Protein-lipid interactions in antipodal plasma membranes of rat colonocytes

The isolation of apical membranes from rat proximal colonic epithelial cells is described. Differential centrifugation yielded a 'crude' membrane fraction which was further purified using sucrose density centrifugation. The final membrane fraction was enriched 20-28-fold over homogenate in alkaline phosphatase and cysteine-sensitive alkaline phosphatase specific activities. Lipid-protein interactions and lipid dynamics examined in apical and basolateral membranes prepared from colonocytes demonstrated: (1) apical membrane, as assessed by steady-state fluorescence polarization studies have a low lipid fluidity; (2) colonic basolateral membranes possess a greater lipid fluidity than apical membranes; (3) compositional differences in these antipodal membranes appear to explain these differences in lipid fluidity; (4) fluorescence polarization studies using diphenylhexatriene detect a thermotropic transition at 21-23 degrees C in apical membranes and liposomes prepared from lipid extracts of these membranes; (5) alkaline phosphatase and L-cysteine-sensitive alkaline phosphatase activities appear to be functionally dependent on the physical state of the apical membrane's lipid.