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Establishment of plasma membrane domains in hepatocytes. I. Characterization and localization to the bile canaliculus of three antigens externally oriented in the plasma membrane

A membrane fraction denoted N2 upper was isolated from homogenates of rat liver by sucrose gradient centrifugation. This fraction, which was enriched 65-fold over the homogenate in 5'-nucleotidase activity, was used as an immunogen in goats. The antisera obtained contained antibodies to three p...

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Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 1983
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Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2112741/
https://www.ncbi.nlm.nih.gov/pubmed/6643579
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description A membrane fraction denoted N2 upper was isolated from homogenates of rat liver by sucrose gradient centrifugation. This fraction, which was enriched 65-fold over the homogenate in 5'-nucleotidase activity, was used as an immunogen in goats. The antisera obtained contained antibodies to three predominant polypeptides in the N2 upper membrane fraction, as shown by crossed immunoelectrophoresis. These polypeptides had molecular weights of 105,000, 110,000, and 160,000 after recovery from the crossed immunoelectrophoretic gels and are denoted PM105, PM110, and PM160. Each was a distinct polypeptide, as shown by the distinct peptide patterns resulting from limited proteolysis in the presence of detergents. The three polypeptides were synthesized by primary cultures of hepatocytes and were externally oriented at the surface of these cells, as shown by their accessibility in situ to iodination catalyzed by lactoperoxidase. They were not detectable in the serum by crossed immunoelectrophoresis. The three antigens were present at very low (PM110) or nondetectable (PM105, PM160) concentrations in intracellular membrane fractions derived from the Golgi and smooth and rough endoplasmic reticulum of liver. The antigens also were reduced in concentration in a plasma membrane fraction most likely derived from the sinusoidal surface of the hepatocyte. The three membrane antigens bind to concanavalin A; hence, they are probably glycoprotein constituents of a discrete domain of the hepatocyte plasma membrane. Immune complexes were isolated after crossed immunoelectrophoresis and injected into rabbits. Each of the antisera obtained was reactive to one of the membrane polypeptides. Sections of fixed rat livers were reacted with each of the antibodies and then the primary antibody was localized by indirect immunocytochemical methods using horseradish peroxidase or colloidal gold as labels. Each of the three antigens was localized by this method to the bile canalicular domain of the hepatocyte plasma membrane.
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spelling pubmed-21127412008-05-01 Establishment of plasma membrane domains in hepatocytes. I. Characterization and localization to the bile canaliculus of three antigens externally oriented in the plasma membrane J Cell Biol Articles A membrane fraction denoted N2 upper was isolated from homogenates of rat liver by sucrose gradient centrifugation. This fraction, which was enriched 65-fold over the homogenate in 5'-nucleotidase activity, was used as an immunogen in goats. The antisera obtained contained antibodies to three predominant polypeptides in the N2 upper membrane fraction, as shown by crossed immunoelectrophoresis. These polypeptides had molecular weights of 105,000, 110,000, and 160,000 after recovery from the crossed immunoelectrophoretic gels and are denoted PM105, PM110, and PM160. Each was a distinct polypeptide, as shown by the distinct peptide patterns resulting from limited proteolysis in the presence of detergents. The three polypeptides were synthesized by primary cultures of hepatocytes and were externally oriented at the surface of these cells, as shown by their accessibility in situ to iodination catalyzed by lactoperoxidase. They were not detectable in the serum by crossed immunoelectrophoresis. The three antigens were present at very low (PM110) or nondetectable (PM105, PM160) concentrations in intracellular membrane fractions derived from the Golgi and smooth and rough endoplasmic reticulum of liver. The antigens also were reduced in concentration in a plasma membrane fraction most likely derived from the sinusoidal surface of the hepatocyte. The three membrane antigens bind to concanavalin A; hence, they are probably glycoprotein constituents of a discrete domain of the hepatocyte plasma membrane. Immune complexes were isolated after crossed immunoelectrophoresis and injected into rabbits. Each of the antisera obtained was reactive to one of the membrane polypeptides. Sections of fixed rat livers were reacted with each of the antibodies and then the primary antibody was localized by indirect immunocytochemical methods using horseradish peroxidase or colloidal gold as labels. Each of the three antigens was localized by this method to the bile canalicular domain of the hepatocyte plasma membrane. The Rockefeller University Press 1983-12-01 /pmc/articles/PMC2112741/ /pubmed/6643579 Text en This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
spellingShingle Articles
Establishment of plasma membrane domains in hepatocytes. I. Characterization and localization to the bile canaliculus of three antigens externally oriented in the plasma membrane
title Establishment of plasma membrane domains in hepatocytes. I. Characterization and localization to the bile canaliculus of three antigens externally oriented in the plasma membrane
title_full Establishment of plasma membrane domains in hepatocytes. I. Characterization and localization to the bile canaliculus of three antigens externally oriented in the plasma membrane
title_fullStr Establishment of plasma membrane domains in hepatocytes. I. Characterization and localization to the bile canaliculus of three antigens externally oriented in the plasma membrane
title_full_unstemmed Establishment of plasma membrane domains in hepatocytes. I. Characterization and localization to the bile canaliculus of three antigens externally oriented in the plasma membrane
title_short Establishment of plasma membrane domains in hepatocytes. I. Characterization and localization to the bile canaliculus of three antigens externally oriented in the plasma membrane
title_sort establishment of plasma membrane domains in hepatocytes. i. characterization and localization to the bile canaliculus of three antigens externally oriented in the plasma membrane
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2112741/
https://www.ncbi.nlm.nih.gov/pubmed/6643579