title: Characterization of a recombinant form of annexin VI for detection of apoptosis.
abstract: We developed a recombinant form of human annexin VI called annexin VI-601 (M(r) 76,224) with the N-terminal extension of Ala-Gly-Gly-Cys-Gly-His to allow ready attachment of fluorescent or radioactive labels. The protein was produced by expression in E. coli and was purified by calcium-dependent membrane binding, anion-exchange chromatography, and heparin-Sepharose affinity chromatography. The protein could be readily labeled with iodoacetamidofluorescein and with (99m)Tc. The protein bound with high affinity to PS-containing phospholipid vesicles and to erythrocytes with exposed phosphatidylserine. Fluorescent annexin VI-601 readily detected apoptosis of Jurkat cells by flow cytometry at much lower calcium concentrations than those required for equivalent detection by annexin V. In vivo administration of radiolabeled protein showed that blood clearance was much slower than annexin V. In conclusion, annexin VI may have advantages over annexin V in certain situations for both in vitro and in vivo detection of apoptosis and therapeutic targeting of PS due to its lower calcium requirement for membrane binding and its higher molecular weight.
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, WA 98195-7110, USA.
authors.forename: Christina
authors.initials: C
authors.lastname: Smith
authors.forename: Ranjana
authors.initials: R
authors.lastname: Mehta
authors.forename: Donald F
authors.initials: DF
authors.lastname: Gibson
authors.forename: Zoia
authors.initials: Z
authors.lastname: Levashova
authors.forename: Francis G
authors.initials: FG
authors.lastname: Blankenberg
authors.forename: Jonathan F
authors.initials: JF
authors.lastname: Tait
mesh_terms: D000818:Animals; D017317:Annexin A6 / Q000096:biosynthesis / Q000097:blood / Q000737:chemistry*; D017209:Apoptosis; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D005453:Fluorescence; D006801:Humans; D019169:Jurkat Cells / Q000166:cytology; D051379:Mice; D008807:Mice, Inbred BALB C; D010743:Phospholipids / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000097:blood / Q000737:chemistry
chemical_list: D017317:Annexin A6; D010743:Phospholipids; D011994:Recombinant Proteins