Demonstration and partial characterisation of phospholipid methyltransferase activity in bile canalicular membrane from hamster liver

Background/Aims: Methylation of phosphatidylethanolamine to phosphatidylcho line predominantly takes place in mitochondrial-associated membrane and the endoplasmic reticulum of the liver. The transport of the phospholipids fro m endoplasmic reticulum to the bile canalicular membrane is via vesicular a nd protein transporters, In the bile canalicular membrane a flippase enzyme helps to transport phosphatidylcholine specifically to the biliary leaflet , The phosphatidylcholine then enters the bile where it accounts for about 95% of the phospholipids. We postulated that the increased proportion of ph osphatidylcholine in the bile canalicular membrane and the bile compared to the transport vesicles may be due to a methyltransferase activity in the b ile canalicular membrane which, using s-adenosyl methionine as the substrat e, converts phosphatidylethanolamine on the cytoplasmic leaflet to phosphat idylcholine, which is transported to the biliary leaflet. The aim of our st udy was to demonstrate and partially characterise methyltransferase activit y in the bile canalicular membrane. Methods: Organelles were obtained from hamster liver by homogenisation and separation by sucrose gradient ultracentrifugation. These, along with phosp hatidylethanolamine, were incubated with radiolabelled s-adenosyl methionin e. Phospholipids were separated by thin-layer chromatography and radioactiv ity was counted by scintigraphy, Results: We demonstrated methyltransferase activity (nmol of SAMe converted /mg of protein/h at 37 degrees C) in the bile canalicular membrane of 0.442 (SEM 0.077, n=8), which is more than twice that found in the microsomes at 0.195 (SEM 0.013, n=8). The Km and pH optimum for the methyltransferase in the bile canalicular membrane and the microsomes were similar (Km 25 and 2 8 mu M, respectively, pH 9.9 for both). The Vmax was different at 0.358 and 0.168 nmol of SAMe converted/mg of protein/h for the bile canalicular memb rane and the microsomes, respectively. Conclusion: The presence of the methyltransferase activity in the bile cana licular membrane may be amenable to therapeutic manipulation.