Simple method of preparation of rat colonocyte apical membranes using polyethylene glycol precipitation

Abstract

Background: Although a number of methods are available for the preparation of brush border membranes (BBM) from the small intestine, very few studies have been performed on the isolation of colonic apical membranes (CAM). Cation precipitation is one of the methods used for the isolation of these membranes, which can result in altered lipid composition as a result of activation of phospholipase. We have earlier established a method for BBM isolation using polyethylene glycol (PEG) precipitation. In the present study, CAM from isolated colonocytes were prepared using PEG and the isolated membranes were characterized.

Methods: The CAM were prepared from isolated rat colonocytes using PEG precipitation and differential centrifugation. Purity was assessed by enrichment of the marker enzymes and contamination by other subcellular particles. Lipid composition and sugar components were analyzed in the isolated membranes.

Results: The CAM showed 11-12-fold enrichment in alkaline phosphatase and 9-10-fold enrichment in gamma-glutamyl transpeptidase as compared with the cell homogenate. The final membrane preparation also showed good vesicle formation as seen by electron microscopy. There was very little contamination of basolateral membranes, microsomes or lysosomes in the final membrane preparation. Analysis of sugars indicated high content of fucose and sialic acid as compared with hexoses. Lipid analysis indicated the presence of various neutral and phospholipids and a cholesterol/phospholipid ratio of 0.65 was seen.

Conclusion: This study has shown a simple method of CAM preparation using PEG precipitation, which is less time-consuming than other methods and comparatively pure.