Rapid purification of glycosyl-phosphatidylinositol-anchored alkaline phosphatase from human neutrophils after up-regulation to the cell surface.
Alkaline phosphatase (APase) belongs to a growing family of membrane-associated proteins tethered to the lipid bilayer via a glycosyl-phosphatidylinositol (GPI) anchor. Human neutrophils contain an intracellular pool of APase associated with a novel membrane-bound compartment. Stimulation of neutrophils with the chemotactic peptide formyl-Met-Leu-Phe (fMLP) leads to rapid up-regulation of essentially all of the APase to sites in continuity with the extracellular medium. Pre-treatment of neutrophils with cytochalasin B (cyto B) followed by fMLP likewise leads to expression of the enzyme on the cell surface and a dramatic alteration in cell morphology, but subsequent internalization of the plasmalemma is minimized. Pre-treatment with cyto B and fMLP has been used for isolation and purification of neutrophil APase. Specifically, neutrophils were treated with phosphatidylinositol-specific phospholipase C to release GPI-anchored proteins from the cell surface. APase was purified from supernatants of these preparations by electrophoresis in a non-denaturing gel system and subsequent electroelution. With this approach we rapidly purified neutrophil APase to homogeneity; this protein was then used for immunization. Immunoblotting, ELISA, and immunocytochemical localization were used to characterize the resulting antibodies.
Department of Cell Biology, Neurobiology and Anatomy, Ohio State University, Columbus.
T J
TJ
Cain
Y
Y
Liu
T
T
Kobayashi
J M
JM
Robinson
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D003571:Cytochalasin B / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D017261:Glycosylphosphatidylinositols / Q000032:analysis* / Q000378:metabolism / Q000502:physiology; D006801:Humans; D006868:Hydrolysis; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000166:cytology / Q000201:enzymology* / Q000648:ultrastructure; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000494:pharmacology; D010738:Type C Phospholipases / Q000494:pharmacology; D015854:Up-Regulation / Q000502:physiology*
D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D003571:Cytochalasin B; D009240:N-Formylmethionine Leucyl-Phenylalanine; D000469:Alkaline Phosphatase; D010727:Phosphoric Diester Hydrolases; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase