Highly purified bile-canalicular vesicles and lateral plasma membranes isolated from rat liver on Nycodenz gradients. Biochemical and immunolocalization studies.
1. A liver canalicular plasma-membrane fraction enriched 115-155-fold in five marker enzymes relative to the tissue homogenate was obtained by sonication of liver plasma membranes followed by fractionation in iso-osmotic Nycodenz gradients. 2. Two lateral-plasma membrane fractions were also collected by this procedure; the lighter-density fraction was still associated with canalicular membranes, as assessed by enzymic and polypeptide analysis. 3. The polypeptide composition of the domain-defined plasma-membrane fractions was evaluated. It was demonstrated by immunoblotting that the 41 kDa alpha-subunit of the inhibitory G-protein, associated in high relative amounts with canalicular plasma-membrane fractions, was partially lost in the last stage of purification; however, this subunit was retained by lateral plasma membranes. 4. Antibodies to the proteins of bile-canalicular vesicles were shown to localize to the hepatocyte surface in thin liver sections examined by immunofluorescent and immuno-gold electron microscopy. Two subsets of antigens were identified, one present on both sinusoidal and canalicular plasma-membrane domains and another, by using antisera pre-absorbed with sinusoidal plasma membranes, that was confined to the bile-canalicular domain.
Ali
N
N
National Institute for Medical Research, Mill Hill, London, U.K.
Aligue
R
R
Evans
W H
WH
D000818:Animals; D001648:Bile Canaliculi / Q000201:enzymology / Q000648:ultrastructure*; D015153:Blotting, Western; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology / Q000648:ultrastructure*; D002499:Centrifugation, Density Gradient; D007472:Iohexol; D008099:Liver / Q000201:enzymology / Q000648:ultrastructure*; D008297:Male; D008565:Membrane Proteins / Q000032:analysis; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D051381:Rats; D011919:Rats, Inbred Strains; D013010:Sonication
D008565:Membrane Proteins; D007472:Iohexol