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

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Preparation and characterization of plasma membrane-enriched fractions from rat pancreatic islets. / Lernmark, ÅKe; Nathans, Anne; Steiner, Donald F.

In: Journal of Cell Biology, Vol. 71, No. 2, 01.11.1976, p. 606-623.

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T1 - Preparation and characterization of plasma membrane-enriched fractions from rat pancreatic islets

AU - Lernmark, ÅKe

AU - Nathans, Anne

AU - Steiner, Donald F.

PY - 1976/11/1

Y1 - 1976/11/1

N2 - 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.

AB - 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.

U2 - 10.1083/jcb.71.2.606

DO - 10.1083/jcb.71.2.606

M3 - Article

C2 - 791956

AN - SCOPUS:0017188694

VL - 71

SP - 606

EP - 623

JO - Journal of Cell Biology

JF - Journal of Cell Biology

SN - 0021-9525

IS - 2

ER -