MEMBRANE LIPID-COMPOSITION AND VESICLE SIZE MODULATE BILIRUBIN INTERMEMBRANE TRANSFER - EVIDENCE FOR MEMBRANE-DIRECTED TRAFFICKING OF BILIRUBIN IN THE HEPATOCYTE

To characterize the mechanisms underlying intracellular bilirubin transport, stopped-flow fluorometry was utilized to study the effects of membrane vesicle size and lipid composition on the kinetics of unconjugated bilirubin movement between model and native hepatocyte membranes. Bilirubin transfer rates declined asymptotically with increasing donor vesicle diameter, due primarily to a 1.4 kcal.mol(-1) decrease in the entropy of activation for the larger vesicles. The incorporation of phosphatidylethanolamine and phosphatidylserine significantly enhanced the dissociation of bilirubin from phosphatidylcholine vesicles. Cholesterol induced a biphasic effect on the transfer rate constant; an initial decrease in rate from 248 to 217 s(-1) associated with cholesterol:phospholipid ratios up to 20% was followed by a dramatic rise to 312 s(-1) as the cholesterol concentration was increased to 70 mol %. The bilirubin dissociation rate from isolated rat liver endoplasmic reticulum (9.1 s(-1)) was significantly slower than for both basolateral and canalicular plasma membranes, which exhibited rate constants of 11.7 and 25.8 s(-1), respectively. Collectively, these data suggest that the cholesterol:phospholipid ratio is the principal determinant of bilirubin dissociation from membranes. We postulate that the inherent cellular membrane cholesterol gradient in the hepatocyte creates a directed flux of bilirubin from the plasma membrane to the endoplasmic reticulum and represents a potential driving force for intracellular bilirubin transport.