Isolation of endocytic and exocytic populations of coated vesicles from perfused rat liver using an acetylcholinesterase mediated density shift technique

Rat liver contains endogenous acetylcholinesterase (AChE). The authors have isolated rat liver coated vesicles (CVs) and incubated them with the Karnovsky-Roots reagents and the AChE substrate, acetylthiocholine. A dense iron-copper precipitate is deposited at hydrolysis sites. When the CVs are subjected to sucrose-Ficoll-D20 density gradient ultracentrifugation, the AChE containing CVs are shifted to a denser region of the gradient. The molecular forms of AChE present in the CVs resemble secretory AChE not serum AChE. CVs isolated from perfused liver, treated with diisopropylfluorophosphate (DFP), to inactivate endogenous AChE, and allowed to resynthesize AChE for 30 minutes, contain AChE which is shown to be exocytic. When 125I-insulin and gal-AChE are co-perfused into DFP treated liver for 3 minutes, the CVs isolated and density shifted, approximately 80% of both Gal-AChE and 125I-insulin are in the shifted fraction which also contains approximately 50% of the CVs. Similar experiments have been carried out using 35S-methionine to label newly synthesized secretory proteins. They have measured the diameter of the endocytic and exocytic CVs and have found that the endocytic CVs are significantly larger than the exocytic CVs and more heterogeneous in size. When the cholesterol/phospholipid ratio was compared in both populations, the endocytic CVs had a significantly higher ratio than the exocytic CVs. Finally both populations were subjected to one dimensional and two dimensional SDS gel electrophoresis and were found to be remarkably similar in protein composition