[6] Purification of calpactins I and II and isolation of N-terminal tail of calpactin I

Publisher Summary Calpactins are Ca2+-binding proteins, which interact with phospholipids and the cytoskeletal proteins actin and spectrin. Calpactins I and II (also termed p34, p36, p39, or lipocortin or annexin) are substrates of protein tyrosine kinases in vivo and are found in cells as either a 38-kDa monomer or an M 90,000 tetramer of heavy (38 kDa) and fight (11 kDa) chain subunits. This chapter outlines the method for the isolation of calpactins I and II from readily available tissue sources (lung and placenta) in quantities (20–100 mg) sufficient for most biochemical studies. This method relies on the CA2+-dependent precipitation of calpactin together with cytoskeletal or membrane fragments, followed by solubilization with EGTA to achieve a high level of purity prior to column chromatography. For the higher yield of calpactin II, a phosphatidylserine affinity column, developed for the purification of protein kinase C, is found to work well. Since the two calpactins described here represent members of a much larger family of proteins with similar structure and function, it may be important in isolating putative homologs from other tissue or cell sources to prove their identity.