Isolation and lipid composition of apical and basolateral membranes of colonic segments of guinea pig

In adapting several methods of membrane isolation we established a successf ul way to purify apical and basolateral membranes of guinea pig colon in a parallel procedure. The conventional purification control by marker enzymes was applied. In addition, luminal membrane proteins were stained with Texa s Red. Apical and basolateral enterocyte membranes were enriched 10- to 12- fold by differential precipitation and via a continuous sorbitol gradient. The membrane fractions were examined with regard to their phospholipid (PL) and fatty acid patterns and to their cholesterol content. Fluorescence pol arization studies were carried out using 1,8-diphenyl-1,3,5-hexatrien. Rema rkable differences in the fatty acid pattern of the proximal and the distal colon were seen. Due to a higher content of oleic acid the saturation inde x of the apical membranes of the proximal colon is lower compared to that o f the apical membranes of the distal colon (0.34 +/- 0.03 vs 0.42 +/- 0.05) . The cholesterol content of the apical membranes of the proximal colon is markedly higher than that of the apical membranes of the distal colon (3.42 +/- 0.14 vs 1.88 +/- 0.29 mol/mol PL). There are no differences in the flu idity of these apical membranes, We assume a balancing mechanism between th e cholesterol content and the amount of saturated PL-fatty acids. (C) 1999 Academic Press.