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 tran sport, stopped-flow fluorometry was utilized to study the effects of m embrane vesicle size and lipid composition on the kinetics of unconjug ated bilirubin movement between model and native hepatocyte membranes. Bilirubin transfer rates declined asymptotically with increasing dono r vesicle diameter, due primarily to a 1.4 kcal.mol(-1) decrease in th e entropy of activation for the larger vesicles. The incorporation of phosphatidylethanolamine and phosphatidylserine significantly enhanced the dissociation of bilirubin from phosphatidylcholine vesicles. Chol esterol induced a biphasic effect on the transfer rate constant; an in itial decrease in rate from 248 to 217 s(-1) associated with cholester ol:phospholipid ratios up to 20% was followed by a dramatic rise to 31 2 s(-1) as the cholesterol concentration was increased to 70 mol %. Th e bilirubin dissociation rate from isolated rat liver endoplasmic reti culum (9.1 s(-1)) was significantly slower than for both basolateral a nd 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 bil irubin dissociation from membranes. We postulate that the inherent cel lular membrane cholesterol gradient in the hepatocyte creates a direct ed flux of bilirubin from the plasma membrane to the endoplasmic retic ulum and represents a potential driving force for intracellular biliru bin transport.