An assay for glycosylphosphatidylinositol-anchor degrading phospholipases.
J Biochem Biophys Methods
; 33(2): 105-15, 1996 Nov 15.
Article
in En
| MEDLINE
| ID: mdl-8951531
ABSTRACT
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.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
Phospholipase D
/
Glycosylphosphatidylinositols
/
Phosphoric Diester Hydrolases
Limits:
Animals
Language:
En
Journal:
J Biochem Biophys Methods
Year:
1996
Type:
Article
Affiliation country:
Brazil