A method for the assay of phosphatidylinositol-specific phospholipase D activity in serum.
A reproducible substrate for the assay of phosphatidylinositol-specific phospholipase D (PIPLD) can be prepared by extracting alkaline phosphatase from placental tissue with n-butanol under alkaline conditions. The alkaline phosphatase thus prepared retains its hydrophobic glycan phosphatidylinositol (GPI) anchor and aggregates into high M(r) forms. Incubation with serum hydrolyses the phosphate inositol linkage by PIPLD action, producing a less lipophilic, non-aggregated isoform of alkaline phosphatase. Three methods of measuring the amount of this isoform produced after a timed incubation with serum are described and compared: two types of phase partitioning systems, and electrophoresis and densitometry of the products after gradient-pore electrophoresis. All give comparable and reproducible measurements of PIPLD; however, the electrophoretic method is preferred for routine analysis.
Raymond
F D
FD
Royal Postgraduate Medical School, Hammersmith Hospital, London, UK.
Fortunato
G
G
Moss
D W
DW
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D004795:Enzyme Stability; D005260:Female; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008970:Molecular Weight; D017830:Octoxynol; D010618:Phenanthrolines / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism*; D010739:Phospholipase D / Q000097:blood*; D010920:Placenta / Q000201:enzymology; D011092:Polyethylene Glycols; D013696:Temperature
D017261:Glycosylphosphatidylinositols; D010618:Phenanthrolines; D010716:Phosphatidylinositols; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D000469:Alkaline Phosphatase; D010739:Phospholipase D; C025205:1,10-phenanthroline