An assay for glycosylphosphatidylinositol-anchor degrading phospholipases.
This paper describes a new approach to assay phospholipases which cleave glycosylphosphatidylinositol using a biotinylated protein substrate coupled to 125I-streptavidin and Triton X-114 phase separation. Substrate preparation with variant surface glycoprotein of Trypamosoma brucei, its characterization and solubilization by glycosylphosphatidylinositol-specific phospholipase C and D are reported. Hydrolysis of substrate exhibited first-order kinetics with respect to enzyme concentration, and the rate constant of the reaction is independent both from substrate concentration and reaction time. This assay was compared with the one using 3H-myristoylated variant surface glycoprotein and proved to be equally suitable to quantitate glycosylphosphatidylinositol-specific phospholipases, with the advantage that avoids biosynthetic labeling. Furthermore, it introduces a basic methodology which can be easily adapted to use other glycosylphosphatidylinositol-anchored proteins as substrates.
Stambuk
B U
BU
Department of Microbiology, Immunology and Parasitology, Universidade Federal de São Paulo, Brasil. bstambuk@mbox1.ufsc.br
Cardoso de Almeida
M L
ML
D000818:Animals; D001426:Bacterial Proteins; D001710:Biotin; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D007457:Iodine Radioisotopes; D007700:Kinetics; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010739:Phospholipase D / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D051381:Rats; D012995:Solubility; D019809:Streptavidin; D013379:Substrate Specificity; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000378:metabolism
D001426:Bacterial Proteins; D017261:Glycosylphosphatidylinositols; D007457:Iodine Radioisotopes; D014643:Variant Surface Glycoproteins, Trypanosoma; D001710:Biotin; D019809:Streptavidin; D010727:Phosphoric Diester Hydrolases; D010739:Phospholipase D; C065017:glycoprotein phospholipase D; D043265:Phosphatidylinositol Diacylglycerol-Lyase