Midportion antibodies stimulate glycosylphosphatidylinositol-specific phospholipase D activity

Limited information is known regarding the regulation, structural features, and functional domains of glycosylphosphatidylinositol-specific phospholip ase D (GPI-PLD, EC 3.1.4.50), Previous studies demonstrated that trypsin cl eavage of GPI-PLD at or near Arg325 and/or Arg589 in bovine serum GPI-PLD w as associated with an increase in enzymatic activity. Since the Arg325 is p redicted to be in a region between the catalytic domain and predicted beta- propeller structure in the C-terminal portion of GPI-PLD (T, A. Springer, P roc. Natl. Acad. Sci, USA 94, 65-72, 1997), we hypothesized that this conne cting region is important for catalytic activity. Trypsin cleavage of human serum GPI-PLD, which has an Arg325 but lacks the Arg589 present in bovine serum GPI-PLD, also increased GPI-PLD activity. Peptide-specific antibodies to residues 275-296 (anti-GPI-PLD275) and a monoclonal antibody, 191, with an epitope encompassing Arg325, also stimulated GPI-PLD activity. Pretreat ing human GPI-PLD with trypsin demonstrated that anti-GPI-PLD275 only stimu lated the activity of intact GPI-PLD. These results suggest that trypsin ac tivation and anti-GPI-PPLD275 may have similar effects on GPI-PLD. Consiste nt with this is the observation that both manipulations decreased the affin ity of GPI-PLD for mixed micelle substrates. These results indicate that th e midportion region of GPI-PLD is important in regulating enzymatic activit y, (C) 1999 Academic Press.