Preparation of a homogeneous coated vesicle fraction from bean leaves

Summary Using a procedure previously developed for suspension-cultured carrot cells, we have been able to isolate two different coated vesicle-containing fractions from green bean leaves (Vicia faba). The two fractions differ in their isopycnic densities in D₂O-Ficoll as well as in their diameters. One of the fractions (the less dense of the two) is almost 100% pure as judged by negative staining. Because of this the polypeptide pattern obtained from SDS-PAGE is most clear and has enabled a clear recognition of clathrin light chains, in addition to the 190 kDa heavy chain coat component. Significantly the 100k Da and 50k Da polypeptides typical of brain coated vesicles are absent from bean leaf coated vesicles. Due to a) the high degree of vacuolation b) the presence of large amounts of ribulose bisphosphate carboxylase in the postmicrosomal supernatant, the yield of coated vesicles from bean leaves, as compared to nongreen plant cells, or to bovine brain tissue is extremely low (1 mg coated vesicles from 2.4 kg leaf tissue).Abbreviations D₂O deuterium oxide - EGTA ethylene glycol-bis ( amino ethyl ether) N,N,N,N tetraacetic acid - MES, 2 (N-morpholino)-ethanesulfonic acid - PMSF phenyl methylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TRIS Tris-hydroxy methyl amino methane