PREPARATION AND CHARACTERIZATION OF AN ENDOGENOUSLY FLUORESCENT ANNEXIN FOR DETECTION OF APOPTOTIC CELLS

Annexin proteins specifically bind anionic phospholipids such as phosp hatidylserine, which are normally confined to the cytoplasmic leaflet of cellular membranes. During programmed cell death, or apoptosis, thi s phospholipid asymmetry is lost, and anionic phospholipids are expose d on the extracellular leaflet of the plasma membrane where they are a ccessible to exogenously added, labeled annexins. Chemically [e.g., fl uoroscein isothiocyanate (FITC)]-modified annexin V has been widely us ed to detect and enumerate apoptotic cells by how cytometry. We prepar ed chimeric proteins containing green fluorescent protein (GFP) fused to annexin V. A chimera containing GFP fused to the C-terminus of anne xin V was soluble and fluorescent, but was unable to bind phospholipid s. In contrast, a chimera containing GFP fused to the N-terminus of an nexin V specifically bound apoptotic cells. GFP-annexin V represents a sensitive and facile alternative to FITC-annexin V for studies of apo ptosis. (C) 1998 Academic Press.