Molecular characterization of the AP180 coated vesicle assembly protein.
Recently, a new clathrin assembly protein (AP180) has been purified from coated vesicles of bovine brain (Ahle & Ungewickell, 1986). This protein has been shown to promote polymerization of clathrin into a homogeneous population of baskets under conditions where pure clathrin does not polymerize by itself. We have purified this protein from coated vesicles by a simpler method than has been reported. The method involves a gel filtration step on a Sephacryl S-300 column, in 0.5 M Tris-HCl, pH 8.0, and a hydroxylapatite column eluted with 10 mM sodium phosphate/0.5 M Tris-HCl, pH 7.0. By running SDS gels over an extended period of time (5-15% gradient gel, 10 mA for the first 12 h followed by 20 mA for the next 3-4 h) after the marker dye entered the electrode buffer, we have been able to separate AP180 from clathrin heavy chain on the gels. This enabled us to determine its stoichiometry to clathrin heavy chains in isolated coated vesicles and assembled baskets, and was helpful in the purification procedure. The apparent molecular weight of the pure protein on SDS gels was about 180,000, yet gel filtration yielded values of about 120,000. Thus, we undertook the molecular weight determination by another independent method, sedimentation equilibrium analysis, and found a molecular weight of 115,000 and a sedimentation coefficient of 3.50 +/- 0.05 S. Circular dichroism data revealed that it has 30% helical structure, 14% beta-structure, 27% beta-sheet, and the rest random peptides.(ABSTRACT TRUNCATED AT 250 WORDS)
Prasad
K
K
Clinical Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
Lippoldt
R E
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D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002942:Circular Dichroism; D002966:Clathrin / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification*; D011108:Polymers; D011487:Protein Conformation; D014461:Ultracentrifugation
D002966:Clathrin; D010750:Phosphoproteins; D011108:Polymers