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Rapid purification of glycosyl-phosphatidylinositol-anchored alkaline phosphatase from human neutrophils after up-regulation to the cell surface

TJ Cain, Y Liu, T Kobayashi and JM Robinson

Department of Cell Biology, Neurobiology and Anatomy, Ohio State University, Columbus.

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.

Volume 41, Issue 9, pp. 1367-1372, 09/01/1993
Copyright © 1993 by The Histochemical Society


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