| Abstract: | Postmicrosomal pellets from plant sources are grossly contaminatedwith nbosomes. Previous purifications of clathrin coated vesicles(CCV) from such subcellular fractions have therefore often involvedan RNase treatment. Performed at 30°C, this step inherentlycarries with it the dangers of proteolysis. We document herea method for CCV isolation which avoids this. Through the inclusionof suitable antiproteases in the homogenizing and subsequentisolation media, we have also been able to improve the qualityof CCV recovered from plant tissues. As a result we have tentativelybeen able to identify clathrin light chains from zucchini hypocotyland pea cotyledon CCV. Similar to light chains from bovine brainthese polypeptides are heat stable, can be solubilized fromneutralized TCA precipitates, bind calcium and clathrin heavychains. However, in contrast to brain CCV the two light chainsof plant CCV are some 10 kDa heavier. Key words: Antiproteases, Ca2+-binding, clathrin coated vesicles, clathrin heavy chains, clathrin light chain(s), heat stability, pea cotyledons, RNase, zucchini hypocotyls |
