Purification and characterization of a molecular weight 100,000 coat protein from coated vesicles obtained from...

A protein designated as a 100-kDa protein on the basis of sodium dodecyl sulfate gel electrophoresis was purified from coated vesicles obtained from bovine brain, with uncoated vesicles as starting material. Two gel filtration steps, one involving 0.5 M tris(hydroxymethyl)aminomethane, pH 8.0, buffer, and the other 0.01 M tris(hydroxymethyl)aminomethane, pH 8.0, and 3 M urea buffer, were employed. The purified protein has a native molecular weight of 114,000 as determined by sedimentation equilibrium analysis. Circular dichroism data showed that the protein has 28% helical structure, 29% beta-structure, and 15% beta-turns, and the rest is random coil. Addition of the purified protein to clathrin results in the polymerization of clathrin to homogeneous size baskets of sedimentation velocity 150 S. A scan of the Coomassie Blue stained electrophoresis gels of the polymerized baskets shows that, for every clathrin trimer, there is approximately one 100-kDa protein molecule.