Rat liver canalicular membrane vesicles. Isolation and topological characterization

Area of Science:

• Biochemistry
• Cell Biology
• Membrane Biology

Background:

• Rat liver canalicular plasma membranes are crucial for bile secretion.
• Understanding the orientation and protein localization of these membranes is vital for studying liver function and disease.

Purpose of the Study:

• To isolate and characterize rat liver canalicular plasma membranes.
• To determine the sidedness (orientation) of the isolated membrane vesicles.
• To localize specific enzymes, gamma-glutamyltransferase and aminopeptidase M, within these membranes.

Main Methods:

• Isolation of canalicular plasma membranes using nitrogen cavitation and calcium precipitation.
• Enrichment analysis of membrane-specific marker enzymes (gamma-glutamyltransferase, aminopeptidase M, alkaline phosphatase).
• Electron microscopy for structural integrity assessment.
• Enzyme activity assays and inhibition studies using specific antisera.
• Limited proteolysis with papain to assess enzyme accessibility.
• Binding studies with antibodies and radiolabeled protein A on isolated hepatocytes.

Main Results:

• The isolated membranes showed significant enrichment (55- to 56-fold) in canalicular enzymes.
• Electron microscopy and osmotic sensitivity confirmed the preparation consisted of right-side-out vesicles.
• Enzyme activity inhibition by specific antisera and antibody-induced density shift supported enzyme localization.
• Selective removal of gamma-glutamyltransferase and aminopeptidase M by papain indicated their luminal location.
• Specific antibody binding to isolated hepatocytes confirmed enzyme accessibility on the outer surface.

Conclusions:

• The study successfully isolated right-side-out oriented rat liver canalicular plasma membrane vesicles.
• Gamma-glutamyltransferase and aminopeptidase M are confirmed to be located on the luminal side of these membranes.
• The findings provide a valuable tool for further research into liver transport and enzyme function.