CHARACTERIZATION OF THE ATP-DEPENDENT TAUROCHOLATE-CARRIER PROTEIN (GP110) OF THE HEPATOCYTE CANALICULAR MEMBRANE

The canalicular domain-specific glycoprotein gp110, which recently has been shown to function as an ATP-dependent taurocholate transporter, has been purified 1800-fold from rat liver plasma membranes. gp110 has been characterised as an integral plasma membrane protein with M(r) of 100000-115000 and pI of 2.5-3.5 and possesses a highly glycosylated and negatively charged extra-cellular domain. The broad range of M(r) and pI values results from the existence of numerous glycoforms composed of sialylated N-glycans. After deglycosylation, the polypeptide has M(r) 48000 and pI 5.0. In primary cultures of rat hepatocytes, gp110 is synthesised with M(r) 110000, while in the presence of tunicamycin the non-glycosylated form has M(r) 48000. In the presence of 1-deoxymannojirimycin, two forms of M(r) 83000 and M(r) 91000 were found, which were converted by endo-beta-N-acetylglucosaminidase H into a single 52000-M(r) band, indicating the existence of two basic glycoforms at the oligomannosyl stage of biosynthesis. gp110 was phosphorylated at serine residues in primary cultures of hepatocytes. The sequences of ten internal peptides of gp110 were identical to the sequence of the high-M(r) form of ecto-ATPase, but ecto-ATPase activity from plasma-membrane extracts was not depleted by anti-(gp110) serum. In contrast, Fab fragments of these antibodies inhibit the aggregation of freshly isolated hepatocytes.