Dataset Information

Preparation and characterization of the plasma membrane of pig lymphocytes.

ABSTRACT: Lymphocyte plasma membrane was isolated from minced pig mesenteric lymph node by differential centrifugation and by centrifuging through a sucrose density gradient. The yield of membrane was approx. 0.1% (dry wt. relative to wet wt. of lymph node). The purified material had a sucrose density of 1.14g/cm(3) and consisted mainly of smooth vesicles. The membrane fraction contained, apart from protein and lipid, 59mug of carbohydrate, 11mug of sialic acid and 28mug of RNA/mg of protein; no DNA was detected. The cholesterol/phospholipid molar ratio was 1.01. Specific activities (mumol of product/h per mg of protein) of 5'-nucleotidase, succinate dehydrogenase, acid phosphatase and glucose 6-phosphatase were 10.1, 0, 0.51 and 0.30 respectively. The membrane vesicles were aggregated by an antiserum against pig lymphocytes and adsorbed the agglutinins to whole lymphocytes present in the antiserum; the membrane fraction was 28 times as effective as whole cells (on a dry wt. basis) in removing the lympho-agglutinins. Antisera against the membrane fraction agglutinated whole lymphocytes. It is concluded that the preparation represents the plasma membrane of small lymphocytes. The plasma membrane of pig thymocytes was isolated by using the same procedure. Its properties were similar to those of the lymphocyte plasma membrane.

SUBMITTER: Allan D

PROVIDER: S-EPMC1179577 | biostudies-other | 1970 Nov

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

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.

Dataset Information

Preparation and characterization of the plasma membrane of pig lymphocytes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets