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 ( K m = 52 nM) and did not degrade either PtdCho or PtdIns. Hydrophobic labeling of the anchor of the substrate with 3‐trifluoromethyl‐3‐( m ‐[ 125 I]iodophenyl)diazirine ([ 125 I]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 [ 125 I]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, o ‐phenanthroline and EGTA. It was stimulated by Ca 2+ in micromolar concentrations indicating that PtdIns‐glycan‐phospholipase D is a Ca 2+ ‐regulated enzyme.