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Preparation and characterization of plasma membrane-enriched fractions from rat pancreatic islets

Lernmark, ÅKe LU orcid ; Nathans, Anne and Steiner, Donald F. (1976) In Journal of Cell Biology 71(2). p.606-623
Abstract

Methods have been developed for the isolation on a semi-micro scale of a plasma membrane-enriched fraction from rat islets of Langerhans. An important feature of these experiments is the use of 125I-labeled wheat germ agglutinin as a specific probe for plasma membrane-containing fractions. The partly purified plasma membrane fraction had a density in sucrose of about 1.10 and was enriched in the activities of 5′-nucleotidase, alkaline phosphatase, sodium-potassium, and magnesium- dependent ATPases and adenylate cyclase. It contained only very low levels of acid phosphatase, cytochrome c oxidase, insulin, and RNA. Further purification was hampered by the relatively small amounts of fresh plasma membrane material that could be obtained... (More)

Methods have been developed for the isolation on a semi-micro scale of a plasma membrane-enriched fraction from rat islets of Langerhans. An important feature of these experiments is the use of 125I-labeled wheat germ agglutinin as a specific probe for plasma membrane-containing fractions. The partly purified plasma membrane fraction had a density in sucrose of about 1.10 and was enriched in the activities of 5′-nucleotidase, alkaline phosphatase, sodium-potassium, and magnesium- dependent ATPases and adenylate cyclase. It contained only very low levels of acid phosphatase, cytochrome c oxidase, insulin, and RNA. Further purification was hampered by the relatively small amounts of fresh plasma membrane material that could be obtained from 16-24 rats in each experiment. When islets were prelabeled with radioactive fucose, the plasma membrane-enriched fraction contained radioactivity at a four- to fivefold higher specific activity than the whole islet homogenate. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of plasma membrane-enriched fractions pooled from several experiments revealed a distinctive pattern of protein bands as compared with other less pure fractions. With respect to rapidity, apparent specificity, and easy reversibility of the labeling of the plasma membrane fraction, 125I-wheat germ agglutinin provides a highly useful tool for the detection of microgram quantities of plasma membrane components which should be applicable to many other systems as well.

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author
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publishing date
type
Contribution to journal
publication status
published
in
Journal of Cell Biology
volume
71
issue
2
pages
606 - 623
publisher
Rockefeller University Press
external identifiers
  • scopus:0017188694
  • pmid:791956
ISSN
0021-9525
DOI
10.1083/jcb.71.2.606
language
English
LU publication?
no
id
637ea260-42de-4d62-b780-0d7eaed7dd2b
date added to LUP
2019-09-18 12:12:13
date last changed
2024-04-02 16:34:08
@article{637ea260-42de-4d62-b780-0d7eaed7dd2b,
  abstract     = {{<p>Methods have been developed for the isolation on a semi-micro scale of a plasma membrane-enriched fraction from rat islets of Langerhans. An important feature of these experiments is the use of 125I-labeled wheat germ agglutinin as a specific probe for plasma membrane-containing fractions. The partly purified plasma membrane fraction had a density in sucrose of about 1.10 and was enriched in the activities of 5′-nucleotidase, alkaline phosphatase, sodium-potassium, and magnesium- dependent ATPases and adenylate cyclase. It contained only very low levels of acid phosphatase, cytochrome c oxidase, insulin, and RNA. Further purification was hampered by the relatively small amounts of fresh plasma membrane material that could be obtained from 16-24 rats in each experiment. When islets were prelabeled with radioactive fucose, the plasma membrane-enriched fraction contained radioactivity at a four- to fivefold higher specific activity than the whole islet homogenate. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of plasma membrane-enriched fractions pooled from several experiments revealed a distinctive pattern of protein bands as compared with other less pure fractions. With respect to rapidity, apparent specificity, and easy reversibility of the labeling of the plasma membrane fraction, <sup>125</sup>I-wheat germ agglutinin provides a highly useful tool for the detection of microgram quantities of plasma membrane components which should be applicable to many other systems as well.</p>}},
  author       = {{Lernmark, ÅKe and Nathans, Anne and Steiner, Donald F.}},
  issn         = {{0021-9525}},
  language     = {{eng}},
  month        = {{11}},
  number       = {{2}},
  pages        = {{606--623}},
  publisher    = {{Rockefeller University Press}},
  series       = {{Journal of Cell Biology}},
  title        = {{Preparation and characterization of plasma membrane-enriched fractions from rat pancreatic islets}},
  url          = {{http://dx.doi.org/10.1083/jcb.71.2.606}},
  doi          = {{10.1083/jcb.71.2.606}},
  volume       = {{71}},
  year         = {{1976}},
}