Isolation and preliminary characterization of clathrin-associated proteins.
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 calmodulin-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 Ca2+-dependent affinity for calmodulin to which they bind in a 1:1 molar ratio. Chymotryptic treatment of coated vesicles selectively cleaved the clathrin-associated proteins into a 15 000-18 000-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.
D002118:Calcium; D002966:Clathrin; D003432:Cross-Linking Reagents; D007120:Immunochemistry; D008565:Membrane Proteins / Q000032:analysis* / Q000302:isolation & purification*; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence
D002966:Clathrin; D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D002118:Calcium