Two molar urea (pH 7.5) and column chromatography on Sepharose 4B were used to separate clathrin (coat protein) from the membrane of coated vesicles from bovine brain. Lytron (polystyrene) particles were used for study of the interaction of ciathrin with contractile proteins. Muscle G-actin, F-actin, and a-actinin were bound by clathrin-coated Lytron particles, while no interaction was found when muscle tropomyosin and serum albumin were tested. Clathrin molecules dispersed in a solution of 20 mM Tris*HCl (pH 7.5) were found to e elongated. When the pH was adjusted from 7.5 to 6.5, clathrin molecules associated into basketlike or cage structures similar in size and shape to those observed in enriched preparations of coated vesicles. Below pH 6.0, cages or baskets became amorphous aggregates. Raising the pH from 6.5 to 8.0, addition of 5-10 mM ATP or EDTA, or addition of 200 mM KCI resulted in the disassembly of baskets and the formation of filamentous arrays of various widths. Because of clathrin's biochemical and biophysical properties, its interaction with contractile proteins, and its presence in the membrane of vesicles of various cell types, we classified clathrin in the group of mechanochemical proteins.

A protein kinase activity was observed in coated vesicles, prepared from bovine brain, that had clathrinassociated protein 2 (CAP2, also known as clathrin light chain 2) as its principal substrate. Coated vesicles were purified by sucrose density gradient centrifugation followed by Sephacryl

Clathrin‐associated proteins were separated from clathrin under various clathrin‐denaturing conditions, i.e. heating, freezing and isoelectric precipitation. The proteins retained biological activity; they were purified further by affinity chromatography on calniodulin‐conjugated CNBr‐Sepharose 4B and used for antibody purification. The affinity‐purified anti‐(clathrin‐associated proteins) antibodies gave a fluorescent dotted pattern in cultured fibroblasts consistent with the known distribution of clathrin. Chemical cross‐linking of pure clathrin‐associated proteins indicated that these polypeptides exist as monomers in solution, each possessing Cazi‐dependent affinity Tor calmodulin to which they bind in a 1: 1 molar ratio. Chymotryptic treatment of coated vesicles selectively cleaved the clathrin‐associated proteins into a 15000–18000‐Mr doublet polypeptide. These subfragments retained their Ca2+‐dependent affinity for calmodulin. Our results support a regulatory role for clathrin‐associated proteins in clathrin assemblies.