mass is 37 kDa. In solutions containing the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), the Stokes radius (2.6 nm), S20,w (3.7 S), and v (0.77 cm3/g) of VSG lipase suggest a molecular mass for the native enzyme of about 47 kDa, part of which may be due to bound CHAPS. Therefore, it is probably monomeric. VSG lipase does not require Ca2+; it is stimulated by chelating agents or dithiothreitol, and it is inhibited by some sulfhydryl reagents. The purified enzyme appears to be highly specific. Under the conditions of our assay, it cleaves the VSG glycolipid, a biosynthetic precursor of the VSG glycolipid, and, to a much lesser extent, 1,2-dimyristoyl-sn-phosphatidylinositol. There was no apparent cleavage of other myristate-containing lipids of trypanosomes or 1-stearoyl-2-arachidonoyl-sn-phosphatidylinositol.
D000818:Animals; D006017:Glycolipids / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D007700:Kinetics; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D011487:Protein Conformation; D014316:Tritium; D014346:Trypanosoma brucei brucei / Q000201:enzymology*; D010738:Type C Phospholipases / Q000302:isolation & purification / Q000378:metabolism*; D014643:Variant Surface Glycoproteins, Trypanosoma
D006017:Glycolipids; D006023:Glycoproteins; D009227:Myristic Acids; D014643:Variant Surface Glycoproteins, Trypanosoma; D019814:Myristic Acid; D014316:Tritium; D010738:Type C Phospholipases