Isolation and characterization of a phosphatidylinositol-glycan-anchor-specific phospholipase D from bovine brain.
In recent years an increasing number of proteins has been shown to be membrane-anchored by a covalently attached PtdIns-glycan residue. In mammalian cells little is known about PtdIns-glycan-specific phospholipases which might play a role in the metabolism of PtdIns-glycan-anchored proteins. In order to identify PtdIns-glycan-specific phospholipases, a rapid and sensitive assay for such enzymes was developed using the PtdIns-glycan-anchored amphiphilic membrane form of acetylcholinesterase as substrate. The rate of product formation was monitored by the increase in soluble hydrophilic acetylcholinesterase in the aqueous phase after separation in Triton X-114. With this assay we established the presence of a PtdIns-glycan-specific phospholipase in bovine brain. This enzyme was soluble and could be partially purified by a heat step followed by chromatography on DEAE-cellulose and by gel filtration on Sepharose CL-6B. PtdIns-glycan-specific phospholipase had a high affinity for the PtdIns-glycan anchor of the substrate (Km = 52 nM) and did not degrade either PtdCho or PtdIns. Hydrophobic labeling of the anchor of the substrate with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine [( 125I]TID) caused a marked decrease in the cleavage rate and methylation of the amino group of the glucosamine residue of the anchor decreased the cleavage rate to zero. Using [125I]TID-labeled substrate, diradylglycerol phosphate was identified as the second product showing that the cleavage specificity of PtdIns-glycan-specific phospholipase was that of a phospholipase D. PtdIns-glycan-specific phospholipase D was inhibited by mercurials, omicron-phenanthroline and EGTA. It was stimulated by Ca2+ in micromolar concentrations indicating that PtdIns-glycan-phospholipase D is a Ca2(+)-regulated enzyme.
Hoener
M C
MC
Abteilung Neurobiochemie, Universität Bern, Switzerland.
Stieger
S
S
Brodbeck
U
U
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D006358:Hot Temperature; D007700:Kinetics; D010739:Phospholipase D / Q000302:isolation & purification / Q000378:metabolism*; D010740:Phospholipases / Q000378:metabolism*; D013379:Substrate Specificity
D010740:Phospholipases; D010739:Phospholipase D; C065017:glycoprotein phospholipase D