Purification and characterization of annexin proteins from bovine lung.

Calcium-dependent association with a detergent-extracted particulate fraction was used as the first step in the purification of a group of phospholipid binding proteins. Elution of the detergent-insoluble fraction with excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) resulted in the release of several soluble proteins, termed calcium-activated proteins or CAPs. In the present paper, we describe the simultaneous purification of these CAPs and characterize their interaction with phospholipid, actin, and calmodulin. Partial sequence analysis has identified the majority of the CAPs as members of the annexin family of calcium and phospholipid binding proteins. Two additional CAPs may be novel proteins, one of which appears to be an annexin protein. All CAPs demonstrated Ca2(+)-dependent binding to phosphatidylserine vesicles but did not bind to phosphatidylcholine vesicles. The majority of CAPs exhibited Ca2(+)-dependent binding to F-actin; however, only CAP-III affected the rate of conversion of G-actin to F-actin. The interaction of CAP-III and lipocortin-85 with F-actin resulted in a Ca2(+)-dependent increase in both light scattering and sedimentation of F-actin under comparatively low centrifugal force. In contrast, only lipocortin-85 caused the formation of F-actin bundles. Although all of the CAPs bound to a calmodulin affinity column in a Ca2(+)-dependent manner, attempts to demonstrate binding of CAPs to native calmodulin were unsuccessful. These studies therefore document the similar behavior of the CAPs toward phospholipid and calmodulin but clearly show that F-actin binding or bundling is not a general property of these proteins. The reported purification procedure should allow further comparative studies of these proteins.