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Characterization of plasma-membrane glycoproteins from functional domains of the rat hepatocyte.

ABSTRACT: Plasma-membrane glycoproteins from the three different functional domains of the rat hepatocyte were radioactively labelled by oxidation with NaIO4, followed by reduction with NaB3H4. Analysis of the radioactively labelled glycoproteins by polyacrylamide-gel electrophoresis revealed the presence of at least 12 major sialoglycoproteins in each different region of the hepatocyte surface. The Mr-110 000 component was homogeneously distributed over the plasma membrane, whereas the Mr-90 000 polypeptide was only located at the sinusoidal face. These radiolabelled glycoproteins were solubilized in 1% Triton X-100, and the soluble fraction was subjected to affinity chromatography on Sepharose-conjugated wheat-germ agglutinin (WGA). The labelled glycoproteins were poorly bound to WGA. Membrane glycoproteins were also labelled by the galactose oxidase/NaB3H4 method. The results show that the polypeptides with apparent Mr 170 000 from the sinusoidal, 230 000 from the canalicular and 170 000 from the lateral membranes were specifically labelled. When the membranes were treated with neuraminidase and galactose oxidase/NaB3H4, the electrophoretic patterns showed changes in the apparent Mr values of the glycoproteins, owing to loss of sialic acid, and a clear increase in labelling in the sinusoidal and canalicular membranes compared with the lateral membranes. When these labelled membranes were solubilized in 1% Triton X-100 and subjected to affinity chromatography on Sepharose-conjugated Ricinus communis agglutinin and/or Lens culinaris agglutinin, the results showed that the former columns efficiently bound the radiolabelled glycoproteins, whereas the latter columns bound poorly. The results show that there is a differential distribution of glycoproteins along the hepatocyte's surface.

SUBMITTER: Enrich C

PROVIDER: S-EPMC1144876 | biostudies-other | 1985 Apr

REPOSITORIES: biostudies-other

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Project description:1. Six rat liver plasma-membrane subfractions of different density and morphological, enzymic and chemical properties were prepared from homogenates by a combination of differential, rate-zonal and density-gradient centrifugation. They consisted of three vesicular 'light' subfractions of density 1.12-1.13 and three 'heavy' subfractions of density 1.16-1.18 containing membrane strips and intercellular junctions. 2. All six subfractions contained a basal adenylate cyclase activity. One of the 'light' subfractions that showed the highest glucagon-stimulated adenylate cyclase activity was identified as deriving form the blood-sinusoidal face of the hepatocyte. This subfraction, unlike the others, was contaminated by Golgi components, as indicated by its morphological properties and the presence of galactosyl- and sialyl-transferase activities. 3. All the six subfractions showed high activities of the following plasma-membrane marker enzymes: 5'-nucleotidase, alkaline phosphodiesterase (nucleotide pyrophosphatase), alkaline phosphatase, leucine naphthylamidase and Mg2+-activated adenosine triphosphatase. A 'light' subfraction that showed the highest specific activities of all the above marker enzymes, but lacked a glucagon-stimulated adenylate cyclase activity, was identified as deriving from the bile-canalicular face of the hepatocyte. 4. The 'heavy' subfractions, which showed generally the lowest activities of the above plasma-membrane enzyme markers, and were characterized by the presence of desmosomes and gap junctions, were taken to originate from the contiguous faces of the hepatocyte. 5. The protein composition of the six subfractions was generally similar, as shown by polyacrylamide-gel electrophoresis. Differences in the amounts of various protein and glycoprotein bands among the subfractions correlated with their morphology, enzymic composition and sialic acid content. 6. Hormonal and histochemical evidence supporting the identification of a bile-canalicular subfraction, a blood-sinusoidal subfraction and contiguous-face subfractions is discussed.

Project description:1. Non-desmosomal plasma membranes enriched in plasma-membrane marker enzymes and in metabolically labelled glycoproteins were isolated on a large scale from up to 500g of pig ear skin slices. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and periodic acid/Schiff staining revealed the presence of four major glycosylated components in the apparent molecular-weight range 150000-80000. 2. A large proportion of the marker enzymes, the d-[(3)H]glucosamine-labelled glycoproteins and the periodic acid/Schiff-stained glycoproteins were solubilized by 1% (w/v) sodium deoxycholate. However, several non-glycosylated proteins, in particular those with mol.wts. 81000, 41000 and 38000 (possibly cytoskeletal components), were relatively resistant to solubilization. 3. The deoxycholate-solubilized membranes were fractionated by lectin affinity chromatography using both concanavalin A-Sepharose 4B and lentil lectin-Sepharose 4B. From 75 to 85% of the applied glycoprotein was recovered from the columns. From 30 to 40% of the recovered glycoprotein was specifically bound by the lectins and was eluted with 2% (w/v) alpha-methyl d-mannoside. The enrichment of labelled glycoproteins in the material bound by the lectins (2.5-fold) was similar with both lectins, although the yield was somewhat greater when lentil lectin was used. The glycoprotein-enriched fraction was also enriched in all the plasma-membrane marker enzymes, indicating their probable glycoprotein nature. 4. The glycoprotein-enriched fraction contained the four major periodic acid/Schiff-stained bands that were detected in the original plasma membrane. They had apparent mol.wts. 147000, 130500, 108000 and 91400. The higher-molecular-weight components contained relatively more d-[(3)H]glucosamine, indicating differences in the sugar composition or in the metabolic turnover of the individual glycoproteins in culture. The material bound by the lectins also contained a number of lower-molecular-weight Coomassie Brilliant Blue-stained components. These were weakly stained by periodic acid/Schiff reagent and were lightly labelled with d-[(3)H]glucosamine, indicating that they contained less carbohydrate than the four major glycoprotein bands. 5. Chloroform/methanol-extracted plasma membranes and isolated glycoproteins had a similar carbohydrate composition, containing sialic acid, hexosamine, fucose, xylose, mannose, galactose and glucose. Glucose was not enriched in the isolated glycoproteins, suggesting that it may be a contaminant. Xylose, however, was enriched in the isolated glycoproteins. It remains to be established whether this sugar, which is not usually found in plasma-membrane glycoproteins, is a genuine constituent of plasma-membrane glycoproteins in the epidermis.

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Characterization of plasma-membrane glycoproteins from functional domains of the rat hepatocyte.

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