Fluorescence polarization studies of rat intestinal microvillus membranes

Abstract

Rat intestinal microvillus membranes and lipid extracts prepared from them have been studied by fluorescence polarization with three lipid-soluble fluorophores: diphenylhexatriene, retinol, and anthroyl-stearate. The degree of fluorescence polarization of diphenylhexatriene, which provides an index of the "microviscosity" of the lipid regions of the membrane, is exceptionally high in microvillus membranes, the highest yet reported in normal biological membranes. Both the membrane proteins and lipids were found to contribute to the high values. With each of the three probes the polarization values are higher in ileal microvillus membranes as compared to membranes from proximal intestinal segments. Temperature-dependence studies of the fluorescence polarization of diphenylhexatriene and anthroylstearate demonstrate a phase transition in microvillus membranes and in liposomes prepared from their lipid extracts at approximately 26+/-2 degrees C. Ambient pH influences markedly the diphenylhexatriene fluorescence polarization in microvillus membranes but has little effect on that of human erythrocyte ghost membranes. The "microviscosity" of jejunal microvillus membranes is maximal at pH 6.5-7.0 and decreases as much as 50% at pH 3.0, an effect which depends largely upon the membrane proteins. Addition of calcium ions to suspensions of microvillus membranes increases the fluorescence polarization of retinol and anthroyl-stearate, but not that of diphenyl-hexatriene. This confirms the localization of the last compound to the hydrophobic interior of the membrane, relatively distant from the hydrophilic head groups of the polar lipids. Microvillus membrane proteins solubilized with Triton X-100 give relatively high fluorescence polarization and intensity values with retinol, suggesting the presence of binding proteins which could play a role in the normal absorptive mechanism for the vitamin.