Purification of annexin I and annexin II from human placental membranes by high-performance liquid chromatography.
Annexin I and annexin II were extracted from human placental membranes with ethylene glycol bis(beta-amino-ethyl ether)-N,N'-tetraacetic acid (EGTA) and purified by high-performance liquid chromatography by measuring their ability to inhibit phospholipase A2 activity in vitro. Neither protein was capable of binding to a DEAE-5PW HPLC column at neutral pH; however, they were resolved through binding to a Mono S column and passage through size-exclusion HPLC columns. Annexin I and its covalently linked dimer (36 and 66 kDa, respectively, by sodium dodecyl sulfate (SDS)-gel electrophoresis) reacted in one-dimensional immunoblots with monoclonal antibodies to annexin I and calpactin II, and with monoclonal and polyclonal antibodies to lipocortin I, confirming that annexin I, calpactin II, and lipocortin I are the same or closely related proteins. Milligram amounts of monomeric annexin I containing negligible amounts of the cross-linked dimeric annexin I were selectively isolated from placental membranes by using buffers containing the sulfhydryl reagent iodoacetic acid. Milligram amounts of cross-linked annexin I were selectively isolated when placental membranes were initially treated with buffers that did not contain iodoacetic acid and then extracted with Triton X-100, suggesting that sulfhydryl-dependent transglutaminase activity contributes to the selective isolation of this protein. A third phospholipase A2-inhibitory protein (35 kDa by SDS-gel electrophoresis) that reacted in immunoblots with monoclonal antibodies to calpactin I and annexin II, indicating their similar identity, was isolated. The procedure employed allows the rapid purification of annexins I and II in milligram amounts from placental membranes within 2 days.
Moore
K G
KG
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510.
Goulet
F
F
Sartorelli
A C
AC
D017305:Annexin A1 / Q000302:isolation & purification*; D017306:Annexin A2 / Q000302:isolation & purification*; D002462:Cell Membrane / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004533:Egtazic Acid; D005260:Female; D006801:Humans; D015151:Immunoblotting; D010920:Placenta / Q000737:chemistry*; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000302:isolation & purification*
D017305:Annexin A1; D017306:Annexin A2; D011257:Pregnancy Proteins; D004533:Egtazic Acid