Partial purification and characterization of the N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase of glycosylphosphatidylinositol anchor biosynthesis in African trypanosomes.
N-Acetylglucosaminylphosphatidylinositol (GlcNAc-PI) de-N-acetylase was solubilized from the bloodstream form of African trypanosomes using Zwittergent 3-14. The solubilized GlcNAc-PI de-N-acetylase was assayed using radiolabeled GlcNAc-PI substrates. The enzyme was partially purified about 140-fold from washed trypanosome membranes using conventional liquid chromatography. The enzyme has a Km of 1.5 microM. Replacement of the di-O-substituted D-myo-inositol of the natural GlcNAc-PI substrate by the L-myo-inositol isomer did not significantly alter the ability of the compound to act as a substrate for the de-N-acetylase, suggesting that the C-2 to C-5 hydroxyl groups of the myoinositol ring do not play a critical role in substrate recognition. A substrate analogue lacking fatty acids was a relatively poor substrate for the enzyme, indicating that the lipid component plays an important role in substrate recognition and/or presentation of the substrate to the enzyme in detergent micelles. Substrate analogues lacking the glycerophosphate component were not recognized by the enzyme, suggesting that this component is important in the substrate recognition process.
Milne
K G
KG
Department of Biochemistry, University of Dundee, United Kingdom.
Field
R A
RA
Masterson
W J
WJ
Cottaz
S
S
Brimacombe
J S
JS
Ferguson
M A
MA
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D003902:Detergents; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis*; D007700:Kinetics; D008823:Micelles; D010716:Phosphatidylinositols / Q000378:metabolism*; D051381:Rats; D012995:Solubility; D013237:Stereoisomerism; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D003902:Detergents; D017261:Glycosylphosphatidylinositols; D008823:Micelles; C081027:N-acetylglucosaminylphosphatidylinositol; D010716:Phosphatidylinositols; D000581:Amidohydrolases; C087696:N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase; D000117:Acetylglucosamine