Thomas A. Brasitus and David Schachter* abstract: Rat intestinal basolateral membranes undergo a reversible lipid thermotropic transition previously characterized by differential scanning calorimetry and fluorescence polarization. Arrhenius studies of these membranes provide evidence for two groups of intrinsic enzyme activities. 5'-Nu-cleotidase and adenylate cyclase (basal, NaF stimulated, and prostaglandin Et stimulated) show a discontinuity in the Arrhenius plot at 28-30 C, corresponding to the peak tem-perature of the lipid transition;(Na++ K+)-dependent adenosine triphosphatase, K+-dependent/>-nitrophenylphosphatase, and magnesium-dependent adenosine tri-phosphatase show a break point at 20-22 C, approximately 5-7 C below the lower critical temperature of the thermotropic transition. Benzylalcohol (50 mM) decreased the lipid transition temperature by approximately 6.0±1.0 C and concomitantly reduced the break point temperatures of the basal and stimulated adenylate cyclase activities by 4-7 C. Preparations of basolateral and microvillus membranes were studied with five fluorescent lipid probes (2-anthroylstearate, 12-anthroylstearate, dansylphosphatidylethanolamine, diphenylhexatriene, and retinol) by means of steady-state fluorescence polarization, and excited-state lifetimes were determined by single photon counting. The fluorescence an-isotropy of each probe was considerably greater in microvillus as compared to basolateral membranes, whereas the excitedstate lifetimes were similar. The results indicate that lipid molecules have greater motional freedom in the basolateral membrane. Composition studies suggest that the difference in lipid dynamics between the membranes results from the higher ratios of protein/lipid (w/w), cholesterol/phospholipid (mol/mol), and sphingomyelin/lecithin (mol/mol) inthe microvillus membrane. e microvillus (luminal) and basolateral (contraluminal) plasma membranes of the enterocyte, thepredominant cell type lining the small intestine, are highly differentiated for the processes of digestion, absorption, and secretion. These an-tipodal membranes, whose function is to regulate the exchange of substances betweenorganism and environment, differ from each other in ultrastructure (Bloom & Fawcett, 1968; Oda, 1976), enzyme activities (Douglas et al., 1972; Lewis et al., 1975; Murer et al., 1974, 1976), transport mechanisms (Murer et al., 1974), electrophysiological properties (Rose & Schultz, 1971; Okada et al., 1977), protein components (Fujita et al., 1973), and lipid composition (Forstner et al., 1968; Douglas et al., 1972; Kawai et al., 1974; Lewis et al., 1975). Although there is considerable information concerning individual proteins which mediate specific functions ineach membranetype (Murer et al., 1974, 1976; Kenny & Booth, 1978), the role of the lipids has been more obscure in the face of increasing recognition thatlipid-protein interactions can influence the protein activities of biological membranes (Fox, 1975; Lee, 1975; Razin, 1975; Melchior & Steim, 1976; Tada et al., 1978; Brasitus et al., 1979). Recently we described studies of the lipid dynamics and lipid-protein interactions in rat intestinal microvillus membranes (Schachter et al., 1976; Schachter & Shinitzky, 1977; Brasitus et al., 1979, 1980). These luminal membranes have characteristically low lipid fluidity, 1 as as-sessed by fluorescence polarization of lipid-soluble fluorophors (Schachter & Shinitzky, 1977), and undergo a reversible lipid thermotropic transition inthe range 23-39 C, with a peak temperature () 2 of 31 C on differential scanning calorimetry (DSC)(Brasitus et al., 1980). Moreover, a number of microvillus membrane …