Midportion antibodies stimulate glycosylphosphatidylinositol-specific phospholipase D activity.
Limited information is known regarding the regulation, structural features, and functional domains of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD, EC 3. 1.4.50). Previous studies demonstrated that trypsin cleavage of GPI-PLD at or near Arg325 and/or Arg589 in bovine serum GPI-PLD was associated with an increase in enzymatic activity. Since the Arg325 is predicted 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, Proc. Natl. Acad. Sci. USA 94, 65-72, 1997), we hypothesized that this connecting 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-PLD(275)) and a monoclonal antibody, 191, with an epitope encompassing Arg325, also stimulated GPI-PLD activity. Pretreating human GPI-PLD with trypsin demonstrated that anti-GPI-PLD(275) only stimulated the activity of intact GPI-PLD. These results suggest that trypsin activation and anti-GPI-PPLD(275) may have similar effects on GPI-PLD. Consistent with this is the observation that both manipulations decreased the affinity of GPI-PLD for mixed micelle substrates. These results indicate that the midportion region of GPI-PLD is important in regulating enzymatic activity.
Deeg
M A
MA
Department of Medicine, Indiana University School of Medicine and the Richard L. Roudebush Veterans Affairs Medical Center, Indianapolis, Indiana 46202-2284, USA. mdeeg@iupui.edu
Bowen
R F
RF
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D020134:Catalytic Domain; D002417:Cattle; D004789:Enzyme Activation / Q000187:drug effects; D000939:Epitopes / Q000737:chemistry; D006801:Humans; D007120:Immunochemistry; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D010739:Phospholipase D / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity; D014357:Trypsin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D010739:Phospholipase D; C065017:glycoprotein phospholipase D; D014357:Trypsin